CN101160525A - Unnatural reactive amino acid genetic code additions - Google Patents

Unnatural reactive amino acid genetic code additions Download PDF

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CN101160525A
CN101160525A CNA2004800211558A CN200480021155A CN101160525A CN 101160525 A CN101160525 A CN 101160525A CN A2004800211558 A CNA2004800211558 A CN A2004800211558A CN 200480021155 A CN200480021155 A CN 200480021155A CN 101160525 A CN101160525 A CN 101160525A
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amino acid
alpha
trna
natural amino
protein
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A·戴特斯
A·T·克罗普
J·W·钦
C·J·安德森
P·G·舒尔茨
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Scripps Research Institute
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Scripps Research Institute
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Abstract

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNAsynthetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Description

The unnatural reactive amino acid genetic code increases
The cross reference of related application
The application is based on the USSN60/479 that is entitled as " expansion the eukaryotic genetic code " that Chin equals submission on June 18th, 2003,931, Chin equals the USSN60/493 that is entitled as " expansion the eukaryotic genetic code " of submission on August 5th, 2003,014 and Chin equal the USSN60/496 that is entitled as " expansion the eukaryotic genetic code " that submitted on August 19th, 2003,548 conventional use patent application.This paper requires these right of priority and interests in first to file in view of the above.
Statement about the invention right of doing in the research and development of federal funding
The present invention finishes under the subsidy of the government-funded of NIH fund numbering GM62159 and the fund DE-FG0300ER45812 of Ministry of Energy.Government has certain right of the present invention.
Invention field
The invention belongs to the translation biochemical field in the eukaryotic.The present invention relates to the production method and the composition of quadrature tRNA, quadrature synzyme and their pairings in eukaryotic.The present invention also relates to the composition, protein of alpha-non-natural amino acid and comprise at eukaryotic and to produce method of protein in the alpha-non-natural amino acid.
Background of invention
From the bacterium to the mankind, the genetic code of each known organism 20 the identical common amino acids of all encoding.The various combination of these 20 identical natural amino acids forms protein, carries out in fact all complicated life processes, from photosynthesis to the signal transduction and immune response.In order to study the 26S Proteasome Structure and Function with modified protein, scientists attempts to handle the amino acid sequence of genetic code and protein.But, be difficult to remove the restriction of forcing by genetic code, be about to the standard element that protein is limited to 20 genetic codings (seldom remove selenocysteine (referring to, for example, A.Bock etc., (1991), Molecular Microbiology 5:515-20) and outside pyrroles's lysine (referring to, for example, G.Srinivasan, Deng, (2002), Science 296:1459-62).
Obtaining some progress aspect these restrictions eliminating, though this progress has been limited and the ability of rationally controlling protein structure and function still just is in the embryonic stage.For example, the chemist developed synthetic and handle the method for small molecule structure and strategy (referring to, for example, E.J.Corey and X.-M.Cheng, " logic of chemosynthesis " (The Logic of Chemical Synthesis) (Wiley-Interscience, New York, 1995)).Complete synthesizing (referring to, for example, B.Merrifield, (1986), Science 232:341-7 (1986)) and semisynthesis (referring to, for example, D.Y.Jackson etc., (1994) Science 266:243-7 and P.E.Dawson and S.B.Kent, (2000), Annual Review of Biochemistry 69:923-60) makes synthetic peptide and small protein become possibility, use the protein that surpasses 10 kilodaltons (kDa) but these methods are limited to.Though mutagenesis is powerful, also be limited to the structural change of limited quantity.Under many circumstances, may in whole protein, competitiveness mix the analogue approaching with common amino acid.Referring to, for example, R.Furter, (1998), Protein Science 7:419-26; K.Kirshenbaum, etc., (2002), ChemBioChem 3:235-7 and V.Doring etc., (2001), Science 292:501-4.
In the ability of attempting expansion manipulation protein structure and function; developed in-vitro method with chemical acylation quadrature tRNA; this method allow external in response to nonsense codon the alpha-non-natural amino acid selectivity is mixed (referring to; for example; J.A.Ellman; Deng, (1992), Science 255:197-200).The amino acid selectivity that will have new construction and physical property is mixed in the albumen, with research protein folding and stability, and biomolecule identification and catalytic action.Referring to, for example, D.Mendel, Deng, (1995), Annual Review of Biophysics and BiomolecularStructure 24:435-462 and V.W.Cornish, Deng (March 31 nineteen ninety-five), Angew Chem.Int.Ed.Engl., 34:621-633.Yet the stoichiometry character of this method has seriously limited the protein content that can produce.
Cell is gone in the alpha-non-natural amino acid microinjection.For example; by the wrong acidylate tetrahymena thermophila tRNA of microinjection chemistry (for example; M.E.Saks; Deng (1996); be used for suppressing and to mix the engineering tetrahymena tRNAGln of protein in the alpha-non-natural amino acid body by nonsense; J.Biol.Chem.271:23169-23175) and corresponding mRNA (for example alpha-non-natural amino acid is introduced in the nicotinoyl acetylcholinergic receptor of xenopus leavis oocytes; M.W.Nowak; Deng (1998); Enzymology method .293:504-529 will be mixed in the ion channel of xenopus leavis oocytes expression system) in the alpha-non-natural amino acid body.This allows to have the unique physics or the side chain amino acid of chemical property by introducing, and the acceptor in the egg mother cell is carried out detailed biophysics research.Referring to, for example, D.A.Dougherty (2000) is as the alpha-non-natural amino acid of protein structure and function probe, Curr.Opin.Chem.Biol.4:645-652.
Unfortunately, but this method is limited to the protein in the cell of microinjection, because relevant tRNA is external chemical acylation, acidylate again is so the albumen productive rate is very low.
For overcoming these restrictions, new component is added in the colibacillary protein biology synthesis machine of prokaryotes (for example, L.Wang, etc., (2001), Science 292:498-500), this allows genetic coding alpha-non-natural amino acid in vivo.Be response amber codon TAG, to have some amino acids of new chemistry, physics or biological property with this method, but comprise that amino acid, ketone group amino acid and the glycosylation amino acid of photoaffinity labeling and light isomery effectively mix in the colibacillary albumen with high fidelity.Referring to, for example, J.W.Chin etc., (2002), Journal ofthe American Chemical Society 124:9026-9027; J.W.Chin and P.G.Schultz, (2002), ChemBioChem 11:1135-1137; J.W.Chin, etc., (2002), PNAS United States of America99:11020-11024: and L.Wang and P.G.Schultz, (2002), Chem.Comm., 1-10.Yet prokaryotic and eukaryotic machine translator are not high conservative; Therefore, add colibacillary biosynthesizing machine component can not through be commonly used to the alpha-non-natural amino acid locus specificity mix in the eukaryotic albumen.For example, the Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA that uses in the Escherichia coli is to being non-orthogonal in eukaryotic.In addition, tRNA is in eukaryotic, but not transcribing by rna plymerase iii in prokaryotic carry out, and this has limited the primary sequence of the tRNA structural gene that can transcribe in eukaryotic.And opposite with prokaryotic, the tRNA in the eukaryotic need export kytoplasm to from the nucleus of transcribing them, to work in translation.At last, eucaryon 80S ribosomes is different with 70S protokaryon ribosomes.Therefore, need the improved biosynthesizing machine component of exploitation, with the expansion the eukaryotic genetic code.The present invention has satisfied these and other needs, and is apparent in this disclosed below summary.
Brief summary of the invention
The invention provides eukaryotic with translation component; for example; quadrature aminoacyl-tRNA synthetase (O-RS) to quadrature tRNA (O-tRNA); and their individual component; they are used for eukaryotic protein biosynthesizing machine, in eukaryotic alpha-non-natural amino acid are mixed in the polypeptied chain of growing.
The present composition comprises and (for example contains quadrature aminoacyl-tRNA synthetase (O-RS); derive from non-eucaryote; as Escherichia coli, bacillus stearothermophilus etc.) eukaryotic (for example; yeast cells (as brewing yeast cell); mammalian cell, vegetable cell, alga cells, fungal cell, insect cell etc.), wherein O-RS in eukaryotic preferably aminoacylation have the quadrature tRNA (O-tRNA) of at least one alpha-non-natural amino acid.Randomly, can two or more OtRNA of aminoacylation in given eukaryotic.In one aspect; the O-RS aminoacylation for example; at least 40%, at least 45%, at least 50%, at least 60%, at least 75%, at least 80% or even 90% or more have an O-tRNA of alpha-non-natural amino acid; with have amino acid sequence, effective as the O-RS of listed sequence in SEQ ID NO.:86 or 45.In one embodiment, O-RS aminoacylation of the present invention has the O-tRNA of alpha-non-natural amino acid, have than O-RS aminoacylation natural amino acid O-tRNA the efficient height for example, at least 10 times, at least 20 times, at least 30 times etc.
In one embodiment, listed any polynucleotide sequence among the SEQ ID NO.:3-35 (for example, any other subgroup of 3-19,20-35 or sequence 3-35), or its complementary polynucleotide sequential coding 0-RS or its part.In another embodiment, O-RS comprises SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86, or any listed amino acid sequence in its conservative variant.In another embodiment; O-RS comprise with the tyrosyl aminoacyl-tRNA synthetase (TyrRS) of natural generation for example, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.5% or how identical amino acid sequence, and comprise two or more amino acid from A-E family.A family comprises valine, isoleucine, leucine, glycocoll, serine, alanine or the threonine on the Tyr37 opposite position with Escherichia coli TyrRS.B family comprises the aspartic acid on the Asn126 opposite position with Escherichia coli TyrRS.C family comprises threonine, serine, arginine, asparagine or the glycocoll on the Asp182 opposite position with Escherichia coli TyrRS.D family comprises methionine, alanine, valine or the tyrosine on the Phel83 opposite position with Escherichia coli TyrRS; E family comprises serine, methionine, valine, halfcystine, threonine or the alanine on the Leul86 opposite position with Escherichia coli TyrRS.
The subgroup of any of these family combination is a feature of the present invention.For example, in one embodiment, O-RS has two or more and is selected from valine, isoleucine, leucine or the threonine that occurs on the Tyr37 opposite position with Escherichia coli TyrRS; With threonine, serine, arginine or the glycocoll on the Asp182 opposite position of Escherichia coli TyrRS; With methionine or the tyrosine on the Phel83 opposite position of Escherichia coli TyrRS; With with the Leul86 opposite position of Escherichia coli TyrRS on serine or the amino acid of alanine.In another embodiment, O-RS comprises that two or more are selected from glycocoll, serine or alanine on the Tyr37 opposite position with Escherichia coli TyrRS, with the aspartic acid on the Asnl26 opposite position of Escherichia coli TyrRS, with the asparagine on the Aspl82 opposite position of Escherichia coli TyrRS, with alanine on the Phel83 opposite position of Escherichia coli TyrRS or valine and/or with the Leul86 opposite position of Escherichia coli TyrRS on methionine, valine, halfcystine or threonine.
In another embodiment, compare with natural amino acid, O-RS has the enzymatic property of one or more improvement or enhancing for alpha-non-natural amino acid.For example, compare, the improvement or the enhancing properties of alpha-non-natural amino acid comprised with natural amino acid, for example, higher k m, low k m, higher k Cat, low k Cat, low k Cat/ k m, higher k Cat/ k mDeng any one.
Eukaryotic also randomly comprises alpha-non-natural amino acid.Eukaryotic (for example randomly comprises quadrature tRNA (O-tRNA); from non-eucaryote; as Escherichia coli, bacillus stearothermophilus and/or analog), O-tRNA identification selection codon wherein, and preferably have the O-tRNA of alpha-non-natural amino acid by the O-RS aminoacylation.In one aspect, O-tRNA mediation alpha-non-natural amino acid mixes in the protein, its efficient be equivalent to comprise listed polynucleotide sequence among the SEQ ID NO.:65 or the tRNA efficient of in the cell of this sequence, processing for example, at least 45%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95% or 99%.On the other hand, O-tRNA comprises the sequence of SEQ ID NO.:65, O-RS comprises and is selected from SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86, and/or the peptide sequence of any one listed amino acid sequence in its conservative variant.
In another embodiment, eukaryotic comprises the nucleic acid of the polynucleotide that contain the polypeptide of interest of encoding, and wherein polynucleotide comprise the selection codon of O-tRNA identification.In one aspect, the productive rate that comprises the polypeptide of interest of alpha-non-natural amino acid be lack the natural generation that obtains the cell of selecting codon from polynucleotide polypeptide of interest for example, at least 2.5%, at least 5%, at least 10%, at least 25%, at least 30%, at least 40%, 50% or more.On the other hand, cell is to have under the situation of alpha-non-natural amino acid the polypeptide productive rate for example at the productive rate that does not have under the situation of alpha-non-natural amino acid to produce polypeptide of interest, less than 35%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2.5% etc.
The present invention also provides the eukaryotic of the nucleic acid of the polynucleotide that comprise quadrature aminoacyl-tRNA synthetase (O-RS), quadrature tRNA (O-tRNA), alpha-non-natural amino acid and contain the polypeptide of interest of encoding.Polynucleotide comprise the selection codon of O-tRNA identification.In addition; in eukaryotic O-RS preferably aminoacylation have the quadrature tRNA (O-tRNA) of alpha-non-natural amino acid; cell is to have under the situation of alpha-non-natural amino acid the polypeptide productive rate for example at the productive rate that does not have under the situation of alpha-non-natural amino acid to produce polypeptide of interest, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2.5% etc.
Comprise that the eukaryotic composition that contains quadrature tRNA (O-tRNA) also is a feature of the present invention.Usually, O-tRNA mediates alpha-non-natural amino acid in vivo and mixes in the protein, the polynucleotide encoding of the selection codon of this protein by containing O-tRNA identification.In one embodiment, O-tRNA mediation alpha-non-natural amino acid mixes in the protein, its efficient be equivalent to comprise listed polynucleotide sequence among the SEQ ID NO.:65 or the tRNA efficient of in the cell of this sequence, processing for example, at least 45%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95% or even 99% or higher.In another embodiment, O-tRNA comprises polynucleotide sequence listed among the SEQ ID NO.:65 or processes from polynucleotide sequence, or its conservative variant.In another embodiment, O-tRNA comprises O-tRNA capable of circulation.
In one aspect of the invention, O-tRNA is a posttranscriptional modification.The present invention also is provided at nucleic acid or its complementary polynucleotide of coding O-tRNA in the eukaryotic.In one embodiment, nucleic acid comprises A frame and B frame.
The present invention has also described the method for production translation component, and for example, O-RSs or O-tRNA/O-RS are to (with the translation component of these methods productions).For example, the invention provides the method for producing quadrature aminoacyl-tRNA synthetase (O-RS), this enzyme in eukaryotic preferably aminoacylation have the quadrature tRNA of alpha-non-natural amino acid.This method comprises, for example, the eukaryotic colony of first kind is just being selected in the presence of alpha-non-natural amino acid, and wherein each is self-contained for eukaryotic: i) a member in aminoacyl-tRNA synthetase (RS) library, ii) quadrature tRNA (O-tRNA), iii) the encode polynucleotide of the polynucleotide of positive selected marker and the negative selectable marker of iv) encoding; Wherein the cell of surviving under just selecting is included in the active RS of aminoacylation quadrature tRNA (O-tRNA) in the presence of the alpha-non-natural amino acid.Bear selection at the cell that does not have under just selecting, to survive under the situation of alpha-non-natural amino acid, to remove the active RS that aminoacylation has the O-tRNA of natural amino acid.This provides aminoacylation preferably to have the O-RS of the O-tRNA of alpha-non-natural amino acid.
In some embodiments, the polynucleotide of the positive selected marker of coding are operably connected to response element, cell also comprise a) coding from response element regulate the transcript regutation protein (for example, eukaryotic transcription is regulated albumen etc.) of transcribing and b) comprise at least a selection codon polynucleotide.The O-tRNA that has alpha-non-natural amino acid by aminoacylation mixes alpha-non-natural amino acid and causes transcribing of positive selected marker in the transcript regutation protein.In one embodiment, transcript regutation protein is transcription activating protein (for example, GAL4 etc.), and selecting codon is the amber terminator codon, for example, wherein the amber terminator codon is positioned at or basically near the DNA of the encoding transcription activator protein part polynucleotide in conjunction with the territory.
Positive selected marker can be in the various molecules any one.In one embodiment, just selecting marks packets to contain the growing nutrient replenishers, in lacking the nutrient culture media of nutritious supplementary pharmaceutical, selecting.In another embodiment, the polynucleotide of the positive selected marker of encoding for example are, (for example, wherein his3 gene code imidzoleglycerol phosphate dehydrogenase is detected by 3-aminotriazole(ATA) (3-AT) that provides and/or analog for ura3, leu2, lys2, lacZ gene, his3.In another embodiment, the encode polynucleotide of positive selected marker comprise the selection codon.
As positive selected marker, negative selectable marker also can be in the various molecules any one.In some embodiments, the polynucleotide of coding negative selectable marker are operably connected to response element, and transcript regutation protein is transcribed from the response element mediation.The O-tRNA that has natural amino acid by aminoacylation mixes natural amino acid and causes transcribing of negative selectable marker in the transcript regutation protein.In one embodiment, the polynucleotide of coding negative selectable marker for example are, the ura3 gene, and negative being chosen in the nutrient culture media that contains 5-fluororotic acid (5-FOA) finished.In another embodiment, be used for the negative nutrient culture media of selecting and comprise the selective agent or the selective agent that can be converted into detectable substance by negative selectable marker.In one aspect of the invention, detectable substance is a noxious material.In one embodiment, the polynucleotide of coding negative selectable marker comprise the selection codon.
In some embodiments, positive selected marker and/or negative selectable marker are included in and fluoresce under the existence of suitable reactants or the polypeptide of catalytic luminescence reaction.In one aspect of the invention, by fluorescence-activated cell sorting (FACS) or by positive selected marker of luminous detection and/or negative selectable marker.In some embodiments, positive selected marker and/or negative selectable marker comprise selection markers or the transcript regutation protein based on affinity.In one embodiment, positive selected marker of same polynucleotide encoding and negative selectable marker.
In one embodiment, the polynucleotide that code book is invented positive selected marker and/or negative selectable marker can comprise at least two selection codons, separately or both can comprise at least two different selection codons or at least two identical selection codons.
The additional levels of selection/screening severity also can be used for the inventive method.In one embodiment, method can comprise, for example, at the step (a) and (b) or (a) and the inactivation synzyme that (b) provides quantity not to wait, wherein the inactivation synzyme that do not wait of quantity provides the selection of additional levels or screens severity.In one embodiment, this method is used to produce the step (a) of O-RS, (b) or step (a) and (b) comprise different selections or screening severity, for example, just and/or the severity of negative selectable marker.This method comprises randomly that the O-RS that aminoacylation is preferably had an O-tRNA of alpha-non-natural amino acid adds and selects wheel, for example, additionally just selecting to take turns, the additional negative combination of selecting wheel or additional positive and negative to select wheel.
In one embodiment, selection/screening comprises one or more plus or minus selection/screenings, and they are selected from, for example, and the change of amino acid permeability, the change of translation efficiency, the change of translation fidelity etc.The sudden change that one or more changes are based in one or more polynucleotide of the right assembly of coded orthogonal tRNA-tRNA synzyme is used to produce albumen.
Usually, RS library (for example, mutant RS library) comprises from for example at least a, from the RS of non-Eukaryotic aminoacyl-tRNA synthetase (RS).In one embodiment, the RS library is from the RS of inactivation, and for example, wherein inactivation RS produces by the active RS that suddenlys change.In another embodiment, inactivation RS comprises amino acid binding pocket and one or more amino acid that contains useful one or more different aminoacids replacement binding pockets, and for example, the amino acid of replacement replaces with alanine.
In some embodiments, the method for producing O-RS also is included on the nucleic acid of coding RS carries out random mutation, locus specificity sudden change, reorganization, chimeric construct or their combination in any, therefore produces mutant RS library.In some embodiments, this method also comprises, for example, (c) separates the nucleic acid of coding O-RS; (d) polynucleotide (for example, by random mutagenesis, site-specific mutagenesis, chimeric construct, reorganization or their combination in any) of generation one group coding sudden change O-RS from nucleic acid; (e) repeating step (a) and/or (b) is up to obtaining the sudden change O-RS that aminoacylation preferably has the O-tRNA of alpha-non-natural amino acid.In one aspect of the invention, step (c)-(e) is carried out twice at least.
Producing the right method of O-tRNA/O-RS also is feature of the present invention.In one embodiment; obtain O-RS as described above; bear selection by eukaryotic colony and obtain O-tRNA first kind; wherein eukaryotic comprises a member in tRNA library, comprises by the cell to a member in the tRNA library of eukaryotic endogenous aminoacyl-tRNA synthetase (RS) aminoacylation with removal.This provides the tRNA storehouse with the eukaryotic quadrature of first kind.In one aspect of the invention, the tRNA library comprises from for example at least a, from the tRNA of non-Eukaryotic tRNA.In another aspect of this invention, aminoacyl-tRNA synthetase (RS) library comprises from for example at least a, from the RS of non-Eukaryotic aminoacyl-tRNA synthetase (RS).In another aspect of this invention, the tRNA library comprises from least a tRNAs from first kind of non-Eukaryotic tRNA.Aminoacyl-tRNA synthetase (RS) library randomly comprises from least a RS from second kind of non-Eukaryotic aminoacyl-tRNA synthetase (RS).In one embodiment, first kind is identical with second kind of non-eucaryote.In addition, first kind can be different with second kind of non-eucaryote.The specificity O-tRNA/O-RS that produces by the inventive method is to also being feature of the present invention.
Another feature of the present invention is the method that production translation component and the translation component that will select/screen are introduced second kind in a kind.For example, in first kind (for example, the eucaryote kind is as yeast etc.) in produce the right method of O-tRNA/O-RS and also comprise the eukaryotic (for example, mammal, insect, fungi, algae, plant etc.) of the nucleic acid of the nucleic acid of coding O-tRNA and coding O-RS being introduced second kind.Second kind can be mixed alpha-non-natural amino acid in the polypeptied chain of growing with the translation component of introducing in vivo, for example, and at translate duration.
In another embodiment; production in eukaryotic preferably the method for quadrature aminoacyl-tRNA synthetase (O-RS) of aminoacylation with quadrature tRNA of alpha-non-natural amino acid comprising: (a) in the presence of alpha-non-natural amino acid to the eukaryotic colony of first kind (for example; the eucaryote kind is as yeast or analog) just select.The eukaryotic of first kind comprises separately: the i) a member in aminoacyl-tRNA synthetase (RS) library, ii) quadrature tRNA (O-tRNA), the polynucleotide of the polynucleotide of the positive selected marker of iii) encoding and the negative selectable marker of iv) encoding.The cell that can survive under just selecting is included in the active RS of aminoacylation quadrature tRNA (O-tRNA) in the presence of the alpha-non-natural amino acid.The cell that to survive under just selecting is not having to bear selection under the situation of alpha-non-natural amino acid, to remove the active RS that aminoacylation has the O-tRNA of natural amino acid, therefore provides aminoacylation preferably to have the O-RS of the O-tRNA of alpha-non-natural amino acid.The nucleic acid of the nucleic acid of coding O-tRNA and the O-RS that encodes is introduced the eukaryotic (for example, mammal, insect, fungi, algae, plant and/or analog) of second kind.When these assemblies are translated in second kind, can be used for alpha-non-natural amino acid is mixed protein of interest or polypeptide in second kind.In one embodiment, O-tRNA and/or O-RS are introduced in the biological eukaryotic of second kind.
In some embodiments; by being born, the eukaryotic colony of first kind selects to obtain O-tRNA; wherein eukaryotic comprises a member in tRNA library, to remove the cell of quilt to a member in the tRNA library of eukaryotic endogenous aminoacyl-tRNA synthetase (RS) aminoacylation.This provides the tRNA storehouse with the eukaryotic quadrature of first kind and second kind.
In one aspect, the present invention includes the composition that contains a kind of albumen, wherein this albumen comprises at least a alpha-non-natural amino acid and at least one posttranslational modification, and wherein at least one posttranslational modification comprises that the molecule that will contain second reactive group is attached at least a alpha-non-natural amino acid that contains first reactive group by [3+2] cycloaddition.
Therefore, the albumen (or polypeptide of interest) with at least a alpha-non-natural amino acid also is feature of the present invention.In some embodiments of the present invention, the albumen with at least a alpha-non-natural amino acid comprises at least one posttranslational modification.In one embodiment, at least one posttranslational modification is that the molecule that will contain second reactive group (for example, the derivant of the derivant of dyestuff, polymkeric substance such as polyglycol, photocrosslinking agent, cytotoxic compound, affinity labeling, biotin, resin, second kind of albumen or polypeptide, metal-chelator, co-factor, fatty acid, carbohydrates, polynucleotide (for example, DNA, RNA etc.) etc.) is attached at least a alpha-non-natural amino acid that contains first reactive group by [3+2] cycloaddition.For example, first reactive group is an alkynyl part (for example, right in the alpha-non-natural amino acid-the propargyloxy phenylalanine) (this group is also referred to as acetylene moiety sometimes), and second reactive group is the azido part.In another embodiment, first reactive group is an azido part (for example, right in the alpha-non-natural amino acid-azido-the L-phenylalanine), and second reactive group is the alkynyl part.In some embodiments, albumen of the present invention comprises at least a alpha-non-natural amino acid (for example, the keto-acid alpha-non-natural amino acid) that contains at least one posttranslational modification, and wherein at least one posttranslational modification comprises sugar moieties.In some embodiments, in eukaryotic, carry out posttranslational modification in the body.
In some embodiments, albumen comprises at least one posttranslational modification by carrying out in the eukaryotic body, and wherein posttranslational modification is not to be undertaken by prokaryotic.The example of posttranslational modification includes but not limited to acetylation, acidylate, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-connection modification etc.In one embodiment, posttranslational modification comprises oligosaccharides connected by the GlcNAc-asparagine and is attached to that (for example, wherein oligosaccharides comprises (GlcNAc-Man) on the asparagine 2-Man-GlcNAc-GlcNAc etc.).In another embodiment, posttranslational modification comprises oligosaccharides (for example, Gal-GalNAc, Gal-GlcNAc etc.) connected by GalNAc-serine, GalNAc-threonine, GlcNAc-serine or GlcNAc-threonine and is attached on serine or the threonine.In some embodiments, albumen of the present invention or polypeptide can comprise secretion or positioning sequence, epi-position mark, FLAG mark, polyhistidine tag, gst fusion protein and/or analog.
Usually, albumen and any available albumen are (for example, treatment albumen, diagnosis albumen, industrial enzyme or their part and/or analog) for example have, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or even at least 99% or more how identical, they comprise one or more alpha-non-natural amino acids.In one embodiment, the present composition comprises protein of interest or polypeptide and excipient (for example, damping fluid, pharmaceutically acceptable excipient etc.).
Protein of interest or polypeptide can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten or more alpha-non-natural amino acids.Alpha-non-natural amino acid can be identical or different, for example, comprise 1,2,3,4,5,6,7,8,9,10 or the albumen of how different alpha-non-natural amino acid in can have 1,2,3,4,5,6,7,8,9,10 or more different loci.In some embodiments, exist at least aly in the natural generation form of protein, replace with alpha-non-natural amino acid but be less than whole concrete amino acid.
The example of a kind of albumen (or polypeptide of interest) includes but not limited to, for example, cell factor, growth factor, growth factor receptors, interferon, interleukin, the inflammation molecule, oncoprotein, peptide hormone, signal transducers, the steroid hormone acceptor, hematopoietin (EPO), insulin, human growth hormone (HGH), α-1 antitrypsin, angiostatin, AHF, antibody, apolipoprotein, apoprotein, atrial natriuretic peptide, atrium natriuresis polypeptide, atrial natriuretic peptide, the C-X-C chemotactic factor (CF), T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit part, cell factor, the CC chemotactic factor (CF), monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, MCP-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-1 β, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 part, the C-kit part, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, DHFR, epithelium neutrophil cell activating peptide-78, GRO α/MGSA, GRO β, GRO γ, MIP-1 α, MIP-1 δ, MCP-1, epidermal growth factor (EGF), epithelium neutrophil cell activating peptide, hematopoietin (EPO), exfoliative toxin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropic hormone, growth factor, growth factor receptors, Hedgehog albumen, haemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumins, ICAM1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon, IFN-α, IFN-β, IFN-γ, interleukin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, Neurturin, neutrophil cell inhibiting factor (NIF), oncostatin M, BMP, oncoprotein, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, human growth hormone (HGH), the multi-effect nutrient factor, albumin A, Protein G, the thermal source exotoxin A, B or C, relaxain, feritin, SCF, soluble complement acceptor I, solubility I-CAM1, soluble interleukin-6 receptor, soluble TNF acceptor, somatomedin, somatostatin, somatotropin, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, the steroid hormone acceptor, superoxide dismutase (SOD), toxic shock syndrome toxin, thymosin, tissue plasminogen activator, TGF (TGF), TGF-α, TGF-β, TNF, tumor necrosis factor, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGEF), urokinase, Mos, Ras, Raf, Met; The albumen that exists in p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, PgR, testosterone receptor, aldosterone receptor, ldl receptor, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-l/LFA-1, hyaluronic acid glycosides (hyalurin)/CD44, cortisone, Genebank or other availability database etc., and/or their part.In one embodiment, polypeptide of interest comprises a transcript regutation protein (for example, transcription activating protein (as GAL4), or transcribe Profilin etc.) or their part.
The GAL4 albumen in the eukaryotic or the composition of its part also are features of the present invention.Usually, GAL4 albumen or its part comprise at least a alpha-non-natural amino acid.
Eukaryotic of the present invention provides the synthetic ability that contains the albumen that the consumption alpha-non-natural amino acid is arranged greatly.For example, the protein concentration of albumen in cell extract, damping fluid, pharmaceutically acceptable excipient and/or analog that production contains alpha-non-natural amino acid is, for example, at least 10 micrograms per litre, at least 50 micrograms per litre, at least 75 micrograms per litre, at least 100 micrograms per litre, at least 200 micrograms per litre, at least 250 micrograms per litre or at least 500 micrograms per litre or higher.In some embodiments, the present composition comprises, for example, and at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms or at least 500 micrograms or the albumen that contains alpha-non-natural amino acid more.
In some embodiments, nucleic acid coding protein of interest or polypeptide (or their part).Usually, this nucleic acid comprises at least one and selects codon, at least two selection codons, at least three selection codons, at least four selection codons, at least five selection codons, at least six selection codons, at least seven selection codons, at least eight selection codons, at least nine selection codons or even ten or more options codon more.
The present invention also is provided at and produces at least a method that contains the protein (and albumen of producing in this way) of at least one alpha-non-natural amino acid in the eukaryotic.Method comprises, for example, cultivates the eukaryotic that contains a kind of nucleic acid in proper culture medium, and this nucleic acid comprises at least one and selects the codon and this albumen of encoding.Eukaryotic also contain have in the cell function and can identification selection the quadrature tRNA (O-tRNA) of codon, and preferably aminoacylation has the quadrature aminoacyl tRNA synthetase (O-RS) of the O-tRNA of alpha-non-natural amino acid, nutrient culture media contains alpha-non-natural amino acid.In one embodiment; the O-tRNA that the O-RS aminoacylation has an alpha-non-natural amino acid as the efficient of the O-RS of listed amino acid sequence in SEQ ID NO.:86 or 45 for example is equivalent to have, and at least 45%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95% or even 99% or higher.In another embodiment, O-tRNA comprises by SEQ ID NO.:64 or 65 or its complementary polynucleotide sequential coding, or processing is from this sequence.In another embodiment, O-RS comprises any listed amino acid sequence in SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86.
In one embodiment, this method also comprises mixes alpha-non-natural amino acid in this albumen, and wherein alpha-non-natural amino acid comprises first reactive group; And with this albumen and the molecule that contains second reactive group (for example, dyestuff, polymkeric substance, for example, the derivant of the derivant of polyglycol, photocrosslinking agent, cytotoxic compound, affinity labeling, biotin, resin, second kind of albumen or polypeptide, metal-chelator, co-factor, fatty acid, carbohydrates, polynucleotide (for example, DNA, RNA etc.) etc.) contact.First reactive group and the reaction of second reactive group are attached to this molecule on the alpha-non-natural amino acid by [3+2] cycloaddition.In one embodiment, first reactive group is alkynyl or azido part, and second reactive group is azido or alkynyl part.For example, first reactive group is an alkynyl part (for example, right in the alpha-non-natural amino acid-the propargyloxy phenylalanine), and second reactive group is the azido part.In another embodiment, first reactive group is an azido part (for example, right in the alpha-non-natural amino acid-azido-the L-phenylalanine), and second reactive group is the alkynyl part.
In some embodiments, encoding proteins comprises treatment albumen, diagnosis albumen, industrial enzyme or their part.In one embodiment, further modify the albumen that this method is produced by alpha-non-natural amino acid.For example, by as nucleophilic-electrophilic reaction, modify alpha-non-natural amino acid via [3+2] cycloaddition etc.In another embodiment, by modifying the protein that this method is produced at least one posttranslational modification (for example, N-glycosylation, O-glycosylation, acetylation, acidylate, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-connection modification etc.) body.
The method of producing screening or selecting transcript regutation protein (and produce screening in this way or select transcript regutation protein) also is provided.Method comprises, for example, selects first polynucleotide sequence, wherein the polynucleotide sequence coding nucleic acid binding domain; With first polynucleotide sequence sudden change, to comprise at least a selection codon.This provides screening or has selected polynucleotide sequence.Method also comprises, for example, selects second polynucleotide sequence, wherein second polynucleotide sequence coding transcription activating domain; Construction is provided, and it comprises the screening that is operably connected to second polynucleotide sequence or selects polynucleotide sequence; With, construction alpha-non-natural amino acid, quadrature tRNA synzyme (O-RS) and quadrature tRNA (Q-tRNA) are introduced cell.Use these assemblies, in response to screening or select selection codon in the polynucleotide sequence, O-RS preferably aminoacylation has the O-tRNA of alpha-non-natural amino acid, and O-tRNA identification selection codon also mixes alpha-non-natural amino acid in the nucleic acid binding domain.This provides screening or has selected transcript regutation protein.
In some embodiments, the compositions and methods of the invention comprise eukaryotic.Eukaryotic of the present invention comprises, for example, and any one of mammalian cell, yeast cells, fungal cell, vegetable cell, insect cell etc.Translation component of the present invention can be from various biologies, for example, non-eucaryote, as prokaryotes (for example, Escherichia coli, bacillus stearothermophilus etc.), or archeobacteria, or for example, eucaryote.
Selection codon of the present invention has been expanded the genetic codon framework of eukaryotic protein biosynthesizing machine.Can use any one of various selection codons among the present invention, comprise terminator codon (for example, amber codon, ochre codon or opal temination codon), nonsense codon, rare codon, four (or more) base codon and/or analog.
The example that can be used for the alpha-non-natural amino acid of composition described herein and method includes, but is not limited to: right-acetyl group-the L-phenylalanine, right-iodo-the L-phenylalanine, O-methyl-L-tyrosine, right-the propargyloxy phenylalanine, right-propargyl-phenylalanine, L-3-(2-naphthyl) alanine, the 3-methylphenylalanine, 0-4-allyl-L-tyrosine, 4-propyl group-L-tyrosine, three-oxy-acetyl-GlcNAc β-serine, the L-DOPA, fluoridize phenylalanine, isopropyl-L-phenylalanine, right-azido-the L-phenylalanine, right-acyl group-the L-phenylalanine, right-benzoyl-the L-phenylalanine, the L-phosphoserine, the phosphono serine, phosphono tyrosine, right-bromophenyl alanine, right-amino-the L-phenylalanine, isopropyl-L-phenylalanine, the amino acid whose non-natural analog of tyrosine; The amino acid whose non-natural analog of glutamine; The non-natural analog of the amino acid of phenylalanine; The amino acid whose non-natural analog of serine; The amino acid whose non-natural analog of threonine; Alkyl, aryl, acyl group, azido, cyano group, halogen, hydrazine, hydrazides, hydroxyl, alkenyl, alkynyl, ether, mercaptan, sulfonyl, selenium, ester, thio-acid, boric acid, borate, phosphoryl, phosphono, phosphine, heterocycle, ketenes, imines, aldehyde, azanol, ketone group or amino amino acid that replaces or their combination in any; Have can photoactivated crosslinking chemical amino acid; The amino acid of spin labeling; Fluorescence amino acid; Metal is in conjunction with amino acid; Metallic amino acid; Radioactivity amino acid; The light cage cover (photocaged) but and/or the amino acid of light isomery; The amino acid that contains biotin or biotin-analog; Contain keto amino acid; The amino acid that contains polyglycol or polyethers; The amino acid that heavy atom replaces; But but the amino acid of chemical cleavage or light cutting; Has the amino acid that prolongs side chain; The amino acid that contains poisonous group; Sugar-substituted amino acid; The amino acid that contains carbon-connection sugar; Amino acid with redox active; Contain alpha-hydroxy acid; Amino thio-acid; α, the disubstituted amino acid of α; Beta-amino acids; Cyclic amino acids except that proline or histidine removes phenylalanine, the aromatic amino acid that tyrosine or tryptophane are outer, and/or analog.
The present invention also provides polypeptide (O-RS) and polynucleotide; for example; 0-tRNA; coding O-RS or its part are (for example; the avtive spot of synzyme) polynucleotide; be used to make up the oligonucleotides of aminoacyl-tRNA synthetase mutant, coding contains one or more protein of interest of selecting codon or the polynucleotide of polypeptide etc.For example, polypeptide of the present invention comprises and (for example comprises SEQ ID NO.:36-63,36-47, any other subgroup of 48-63 or 36-63) polypeptide of any listed amino acid sequence and/or in 86, (for example comprise by SEQ ID NO.:3-35,3-19, any other subgroup of 20-35 or sequence 3-35) polypeptide of the amino acid sequence of any listed polynucleotide sequence coding in, with polypeptide with antibody specific immune activity, this antibody is to (for example comprising SEQ ID NO.:36-63,36-47, any other subgroup of 48-63 or 36-63) polypeptide of any listed amino acid sequence or comprise SEQ ID NO.:3-35 (for example, 3-19 and/or in 86, any other subgroup of 20-35 or sequence 3-35) polypeptide of the amino acid sequence of arbitrary listed polynucleotide sequence coding is special in.
Polypeptide of the present invention comprises that also the tyrosyl aminoacyl-tRNA synthetase (TyrRS) (for example, SEQ ID NO.:2) with natural generation has the polypeptide of at least 90% same acid sequence and comprises two or more amino acid whose polypeptide in the A-E family (above-mentioned).Similarly, polypeptide of the present invention also randomly comprises and (for example contains SEQ ID NO.:36-63, any other subgroup of 36-47,48-63 or 36-63) polypeptide of at least 20 continuous amino acids of any one and the two or more aminoacid replacement described in A-E family and/or in 86.The amino acid sequence that contains the conservative variant of arbitrary aforementioned polypeptides is also included as polypeptide of the present invention.
In one embodiment, composition comprises polypeptide of the present invention and excipient (for example, damping fluid, water, pharmaceutically acceptable excipient etc.).The present invention also provides antibody or the antiserum that has specific immune activity with polypeptide of the present invention.
Polynucleotide also are provided among the present invention.Polynucleotide of the present invention comprise those polynucleotide with one or more selection codon code book invention protein of interest or polypeptide.In addition, polynucleotide of the present invention comprise, for example, contain the polynucleotide of any one listed nucleotide sequence among SEQ ID NO.:3-35 (for example, any other subgroup of 3-19,20-35 or sequence 3-35), the 64-85; Polynucleotide with this polynucleotide sequence complementation or this polynucleotide sequence of encoding; And/or coding contains the polynucleotide of the polypeptide of any one listed amino acid sequence in SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86 or its conservative variant.Polynucleotide of the present invention also comprise the polynucleotide of code book invention polypeptide.Similarly, surpassing basically with the nucleic acid of above-mentioned multi-nucleotide hybrid under highly rigorous condition, the nucleic acid of total length is polynucleotide of the present invention.
Polynucleotide of the present invention also comprise the polynucleotide of coded polypeptide; this polypeptide comprise with the tyrosyl aminoacyl-tRNA synthetase (TyrRS) of natural generation (for example; SEQ ID NO.:2) at least 90% identical amino acid sequence and comprise two or more sudden changes in the A-E family (above-mentioned).With above-mentioned polynucleotide and/or contain the identical polynucleotide of the polynucleotide at least 70% (or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% or more) of the conservative variant of arbitrary above-mentioned polynucleotide and be also included within the polynucleotide of the present invention.
In some embodiments, carrier (for example, plasmid, clay, bacteriophage, virus etc.) comprises polynucleotide of the present invention.In one embodiment, carrier is an expression vector.In another embodiment, expression vector comprises the promoter that is operably connected to one or more polynucleotide of the present invention.In another embodiment, cell contains the carrier that comprises polynucleotide of the present invention.
On the other hand, the method that the invention provides compound compositions and produce described compound.For example, compound comprises, for example, alpha-non-natural amino acid (as right-(propargyloxy)-phenylalanine (for example, among Figure 11 1), azido dyestuff (as shown in chemical constitution 4 and chemical constitution 6), alkynyl polyglycol (for example shown in the chemical constitution 7), wherein n for example is, 50 and 10,000,75 and 5,000,100 and 2,000, integer between 100 and 1,000 grade etc.In embodiments of the present invention, the molecular weight of alkynyl polyglycol is, for example about 5,000 to about 100, and 000Da, about 20,000 is to about 50, and 000Da, about 20,000 is to about 10, and 000Da (for example, 20,000Da).
Figure A20048002115500231
The various compositions that contain these compounds also are provided, for example, have had protein and cell.In one aspect, composition comprises right-(propargyloxy)-phenylalanine alpha-non-natural amino acid also comprises quadrature tRNA.Alpha-non-natural amino acid can be attached to (for example) quadrature tRNA, for example, be covalently bound to quadrature tRNA, be covalently bound to the 3 ' OH of terminal ribose of quadrature tRNA or 2 ' OH etc. by amino-acyl bond with covalent manner.
In one aspect of the invention, the protein that contains the azido dyestuff (for example, chemical constitution 4 or chemical constitution 6) also comprise at least a alpha-non-natural amino acid (for example, alkynyl amino acid), wherein the azido dyestuff is attached on the alpha-non-natural amino acid by [3+2] cycloaddition.
In one embodiment, a kind of albumen contains the alkynyl polyglycol of chemical constitution 7.In another embodiment, said composition also comprises at least a alpha-non-natural amino acid (for example, azido amino acid), and wherein the alkynyl polyglycol is attached on the alpha-non-natural amino acid by [3+2] cycloaddition.
The method that has comprised synthetic all cpds among the present invention.The method of synthetic right-(propargyloxy) phenylalanine compound for example, is provided.For example, this method comprises that (a) is with uncle N--butoxy carbonyl-tyrosine and K 2CO 3Be suspended in the dry DMF; (b) propargyl bromide is added in the reaction mixture of (a), alkanisation hydroxyl and carboxylic group produce the protection intermediate compound with following structure:
Figure A20048002115500241
(c) will protect intermediate compound in MeOH, to mix, and make amine moiety go protection with anhydrous HC1, thus synthetic right-(propargyloxy) phenylalanine compound.In one embodiment, this method comprises that also (d) is dissolved in NaOH and the MeOH aqueous solution stirring at room with right-(propargyloxy) phenylalanine HCl; (e) pH is adjusted to 7; (f) right-(propargyloxy) phenylalanine compound of precipitation.
The method of synthetic azido dyestuff also is provided.For example, method comprises: the dye composition that contains sulfonyl halogenide part (a) is provided; (b) in the presence of 3-azido propylamine and triethylamine, dye composition is heated to room temperature, the amine moiety of 3-azido propylamine is coupled to the halogen position of dye composition, thereby synthesize the azido dyestuff.In one embodiment, this dye composition contains dansyl Cl, and the azido dyestuff contains the composition of chemical constitution 4.In one aspect, this method also comprises purifying azido dyestuff from reaction mixture.
In another embodiment, the method for synthetic azido dyestuff comprises that (a) provides and contains the amine dye composition; (b) will contain the amine dye composition and in suitable solvent, mix with carbodiimide and 4-(3-azido propyl group carbamyl)-butyric acid, with the amine moiety coupling of the carbonyl and the dye composition of acid, thus synthetic azido dyestuff.In one embodiment, carbodiimide comprises 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCI).In one aspect, contain the amine dyestuff and comprise fluorescein amine (fluoresceinamine), suitable solvent comprises pyridine.For example, contain the amine dyestuff and comprise fluorescein amine, the azido dyestuff comprises the composition of chemical constitution 6.In one embodiment, this method also comprises (c) precipitation azido dyestuff; (d) use the HC1 washing precipitation; (e) with washed resolution of precipitate in EtOAc; (f) precipitation azido dyestuff in hexane.
The method of synthetic propargyl acid amides polyglycol also is provided.For example, this method comprises at room temperature propargyl amine and polyglycol (PEG)-hydroxysuccinimide eater at organic solvent (for example, CH 2Cl 2) middle reaction, the propargyl acid amides polyglycol of generation chemical constitution 7.In one embodiment, this method also comprises with ethyl acetate precipitation propargyl acid amides polyglycol.In one aspect, this method also is included in the methyl alcohol crystallization propargyl acid amides polyglycol again; Desciccate under the vacuum.
Kit also is a feature of the present invention.For example, provide the kit of producing the protein that comprises at least a alpha-non-natural amino acid in cell, wherein this kit comprises a container that contains the polynucleotide sequence of the polynucleotide sequence of coding O-tRNA or O-tRNA and encode O-RS or O-RS.In one embodiment, this kit also comprises at least a alpha-non-natural amino acid.In another embodiment, this kit also comprises the illustrative material of producing this protein.
Definition
Before describing the present invention in detail, should understand and the invention is not restricted to concrete device or biosystem, they can change certainly.Also should understand term used herein is only to be the purpose of description embodiment, does not plan to limit.Comprise plural number as singulative used in this specification and the appended claims " ", " a kind of " and " being somebody's turn to do ", unless have clearly regulation in being somebody's turn to do.Therefore, for example, the formulation of " cell " comprises the combination of two or more cells; The formulation of " bacterium " comprises the potpourri of bacterium etc.
Unless have other to limit in this paper or the following instructions remainder, the implication of all technology used herein and scientific terminology is identical with the implication of one skilled in the art's common sense of the present invention.
Homology: when protein and/or protein sequence natively or artificially during, then their " homologies " from common ancestor protein or protein sequence.Similarly, when nucleic acid and/or nucleotide sequence natively or artificially during, then their " homologies " from common ancestors' nucleic acid and/or nucleotide sequence.For example, can modify the nucleic acid of any natural generation, make it comprise one or more selection codons by any feasible method of mutagenesis.When the expression of nucleic acid of this mutagenesis, its coding contains the polypeptide of one or more alpha-non-natural amino acids.Certainly, this mutation process can also change one or more standard cipher, thereby also changes one or more standard amino acids in the mutain of gained.
Homology is inferred by the sequence similarity between two or more nucleic acid or the albumen (or its sequence) usually.Be used for determining between the sequence of homology that the accurate percentage of similarity becomes with nucleic acid and albumen and also has arguement, but usually will be less be used for determining homology to 25% sequence similarity.The sequence similarity of higher level, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or higher, also can be used for determining homology.This paper has described the method (for example, using the BLASTP and the BLASTN of default parameters) of the common available row of sequencing really similarity percentage.
Quadrature: term used herein " quadrature " refers to cell or the endogenic corresponding molecule of translation system or can not compare with the molecule that cell endogenous assembly one works; the molecule (for example, quadrature tRNA (O-tRNA) and/or quadrature aminoacyl tRNA synthetase (O-RS)) that works with the endogenous assembly one of cell has the efficient of reduction.Under the situation that refers to tRNA and aminoacyl-tRNA synthetase; just interdigital can not or efficient reduce; for example; the efficiency ratio endogenous tRNA that quadrature tRNA and endogenous tRNA synzyme one work and the efficient of endogenous tRNA synzyme are low, or the efficient that works of the efficiency ratio endogenous tRNA synzyme that works of quadrature aminoacyl-tRNA synthetase and endogenous tRNA one and endogenous tRNA one is less than 20%, less than 10%, less than 5% or less than 1%.Quadrature molecule in the cell lacks function endogenous complementary molecule.For example, low by the efficiency ratio of quadrature tRNA in any endogenous RS aminoacylation cell of cell by the efficient of endogenous RS aminoacylation endogenous tRNA, or or even zero.In another embodiment, in interested cell, the efficiency ratio of any endogenous tRNA of quadrature RS aminoacylation is low by the efficient of endogenous RS aminoacylation endogenous tRNA, or or even zero.Second quadrature molecule can be introduced cell, work with first quadrature molecule one.For example, quadrature tRNA/RS is to comprising the complementary component of introducing, they play a role in cell together, and its efficient is equivalent to (for example, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% or higher) of the endogenous right efficient of corresponding tRNA/RS.
Complementary: term " complementation " refer to can-quadrature that works is to, O-tRNA and O-RS assembly, for example, wherein O-RS makes the O-tRNA aminoacylation.
Aminoacylation preferably: term " preferably aminoacylation " refers to the tRNA of the natural generation of O-RS aminoacylation or the parent material that is used to produce O-tRNA is compared; with for example, 70%, 75%, 85%, 90%, 95% 99% or higher efficient aminoacylation have the O-tRNA of alpha-non-natural amino acid.Mix alpha-non-natural amino acid in the polypeptied chain of growing with high fidelity, for example, for given selection codon efficient greater than 75%, for given selection codon efficient be higher than about 80%, for given selection codon efficient greater than about 90%, for given selection codon efficient greater than about 95% or for given selection codon efficient greater than about 99% or higher.
Select codon: term " selection codon " refers in translation process to be discerned by O-tRNA and the codon do not discerned by endogenous tRNA.Selection codon on the anti-password subring identification of the O-tRNA mRNA also mixes its amino acid on this site of polypeptide, for example, and alpha-non-natural amino acid.Select codon to comprise, for example, nonsense codon, as terminator codon, as, amber, ochre and opal codon; Four or polybase base codon more; Rare codon; From codon natural or non-natural base-pair and/or analog.
Inhibition type tRNA: inhibition type tRNA is in given translation system, for example, and by the tRNA that changes the reading of mRNA (mRNA) in response to the mechanism of selecting codon that amino acid is mixed polypeptied chain is provided.For example, inhibition type tRNA can pass through, and for example terminator codon, four base codons, rare codon and/or analog are read.
TRNA capable of circulation: term " tRNA capable of circulation " refers to the tRNA of aminoacylation, available amino end acid (for example, alpha-non-natural amino acid), and with this amino acid, for example alpha-non-natural amino acid mixes one or more polypeptied chains at translate duration, repeatedly aminoacylation again.
Translation system: term " translation system " refers to the amino acid of natural generation is mixed the set group of the component in the polypeptied chain (albumen) of growing.The component of translation system can comprise, for example, and ribosomes, tRNA, synzyme, mRNA, amino acid etc.Can be (for example with assembly of the present invention, ORS, OtRNAs, alpha-non-natural amino acid etc.) join external or body in translation system for example, eukaryotic, for example, in yeast cells, mammalian cell, vegetable cell, alga cells, fungal cell, insect cell and/or the analog.
Alpha-non-natural amino acid: term used herein " alpha-non-natural amino acid " refers to not be arbitrary amino acid, modified amino acid and/or the amino acid analogue of one of amino acid, selenocystein or pyrroles's lysine of 20 kinds of common natural generations.
Derive from: term used herein " derives from " and refers to from concrete molecule or bio-separation or use the assembly of making from concrete molecule or biological information.
Inactivation RS: term used herein " inactivation RS " refers to make its no longer synzyme of its natural cognate tRNA of available amino end acid aminoacylation through sudden change.
Just selecting or selection markers: term used herein " is just being selected or selection markers " when referring to exist, for example to express, activation etc., identifies the mark of the cell with positive selected marker in can the cell of never positive selected marker.
The negative selection or selection markers: term used herein " the negative selection or selection markers " when referring to exist, is for example expressed, activation etc., can identify the mark (for example, comparing with the cell with required character) of the cell with required character.
The intelligencer: term used herein " intelligencer " refers to be used for select the assembly of the target assembly of interested system.For example, the intelligencer (for example can comprise the fluorescent screening mark, green fluorescent protein), luminescent marking (for example, firefly luciferase albumen), based on the selection markers or the selectable marker gene of affinity, as his3, ura3, leu2, lys2, lacZ, β-gal/lacZ (beta galactosidase), Adh (alcohol dehydrogenase) etc.
Eucaryote: term used herein " eucaryote " refers to belong to systematic growth territory eucaryote, for example, animal (for example, mammal, insect, reptile, bird etc.), the biology of infusorian, plant (for example, monocotyledon, dicotyledon, algae etc.), fungi, yeast, flagellate, microsporidian, protobiont etc.
Non--eucaryote: term used herein " non--eucaryote " refers to non-eucaryote.For example, (for example belong to eubacteria, Escherichia coli, thermus thermophilus, bacillus stearothermophilus etc.) the systematic growth territory, or the non-eucaryote in archeobacteria (for example, rich salt bacterium of Methanococcus jannaschii, hot autotrophic methane bacteria, Halobacterium such as Wo Shi and salt bacillus specie NRC-1, the ancient green-ball bacterium of flicker, fierce fireball bacterium, hole Yue Shi fireball bacterium, the hot bacterium of quick gas etc.) systematic growth territory.
Antibody: term used herein " antibody " includes but not limited to basically by one or more immunoglobulin gene encoded polypeptides, or its fragment, its specificity in conjunction with and discriminance analysis thing (antigen).Example comprises polyclone, monoclonal, chimeric and single-chain antibody etc.The term " antibody " that the present invention uses also comprises the fragment of immunoglobulin (Ig), comprises that Fab fragment and expression library comprise the fragment that phage display produces.Antibody structure and term referring to, for example, Paul, " basic immunology " (Fundamental Immunology), the 4th edition, 1999, Raven Press, New York.
Conservative variant: term " conservative variant " refers to translation component, for example, conservative variant O-tRNA or conservative variant O-RS, its function class like conservative variant based on assembly, for example, O-tRNA or O-RS, but change in the sequence.For example, O-RS has the complementary O-tRNA or the conservative variant O-tRNA of alpha-non-natural amino acid with aminoacylation, though O-tRNA and conservative variant O-tRNA do not have identical sequence.Conservative variant for example can have in sequence, and as long as a kind of variation, two kinds of variations, three kinds of variations, four kinds of variations or five kinds or more the variation are conservative variant and O-tRNA or O-RS complementation accordingly.
Selective agent or selective agent: the reagent of certain component can be selected/screen to term used herein " selective agent or selective agent " when referring to exist from colony.For example, select or selective agent includes but not limited to, for example, nutrients, microbiotic, optical wavelength, antibody, the polynucleotide of expression (for example, transcript regutation protein) etc.Selective agent can with, for example, concentration, intensity etc. and different.
Detectable substance: term used herein " detectable substance " refers to can select/screen the reagent of certain component from colony when activation, change, expression etc.For example, detectable substance can be a chemical reagent, for example, 5-fluororotic acid (5-FOA), it under certain conditions, for example, the expression of URA3 reporter becomes detectable, for example, can kill the toxic products of the cell of expressing the URA3 reporter.
Accompanying drawing is briefly described
Fig. 1, A, B and C group for example is generally used for expanding eukaryotic with graphic extension, the positive and negative selection scheme of saccharomyces cerevisiae genetic code, the A picture group explains orally the activated transcription of contract quotation dao gene, and it is that amber by TAG codon among the GAL4 suppresses to drive.The striped frame is pointed out DNA in conjunction with the territory, and dash box is pointed out main and hidden activation domain.The example of B group profile reporter, for example, the HIS3 among the MaV203, LacZ, URA3.The C picture group is separated the plasmid that explanation can be used for selection scheme, for example, and pEcTyrRS/tRNA CUAAnd pGADGAL4xxTAG.
The EcTyrRS and the tRNA of Fig. 2 explanation first generation GAL4 reporter on selective medium CUAThe dependence phenotype.DB-AD is that GAL4DNA is in conjunction with the fusion between territory and activation domain.DB-TAG-AD has the TAG codon that replaces the tyrosine codon between synthetic linker DB and AD.A5 is the inactivation type of EcTyrRS, and wherein 5 residues in the avtive spot sport alanine.
Fig. 3, the EcTyrRS and the tRNA of A and B group profile second generation GAL4 reporter on selective medium CUAThe dependence phenotype.The striped frame is pointed out DNA in conjunction with the territory, and dash box is pointed out main and hidden activation domain.The construction that has the single amino acids sudden change among the A group profile GAL4.The construction that has two amino acid mutations among the B group profile GAL4.
Fig. 4 A, B and C group profile are with or without the pGADGAL4 (T44TAG, R11OTAG) of EcTyrRS, and the various reporters among the MaV203.A group be presented at X-gal ,-Ura or-Leu ,-result of Trp under existing.B organizes the result under the 3-AT existence that is presented at variable concentrations.C organizes the result under the 5-FOA existence that is presented at different percentages.
Fig. 5 A and the various GAL4 mutant of B group profile ONPG hydrolysis, for example, wherein residue T44 (A) and R110 (B) allow the site.The ONPG hydrolysis that the A group profile is measured with various types of sudden changes on the T44 site.The ONPG hydrolysis that the B group profile is measured with various types of sudden changes on the Rl10 site.' GAL4 ' MaV203 of pCL1 that has been transfection exceeds scale～600 ONPG hydrolysis units.' not having ' is to use the plasmid of coding GAL4DB and GAL4AD to transform MaV203 respectively.
Fig. 6 has shown active EcTyrRS clone's selection.The pEcTyrRS-tRNA that will contain 1:10 CUA: pA5-tRNA CUAThe MaV203 of potpourri is with 10 3Dilutability is plated on (Leu ,-Trp) dull and stereotyped (left side) or (-His+50mM3-AT) dull and stereotyped (right side) covers with XGAL and to handle for Leu ,-Trp.
Fig. 7, A and B group.The A group profile combines the three-dimensional view of avtive spot of the bacillus stearothermophilus tyrosyl-tRNA synthetase of tyrosine.The residue that has shown sudden change, and with from Escherichia coli tyrosyl-tRNA synthetase Tyr 37(bacillus stearothermophilus TyrRS residue Tyr 34), Asn 126(Asn 123), Asp 182(Asp 176), Phe 183(Phe 177) and Leu 186(Leu 180) residue corresponding.B group profile alpha-non-natural amino acid example (from left to right) is right-structural formula of acetyl group-L-phenylalanine (1), right-benzoyl-L-phenylalanine (2), right-azido-L-phenylalanine (3), 0-methyl-L-tyrosine (4) and right-iodo-L-tyrosine (5).
Fig. 8, A, B, C and D group.The A group profile can be used for selecting/screening the carrier of quadrature tRNA and report that the quadrature amino acyl synthetase or the quadrature tRNA/RS of construction, eukaryotic are right.The B group profile contains the phenotype of GAL4 response type HIS3, URA3 and lacZ response type intelligencer's yeast, on selective medium, in response to active (TyrRS) or inactivation (A5RS) aminoacyl-tRNA synthetase.The example of a selection scheme of C group profile at eukaryotic for example is used for, and UAA selects the additional amino acid whose mutant synzyme of coding in the saccharomyces cerevisiae of alpha-non-natural amino acid.The D group profile is right from having-selection of acetyl group-L-phenylalanine the phenotype of separated yeast.
Fig. 9 illustrates (33TAG) protein expression of HIS in saccharomyces cerevisiae of human superoxide dismutase (hSOD), and its genetic coding alpha-non-natural amino acid is as shown in Fig. 7 B group.
Figure 10, the tandem mass spectrum analysis of the tryptic peptide VY*GSIK that contain alpha-non-natural amino acid (be designated as Y*) (SEQ ID NO:87) of A-H group profile as shown in Fig. 7 B group.The A group profile has the tandem mass spectrum analysis of the tryptic peptide (1) of alpha-non-natural amino acid p-acetyl group-L-phenylalanine.The tandem mass spectrum analysis of the tryptic peptide (2) of the B group profile has alpha-non-natural amino acid right-benzoyl-L-phenylalanine.The tandem mass spectrum analysis of the tryptic peptide (3) of the C group profile has alpha-non-natural amino acid right-azido-L-phenylalanine.The D group profile has the tandem mass spectrum analysis of the tryptic peptide (4) of alpha-non-natural amino acid neighbour-methyl-L-tyrosine.The tandem mass spectrum analysis of the tryptic peptide (5) of the E group profile has alpha-non-natural amino acid right-iodo-L-tyrosine.The F group profile has the tandem mass spectrum analysis of the tryptic peptide of tryptophane (W) in the Y* position.The G group profile has the tandem mass spectrum analysis of the tryptic peptide of tyrosine (Y) in the Y* position.The H group profile has the tandem mass spectrum analysis of the tryptic peptide of leucine (L) in the Y* position.
Figure 11 illustrates the example of two kinds of alpha-non-natural amino acids, and (1) right-and propargyloxy phenylalanine and (2) are right-the triazobenzene alanine.
Figure 12, the expression of A, B and C group profile SOD under the alpha-non-natural amino acid shown in Figure 11 1 and 2 existence or non-existent situation.The blue dyeing experiment of A group profile Gelcode.The B group profile Western Blot experiment of anti--SOD antibody.The C group profile Western Blot experiment of anti--6xHis antibody.
Figure 13, A, B and C group profile are by the protein of [3+2] cycloaddition mark.The dye marker 3-6 that the A group profile is synthetic.Reaction between B group profile SOD and dyestuff.The blue dyeing of fluorescent scanning and Gelcode in the C group profile gel.
Figure 14 illustrates eukaryotic, as lacking on the SD nutrient culture media of uracil, in 1 or 2 existence shown in Figure 11 or not with the growth of synthase mutant transformed saccharomyces cerevisiae cell.
Figure 15, A and B group profile contain the tandem mass spectrum analysis of the tryptic peptide VY*GSIK (SEQ ID NO:87) of nitrine (Az) (A group) or alkynes (Al) (B group) alpha-non-natural amino acid in the Y* position, show their expectation fragment ions quality.Arrow shows b (indigo plant) and y (red) ionization series of observing each peptide.
Figure 16 graphic extension is alpha-non-natural amino acid, as just mixing in right-propargyloxy phenylalanine body in the growth polypeptide chain, and [3+2] cycloaddition reaction by this alpha-non-natural amino acid, with the biological conjugation of organic molecule.
Figure 17, A, B and C group profile contain the protein of alpha-non-natural amino acid with [3+2] cycloaddition PEGization.A group profile propargyl acid amides PEG in the presence of Cu (I) and phosphate buffer (PB) with contain the amino acid whose protein of azido (for example, N 3-SOD) reaction.The B group profile is by the gel analysis PEGYLATION OF PROTEINS.C group profile propargyl acid amides PEG's is synthetic.
Detailed Description Of The Invention
In eukaryotic, surmount the ability of the direct genetic modification protein structure of chemical restriction that genetic code forces, will provide powerful molecular tool, to survey or the manipulated cell process. The invention provides the amino acid no purpose translation component that in eukaryotic, can expand genetic coding. These comprise tRNAs (for example, quadrature tRNAs (O-tRNAs)), aminoacyl-tRNA synthetase (for example, quadrature synzyme (O-RS)), O-tRNA/O-RS to and alpha-non-natural amino acid.
Usually, can effective expression and process O-tRNA of the present invention, it brings into play function in eukaryotic translation, but not by host's aminoacyl-tRNA synthetase aminoacylation significantly. In response to selecting codon, O-tRNA of the present invention is delivered to alpha-non-natural amino acid on the polypeptide chain of growing at the mRNA translate duration, do not encode any one of 20 common seed amino acids of this alpha-non-natural amino acid.
O-RS of the present invention in eukaryotic preferably aminoacylation the present invention have the O-tRNA of alpha-non-natural amino acid, but any kytoplasm host's of aminoacylation tRNA not. And the specificity of aminoacyl-tRNA synthetase of the present invention makes it accept alpha-non-natural amino acid and refuses any Endogenous Amino Acids. The polypeptide that comprises example O-RS or its partial amino-acid series also is feature of the present invention. In addition, the polynucleotides of coding translation component, O-tRNA, O-RS and part thereof are features of the present invention.
The present invention also provides to produce alpha-non-natural amino acid is used for eukaryotic required translation component, as O-RS and or quadrature to the method for (quadrature tRNA and quadrature aminoacyl-tRNA synthetase), (and the translation component of being produced by described method). For example, from colibacillary tyrosyl-tRNA synthetase/tRNACUATo being O-tRNA/O-RS of the present invention pair. In addition, the present invention also is described in the method for selection/screening translation component in the eukaryotic, in case select/screening, just can use those assemblies at different eukaryotics (eukaryotic that is used for selection/screening). For example, producing the selection/screening technique that is used for eukaryotic translation component can be at yeast, for example, carry out in the saccharomyces cerevisiae, then those can be selected assembly be used for other eukaryotic, for example, other yeast cells, mammalian cell, insect cell, plant cell, fungal cell etc.
The present invention also provides and produced method of protein in eukaryotic, and wherein this albumen contains alpha-non-natural amino acid. Produce this albumen with translation component of the present invention. The present invention also provides the protein that comprises alpha-non-natural amino acid (with the albumen of being produced by the inventive method). Interested albumen or polypeptide also can comprise posttranslational modification, for example, add modification by [3+2] cycloaddition or nucleophilic-electrophilic reaction, and this can not be undertaken by prokaryotic, etc. In some embodiments, the present invention also comprises the method (with the albumen of being produced by described method) of transcribing adjusting albumen with alpha-non-natural amino acid production. The composition that comprises the albumen that contains alpha-non-natural amino acid also is feature of the present invention.
The kit of producing albumen or polypeptide with alpha-non-natural amino acid also is feature of the present invention.
Quadrature aminoacyl-tRNA synthetase (O-RS)
For the alpha-non-natural amino acid specificity is incorporated in interested albumen or the polypeptide, in eukaryotic, change the substrate specificity of synzyme so that only have required alpha-non-natural amino acid, and 20 any uncommon seed amino acids add tRNA. If the quadrature synzyme mixes, it will cause being mixed with natural and mutein alpha-non-natural amino acid at target position. The invention provides composition and the method for production quadrature aminoacyl-tRNA synthetase that has the substrate specificity of modification for concrete alpha-non-natural amino acid.
The eukaryotic that comprises quadrature aminoacyl-tRNA synthetase (O-RS) is feature of the present invention. O-RS in eukaryotic preferably aminoacylation have the quadrature tRNA (O-tRNA) of alpha-non-natural amino acid. In some embodiments, O-RS utilizes more than an alpha-non-natural amino acid, for example, two or more, three or more etc. Therefore, O-RS of the present invention can have with the different alpha-non-natural amino acids ability of aminoacylation O-tRNA preferably. Put into cell and/or provide additional control level by selecting to put into cell with the alpha-non-natural amino acid of the difference amount of mixing by the combination of selecting which alpha-non-natural amino acid or alpha-non-natural amino acid.
Compare with natural amino acid, O-RS of the present invention randomly has the enzymatic property of one or more improvement or enhancing to alpha-non-natural amino acid. These character comprise, for example, with the amino acid of natural generation as, one of 20 kinds of known common amino acids are compared, to the higher k of alpha-non-natural amino acidm, low km, higher kcat, low kcat, low kcat/k m, higher kcat/k mDeng.
Randomly, the polypeptide that O-RS can be by comprising O-RS and/or the polynucleotides by coding O-RS or its part offer eukaryotic. For example, such as any one listed polynucleotide sequence coding O-RS or its part in SEQ ID NO.:3-35 (for example, any other subgroup of 3-19,20-35 or sequence 3-35) or its complementary polynucleotide sequence. In another embodiment, O-RS comprises such as SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86, or the amino acid sequence of their conservative variant. Referring to for example, this paper table 5,6 and 8 and embodiment 6 be used for the sequence of example O-RS molecule.
O-RS also can comprise with the tyrosyl aminoacyl-tRNA synthetase (TyrRS) of natural generation (for example; listed among the SEQ ID NO.:2) amino acid sequence for example; at least 90%, at least 95%, at least 98%, at least 99% or even at least 99.5% identical amino acid sequence, comprise two or more amino acid of A-E family. A family comprises valine, isoleucine, leucine, glycine, serine, alanine or the threonine on the Tyr37 opposite position with Escherichia coli TyrRS; B family comprises the aspartic acid on the Asn126 opposite position with Escherichia coli TyrRS; C family comprises threonine, serine, arginine, asparagine or the glycine on the Asp182 opposite position with Escherichia coli TyrRS; D family comprises methionine, alanine, valine or the tyrosine on the Phe183 opposite position with Escherichia coli TyrRS; E family comprises serine, methionine, valine, cysteine, threonine or the alanine on the Leu186 opposite position with Escherichia coli TyrRS. Any subgroup combination of these families is features of the present invention. For example, in one embodiment, O-RS has valine, isoleucine, leucine or the threonine on two or more Tyr37 opposite positions that are selected from appearance and Escherichia coli TyrRS; With threonine, serine, arginine or the glycine on the Asp182 opposite position of Escherichia coli TyrRS; With methionine or the tyrosine on the Phe183 opposite position of Escherichia coli TyrRS; With with the Leu186 opposite position of Escherichia coli TyrRS on serine or the amino acid of alanine. In another embodiment, O-RS comprises that two or more are selected from glycine, serine or alanine on the Tyr37 opposite position with Escherichia coli TyrRS, with the aspartic acid on the Asn126 opposite position of Escherichia coli TyrRS, with the asparagine on the Asp182 opposite position of Escherichia coli TyrRS, with alanine or the valine on the Phe183 opposite position of Escherichia coli TyrRS, and/or with the Leu186 opposite position of Escherichia coli TyrRS on the amino acid of methionine, valine, cysteine or threonine. Also referring to, for example, the table 4 of this paper, table 6 and table 8.
Except O-RS, eukaryotic of the present invention also can comprise annexing ingredient, for example, and alpha-non-natural amino acid. Eukaryotic (for example also comprises quadrature tRNA (O-tRNA); from non-eucaryote; such as Escherichia coli, bacillus stearothermophilus and/or analog), O-tRNA identification selection codon wherein, and by O-RS preferably aminoacylation have the O-tRNA of alpha-non-natural amino acid. The nucleic acid that also can have the polynucleotides that comprise the polypeptide of interest of encoding in the cell, wherein polynucleotides comprise the selection codon of O-tRNA identification, or one or more the combination in these.
In one aspect, O-tRNA mediation alpha-non-natural amino acid mixes in the protein, its efficient be equivalent to comprise SEQ ID NO.:65 listed comprise or process from the tRNA of polynucleotide sequence efficient for example, at least 45%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95% or 99%. On the other hand, O-tRNA comprise SEQ ID NO.:65 and O-RS comprise SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86 and/or their conservative variant in any one listed peptide sequence. Also referring to, for example, be used for the sequence of example O-RS and O-tRNA molecule among this paper table 5 and the embodiment 6.
In one embodiment; eukaryotic comprises quadrature aminoacyl-tRNA synthetase (O-RS), quadrature tRNA (O-tRNA), alpha-non-natural amino acid and contains the nucleic acid of the polynucleotides of the polypeptide of interest of encoding, and wherein polynucleotides comprise the selection codon of O-tRNA identification. O-RS in eukaryotic preferably aminoacylation have the quadrature tRNA (O-tRNA) of alpha-non-natural amino acid; cell is the production polypeptide of interest in the situation that does not have alpha-non-natural amino acid; its productive rate for example is equivalent in the presence of alpha-non-natural amino acid the polypeptide productive rate, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2.5% etc.
Be that the method for the production O-RS of feature of the present invention randomly comprises from the framework of wild type synzyme and produces sudden change synzyme storehouse, then with respect to 20 common seed amino acids the specificity of alpha-non-natural amino acid selected sudden change RS based on them. In order to separate described synzyme, system of selection is: (i) responsive, because can be low from the activity of the required synzyme of the first run, number be little; (ii) " adjustable " selects stringency because need to change in different selection wheels; (iii) general, so that these methods can be used for different alpha-non-natural amino acids.
Production in eukaryotic preferably the method for quadrature aminoacyl-tRNA synthetase (O-RS) of aminoacylation with quadrature tRNA of alpha-non-natural amino acid generally comprise the combination that application is just being selected, the negative selection then. In just selecting, introduce the inhibition of selecting codon in positive mark's nonessential site eukaryotic is survived under positive selection pressure. In the presence of alpha-non-natural amino acid, thereby the survivaling cell coding adds the active synzyme of the quadrature inhibition type tRNA with alpha-non-natural amino acid. In negative the selection, select the inhibition of codon to remove in the nonessential site introducing of negative marker and have the specific synzyme of natural amino acid. During positive and negative is selected the cell coding of survival only (or at least preferably) aminoacylation (adding) have the synzyme of the quadrature inhibition type tRNA of alpha-non-natural amino acid.
For example, the method comprises: (a) just select, in the presence of alpha-non-natural amino acid, the biological eukaryotic colony of the first kind, wherein eukaryotic respectively comprises: the i) a member in aminoacyl-tRNA synthetase (RS) library, ii) quadrature tRNA (O-tRNA), iii) coding positive selected marker polynucleotides, and iv) coding negative selectable marker polynucleotides; The cell of wherein surviving in just selecting is included in the active RS of aminoacylation quadrature tRNA (O-tRNA) in the presence of the alpha-non-natural amino acid; The cell that (b) will survive in just selecting is born selection in the situation that does not have alpha-non-natural amino acid; have the active RS of the O-tRNA of natural amino acid to remove aminoacylation, thereby provide aminoacylation preferably to have the O-RS of the O-tRNA of alpha-non-natural amino acid.
Positive selected marker can be any one in the various molecules. In one embodiment, positive selected marker is the product that nutritional supplement is provided for growth, and selects at the culture medium that lacks nutritional supplement. The example of the polynucleotides of the positive selected marker of encoding includes but not limited to, for example, based on the reporter of the amino acid nutrient defective of replenishing cell, his3 gene (for example, his3 gene code imidzoleglycerol phosphate dehydrogenase wherein is by 3-aminotriazole(ATA) (3-AT)), the detection such as ura3 gene, leu2 gene, lys2 gene, lacZ gene, adh gene. Referring to, for example, G.M.Kishore and D.M.Shah, (1988), as the amino acid bio synthetic inhibitor (Amino acid biosynthesis inhibitors as herbicides) of herbicide, Annual Review of Biochemistry 57:627-663. In one embodiment, the hydrolysis by o-nitrophenyl-β-D-galactolipin pyranoside (ONPG) detects the lacZ generation. Referring to, for example, I.G.Serebriiskii and E.A.Golemis, (2000), the purposes of lacZ in the research gene function: be used for the evaluation (Uses of lacZ to study gene function:evaluation of beta-galactosidase assays employed in the yeast two-hybrid system) of the beta galactose glycosides mensuration of yeast two-hybrid system, Analytical Biochemistry 285:1-15. Additional positive selected marker comprises; for example, luciferase, green fluorescent protein (GFP), YFP, EGFP, RFP, antibiotics resistance gene product are (for example; chloramphenicol acetyltransferase (CAT)), transcribe and regulate albumen (for example, GAL4) etc. The polynucleotides of the positive selected marker of encoding randomly comprise the selection codon.
The polynucleotides of the positive selected marker of coding can be operably connected to response element. Also can exist coding from response element regulate transcribe transcribe adjusting albumen, and comprise the additional polynucleotides that at least one selects codon. The O-tRNA that has alpha-non-natural amino acid by aminoacylation mixes alpha-non-natural amino acid and transcribes transcribing of polynucleotides (for example, the reporter) of regulating the positive selected marker that causes in the albumen encoding. For example, see Figure 1A. Select codon randomly to be positioned at encoding transcription and regulate the DNA of albumen in conjunction with the polynucleotides in territory or basically near its part.
Also the polynucleotides of coding negative selectable marker can be operably connected to response element, regulate protein mediated transcribing by transcribing. Referring to, for example, A.J, DeMaggio etc., (2000), yeast division-crossing system (The yeast split-hybrid system), Method Enzymol.328:128-137; H.M.Shih etc., (1996), positive heredity selects to destroy protein-protein interaction: identify to stop the CREB sudden change (A positive genetic selection for disrupting protein-protein interactions:identification of CREB mutations that prevent association with the coactivator CBP) of being combined with coactivator CBP, Proc.Natl.Acad.Sci.U.S.A.93:13896-13901; M.Vidal, Deng, (1996), characterize mammalian proteins-protein-interacting territory (Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system) with the reverse two-hybrid system heredity of yeast, [comment], Proc.Natl.Acad.Sci. U.S.A.93:10321-10326; And M.Vidal, Deng, (1996), detecting protein-protein with reverse double cross and single crosses system dissociates and DNA-protein-interacting (Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions), [comment], Proc.Natl.Acad.Sci.U.S.A.93:10315-10320. The O-tRNA that has natural amino acid by aminoacylation mixes natural amino acid to transcribe to regulate in the albumen and causes transcribing of negative selectable marker. Negative selectable marker randomly comprises the selection codon. In one embodiment, positive selected marker of the present invention and/or negative selectable marker can comprise at least two selection codons, they each or two can contain at least two kinds of different selection codons or at least two kinds of identical selection codons.
Transcribing and regulating albumen is to be operably connected to the molecule that the sequence of response element is transcribed with nucleotide sequence (for example, response element) in conjunction with (directly or indirectly) and adjusting. Transcribe regulate albumen can be transcription activating protein (for example, GAL4, nuclear hormone receptor, AP1, CREB, LEF/tcf family member, SMADs, VP16, SP1 etc.), (for example transcribe CKIs, nuclear hormone receptor, Groucho/tle family, Engrailed family etc.) or the albumen (for example, LEF/tcf, homology frame albumen etc.) that can have according to environment two kinds of activity. Response element generally be transcribe regulate the discernible nucleotide sequence of albumen or with transcribe the additives of regulating the consistent effect of albumen.
Transcribing and regulating another example of albumen is transcription activating protein, and GAL4 is (referring to for example, Figure 1A). Referring to, for example, A.Laughon, Deng, (1984), evaluation is by two kinds of albumen (Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene) of saccharomyces cerevisiae GAL4 gene code, Molecular ﹠Cellular Biology 4:268-275; A.Laughon and R.F.Gesteland, (1984), the primary structure of saccharomyces cerevisiae GAL4 gene (Primary structure of the Saccharomyces cerevisiae GAL4 gene), Molecular﹠Cellular Biology 4:260-267; L.Keegan, Deng, (1986), regulate the transcribing of albumen-mobilizing function DNA isolation in conjunction with (Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein), Science 231:699-704 from eucaryon; And M.Ptashne, (1988), the eukaryotic transcription activator protein is (the How eukaryotic transcriptional activators work) how to work, Nature 335:683-689. Terminal 147 amino acid of the N-of these 881 amino acid whose albumen form specifically DNA in conjunction with dna sequence dna in conjunction with territory (DBD). Referring to, for example, M.Carey, etc., (1989), amino-terminal fragment of GAL4 and DNA are combined into dimer (An amino-terminal fragment of GAL4 binds DNA as a dimer), J.Mol.Biol.209:423-432; And E.Giniger, etc., (1985), GAL4, the positive specific DNA of albumen of regulating of one primary yeast is in conjunction with (Specific DNA binding of GAL4, a positive regulatory protein of yeast), Cell 40:767-774. This DBD is connected to 113 amino acid activation territories (AD) of C-end by interleaving protein sequence, when this activation domain can activated transcription when DNA is combined. Referring to, for example, J.Ma and M.Ptashne, (1987), the deletion analysis of GAL4 defines two kinds of transcriptional activation sections (Deletion analysis of GAL4 defines two transcriptional activating segments), Cell 48:847-853: and J.Ma and M.Ptashne, (1987), 30 amino acid (The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80) of GAL80 identification GAL4 carboxyl-end, Cell 50:137-142. By amber codon is placed, for example, contain the terminal DBD of N-of the single polypeptide of the terminal DBD of N-of GAL4 and the terminal AD of its C-, suppress to be connected with the transcriptional activation by GAL4 (Fig. 1, A organizes) by the right amber of O-tRNA/O-RS. The positive and negative that the reporter that GAL4 activates can be used for carrying out with gene is selected (Fig. 1, B group).
Be used for the negative culture medium of selecting and comprise selective agent or the selective agent that is converted into detectable substance by negative selectable marker. In one aspect of the invention, this detectable substance is noxious material. The coding negative selectable marker polynucleotides can be, for example, the ura3 gene. For example, the URA3 reporter can be placed under the control of the promoter that contains the GAL4DNA binding site. For example, when producing negative selectable marker with the polynucleotides translation of selecting codon coding GAL4, GAL4 activates transcribing of URA3. Finish negative the selection at the culture medium that contains 5-fluororotic acid (5-FOA), the gene outcome of ura3 gene can change into the 5-fluororotic acid detectable substance (for example, the noxious material of cell killing). Referring to, for example, J.D.Boeke, Deng, (1984), in yeast, just selecting to lack the mutant of orotidine-5 '-phosphate decarboxylase activity: 5-fluororotic acid resistance (A positive selection for mutants lacking orotidine-5 '-phosphate decarboxylase activity in yeast:5-fluoroorotic acid resistance), Molecular﹠General Genetics 197:345-346); M.Vidal, Deng, (1996), characterize mammalian proteins-protein-interacting territory (Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system) with the reverse two-hybrid system heredity of yeast, [comment], Proc.Natl.Acad.Sci.U.S.A.93:10321-10326; And M.Vidal, Deng, (1996), detecting protein-protein with reverse double cross and single crosses system dissociates and DNA-protein-interacting (Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions), [comment], Proc. Natl.Acad.Sci.U.S.A.93:10315-10320. Also referring to Fig. 8 C.
As positive selected marker, negative selectable marker also can be any one of various molecules. In one embodiment, positive selected marker and/or negative selectable marker are to fluoresce in the presence of suitable reactant or polypeptide that catalytic luminescence reacts. For example; negative selectable marker includes but not limited to; for example, luciferase, green fluorescent protein (GFP), YFP, EGFP, RFP, antibiotics resistance gene product (such as, chloramphenicol acetyltransferase (CAT)), lacZ gene outcome, transcribe and regulate albumen etc. In one aspect of the invention, by fluorescence-activated cell sorting (FACS) or by the positive selected marker of luminous detection and/or negative selectable marker. In another embodiment, positive selected marker and/or negative selectable marker comprise the selection markers based on affinity. The positive selected marker of same polynucleotides codified and negative selectable marker.
Select/screen the additional levels of stringency also to can be used for the inventive method. This selection or screening stringency can produced difference on one or two steps of O-RS method. This can comprise, for example, is just changing coding and/or the amount of response element in the polynucleotides of negative selectable marker, and the inactivation synzyme that quantity is not waited joined in the step or two steps of step, changes the amount etc. of the selection/selective agent of use. Also can carry out additional wheel just and/or negative the selection.
Select or screening also can comprise one and or multiple plus or minus is selected or screening, comprise, for example, the change of the change of amino acid permeability, the change of translation efficiency, translation fidelity etc. Usually, one or more changes are based on and comprise or encode for the production of the sudden change in one or more polynucleotides of the right assembly of the quadrature tRNA-tRNA synzyme of albumen.
Can from excessive inactivation synzyme, select fast active synzyme with model enrichment research. Can just carry out and/or negative Research on Model Selection. The eukaryotic that for example, will contain possible active aminoacyl-tRNA synthetase mixes with the inactivation aminoacyl-tRNA synthetase of excessive different multiples. Carry out between the cell that the ratio comparison is grown in Nonsele ctive culture media, for example, the X-GAL overlay measurement, with at selective medium (for example, do not exist in the situation of histidine and/or uracil) in carry out in growth and the cell that can survive, for example, X-GAL measures. Select for the negative norm type, possible active aminoacyl-tRNA synthetase is mixed with the inactivation aminoacyl-tRNA synthetase of excessive different multiples, with the negative material of selecting, for example, 5-FO selects.
Usually, RS library (for example, mutant RS library) contain from as from the RS of non-Eukaryotic at least a aminoacyl-tRNA synthetase (RS). In one embodiment, the RS library for example, is wherein passed through from inactivation RS, such as at the avtive spot of synzyme, in editor's mechanism site of synzyme, produce inactivation RS in different loci by the active RS that suddenlys change in conjunction with the modes such as not same area of synzyme. For example, the avtive spot residue of RS is sported, for example, alanine residue. With the polynucleotides of the RS of coding alanine mutation as template, take with alanine residue mutagenesis as all 20 amino acid. Select/screen mutant RS library to produce O-RS. In another embodiment, inactivation RS comprises the amino acid binding pocket, replaces the amino acid that one or more contain binding pocket with one or more different aminoacids. In one embodiment, the amino acid of replacement replaces with alanine. Randomly, with the polynucleotides of the RS of coding alanine mutation as template, take with alanine residue mutagenesis as all 20 amino acid, the row filter/selection of going forward side by side.
The method of producing O-RS also can comprise with various induced-mutation techniques known in the art produces the RS library. For example, can pass through mutation site-specific, random point mutation, homologous recombination, DNA reorganization or other recurrence method of mutagenesis, chimeric construct or their any combination and produce sudden change RS. For example, can be from two or more other, for example less, less different " Ya Wenku " produces mutant RS library. In case synzyme carries out positive and negative selection/screening strategy, further these synzyme of mutagenesis just. The nucleic acid that for example, can separate the O-RS that encodes; Can produce from this nucleic acid the polynucleotides (for example, by random mutagenesis, site-specific mutagenesis, restructuring or their any combination) of the O-RS of group coding sudden change; With, can repeat independent step or the combination of these steps, until obtain the sudden change O-RS that aminoacylation preferably has the O-tRNA of alpha-non-natural amino acid. In one aspect of the invention, these steps are carried out twice at least.
Can at WO2002/086075, be entitled as and find the more details of producing O-RS in " for the production of the right method and composition of quadrature tRNA-aminoacyl tRNA synthetase ". Also referring to, Hamano-Takaku etc., (2000) the mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the alpha-non-natural amino acid azatyrosine than tyrosine more effective (A mutant Escherichia coli Tyrosyl-tRNA Synthetase utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine), Journal of Biological Chemistry, 275 (51): 40324-40328; Kiga etc. (2002), the alpha-non-natural amino acid locus specificity mixed engineering colon bacillus tyrosyl-tRNA synthetase and the application in Wheat Germ Cell-free System (An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell free system) thereof in the albumen in eukaryotic translation, PNAS 99 (15): 9715-9723; With Francklyn etc., (2002), aminoacyl-tRNA synthetase: accomplished performer in the translation arenas of variation (Aminoacyl-tRNA synthetases:Versatile players in the changing theater of traslation); RNA, 8:1363-1372.
Quadrature tRNAs
The invention provides the eukaryotic that comprises quadrature tRNA (O-tRNA). This quadrature tRNA mediates in the protein of the polynucleotide encoding that mixes the selection codon that contains O-tRNA identification in the alpha-non-natural amino acid body. In some embodiments, O-tRNA mediation alpha-non-natural amino acid of the present invention mixes in the protein, its efficient be equivalent to contain the listed polynucleotide sequence of SEQ ID NO.:65 or the tRNA efficient of in the cell of this sequence, processing for example, at least 40%, at least 45%, at least 50%, at least 60%, at least 75%, at least 80% or even 90% or higher. Table 5 referring to this paper.
The example of O-tRNA of the present invention is SEQ ID NO.:65 (referring to embodiment 6 and the table 5 of this paper). SEQ ID NO.:65 is before the montage/transcripton of processing, and it is randomly processed in cell, for example, adopts the endogenous of cell to shear and processing machine, modifies and forms active O-tRNA. Usually, the colony of the front transcripton of this montage forms colony's (active tRNA can be one or more activity forms) of active tRNA in cell. The present invention also comprises the conservative variant of O-tRNA and its cell elaboration products. For example, the conservative variant of O-tRNA comprises function class like the O-tRNA of SEQ ID NO.:65 and keeps tRNA L-shape structure such as form processing, but do not have identical sequence those molecules of (being different from wild type tRNA molecule). Usually, O-tRNA of the present invention is O-tRNA capable of circulation because O-tRNA can be in vivo aminoacylation again, in response to selecting codon to mediate again in the protein that alpha-non-natural amino acid mixes polynucleotide encoding.
TRNA in eucaryote and not transcribing by rna plymerase iii in prokaryotes carries out, and this polymerase is to restricting at the primary sequence of the tRNA of eukaryotic transcription structural gene. In addition, in eukaryotic, tRNA need to be outputed to the place of transcribing them from nuclear is kytoplasm, to play a role in translation. Nucleic acid or its complementary polynucleotide of code book invention O-tRNA also are features of the present invention. In one aspect of the invention, the nucleic acid of code book invention O-tRNA comprises the internal promoter sequence, and for example, the A frame (for example, TRGCNNAGY) and B frame (for example, GGTTCGANTCC, SEQ ID NO:88). O-tRNA of the present invention also can be posttranscriptional modification. For example, the posttranscriptional modification of tRNA gene comprises with RNA enzyme P and 3 '-endonuclease and removes respectively 5 '-and 3 '-flanking sequence in eucaryote. Adding 3 '-CCA sequence also is the posttranscriptional modification of tRNA gene in the eucaryote.
In one embodiment, bear selection by the eukaryotic colony with the first kind and obtain O-tRNA, wherein eukaryotic contains a member in tRNA library. The cell that contains by to a member in the tRNA library of eukaryotic endogenous aminoacyl-tRNA synthetase (RS) aminoacylation has been removed in negative selection. This provides the tRNA storehouse with the eukaryotic quadrature of the first kind.
In addition, above-mentioned alpha-non-natural amino acid is mixed in the polypeptide method or with other Combination of Methods in, can use trans translation system. This system comprises and is present in the molecule that Escherichia coli are called tmRNA. Relate to alanyl tRNA on this RNA molecular structure, by alanyl synzyme aminoacylation. Difference between tmRNA and the tRNA is that anticodon loop is replaced by special large sequence. This sequence allows ribosomes to continue translation as template in suspended sequence with the ORFs of coding in the tmRNA. In the present invention, can produce with quadrature synzyme aminoacylation and be loaded with the quadrature tmRNA of alpha-non-natural amino acid preferably. By by this system's open gene, ribosomes is ended work in the specificity site; Alpha-non-natural amino acid is introduced this site, and then the sequence with coding in the quadrature tmRNA continues translation.
Other method of Restruction quadrature tRNA can; for example; International Patent Application WO 2002/086075 is entitled as in " for the production of the right method and composition of quadrature tRNA-aminoacyl tRNA synthetase (Methods and compositions for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs) " and finds. Also referring to Forster etc., (2003) by translation from the beginning the genetic code sequencing of design intend peptide synthetase (Programming peptidomimetic synthetases by translating genetic codes designed de novo) PNAS 100 (11): 6353-6357; With Feng etc., (2003), the tRNA that changes expansion tRNA synzyme by monamino acid identifies (Expanding tRNA recognition of a tRNA synthetase by a single amino acid change), and PNAS 100 (10): 5676-5681.
Quadrature tRNA and quadrature aminoacyl-tRNA synthetase pair
Quadrature is to by O-tRNA, and for example, inhibition type tRNA, frameshit tRNA etc. form with O-RS. O-tRNA is by endogenous synzyme acidylate, and can mediate alpha-non-natural amino acid and mix in the protein that contains the polynucleotide encoding of the selection codon of identification in the O-tRNA body. In eukaryotic, O-RS identification O-tRNA and preferably aminoacylation have the O-tRNA of alpha-non-natural amino acid. The present invention also comprises and produces the right method of quadrature and the quadrature produced of method pair thus, and is used for eukaryotic quadrature to composition. In eukaryotic, the right generation of a plurality of quadrature tRNA/ synzyme can allow to mix simultaneously a plurality of alpha-non-natural amino acids with different codons.
In eukaryotic, can suppress right such as nonsense by striding kind of aminoacylation with poor efficiency from different biologicalinputs pair, produce quadrature O-tRNA/O-RS pair. In eukaryotic, O-tRNA and O-RS effectively express and process, and O-tRNA exports kytoplasm to effectively from nuclear. For example, one described to being from colibacillary tyrosyl-tRNA synzyme/tRNACUATo (referring to, for example, H.M.Goodman, etc., (1968), Nature 217:1019-24; And D.G.Barker, etc., (1982), FEBS Letters 150:419-23). When expressing in the kytoplasms at saccharomyces cerevisiae both, the Escherichia coli tyrosyl-tRNA synthetase is its related Escherichia coli tRNA of aminoacylation effectivelyCUA, but aminoacylation saccharomyces cerevisiae tRNA not. Referring to, for example, H.Edwards and P.Schimmel, (1990), Molecular ﹠Cellular Biology 10:1633-41; And H.Edwards, etc., (1991), PNAS United States of America 88:1153-6. In addition, Escherichia coli tyrosyl tRNACUABe the saccharomyces cerevisiae aminoacyl-tRNA synthetase poor substrate (referring to, for example, V.Trezeguet, etc., (1991), Molecular﹠Cellular Biology 11:2744-51), but in the protein translation of saccharomyces cerevisiae effective performance function. Referring to, for example, H.Edwards and P.Schimmel, (1990) Molecular﹠Cellular Biology 10:1633-41; H.Edwards, etc., (1991), PNAS United States of America 88:1153-6; And V.Trezeguet, etc., (1991), Molecular﹠Cellular Biology 11:2744-51. And Escherichia coli TyrRS does not have editor's mechanism of proofreading and correct the alpha-non-natural amino acid that is connected to tRNA.
O-tRNA and O-RS can be in the various biologies natural generation maybe can be that the tRNA of natural generation and/or RS sudden change obtain, it has produced tRNA library and/or RS library. Referring to the part that is entitled as " source and host " herein. In various embodiments, O-tRNA and O-RS are from least a biology. In another embodiment, O-tRNA is from the natural generation tRNA of natural generation or sudden change in the first biology, and O-RS is from the natural generation RS of natural generation or sudden change in the second biology. In one embodiment, the first and second non-eucaryotes are identical. In addition, the first and second non-eucaryotes can be different.
Referring to the method for producing O-RS and O-tRNA in the part that is entitled as " quadrature aminoacyl-tRNA synthetase " and " O-tRNA " herein. Also referring to International Patent Application WO 2002/086075, be entitled as " producing the right method and composition of quadrature tRNA-aminoacyl tRNA synthetase " (Methods and compositions for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs).
Fidelity, efficient and productive rate
Fidelity refers to desired molecule, for example, and the degree of accuracy of desired location in the polypeptide that alpha-non-natural amino acid or amino acid incorporation are being grown. Translation component of the present invention is mixed alpha-non-natural amino acid in the protein with high fidelity in response to selecting codon. For example, use assembly of the present invention, required alpha-non-natural amino acid (is for example just mixed in the growth polypeptide chain efficient of desired location, in response to selecting codon) efficient that is equivalent to unwanted specificity natural amino acid is mixed E desired location in the growth polypeptide chain for example, greater than 75%, greater than 85%, greater than 95% or even greater than 99% or higher.
Efficient also can refer to compare with corresponding contrast, and the O-RS aminoacylation has the degree of the O-tRNA of alpha-non-natural amino acid. Can limit O-RS of the present invention by their efficient. In some embodiments of the present invention, an O-RS is compared with another O-RS. For example; the O-RS aminoacylation O-tRNA efficient that the efficient that O-RS aminoacylation of the present invention has the O-tRNA of alpha-non-natural amino acid is equivalent to have SEQ ID NO.:86 or 45 listed amino acid sequences (or in the table 5 another specificity RS) for example, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95% or even 99% or higher. In another embodiment, the efficient that O-RS aminoacylation of the present invention has an O-tRNA of alpha-non-natural amino acid has at least 10 times of the efficient height of the O-tRNA of natural amino acid, at least 20 times, at least 30 times etc. than O-RS aminoacylation.
Use translation component of the present invention, the productive rate that contains the polypeptide of interest of alpha-non-natural amino acid be lack the cell of selecting codon the polypeptide of interest that obtains natural generation from polynucleotides productive rate for example, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, 50% or higher. On the other hand, the productive rate of cell production polypeptide of interest in the situation that does not have alpha-non-natural amino acid is for example to produce the polypeptide productive rate in the presence of alpha-non-natural amino acid, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2.5% etc.
Source and host living beings
Orthogonal translation assembly of the present invention is used for eukaryotic or translation system generally from non-eucaryote. For example, quadrature O-tRNA can be from non-eucaryote, for example, eubacteria, such as Escherichia coli, thermus thermophilus, bacillus stearothermophilus etc., or archeobacteria, such as Methanococcus jannaschii, hot autotrophic methane bacteria, Halobacterium such as the rich salt bacterium of Wo Shi and salt bacillus specie NRC-1, the ancient green-ball bacterium of flicker, fierce fireball bacterium, hole Yue Shi fireball bacterium, the hot bacterium of quick gas etc., quadrature O-RS can be from non-eucaryote, for example, eubacteria, such as Escherichia coli, thermus thermophilus, bacillus stearothermophilus etc., or archeobacteria, such as Methanococcus jannaschii, hot autotrophic methane bacteria, Halobacterium such as the rich salt bacterium of Wo Shi and salt bacillus specie NRC-1, the ancient green-ball bacterium of flicker, fierce fireball bacterium, hole Yue Shi fireball bacterium, the hot bacterium of quick gas etc. In addition, also can use the eucaryon source, for example, plant, algae, protist, fungi, yeast, animal are (for example, mammal, insect, arthropod etc.) etc., for example, wherein assembly and interested cell or translation system quadrature, or their are modified (for example, sudden change) be and cell or translation system quadrature.
The independent assembly that O-tRNA/O-RS is right can be from identical biology or different biological. In one embodiment, O-tRNA/O-RS is to from identical biology. For example, O-tRNA/O-RS is to can be from colibacillary tyrosyl-tRNA synthetase/tRNACUARight. In addition, the O-tRNA that O-tRNA/O-RS is right and O-RS are randomly from different biological.
Can in eukaryotic, select or screen and/or use quadrature O-tRNA, O-RS or O-tRNA/O-RS pair, to produce polypeptide with alpha-non-natural amino acid. Eukaryotic can be from any one of various sources, for example, plant (for example, higher plant, such as monocotyledon or dicotyledon), algae, protist, fungi, yeast (for example, saccharomyces cerevisiae), animal (for example, mammal, insect, arthropod etc.) etc. Eukaryotic composition with translation component of the present invention also is feature of the present invention.
The present invention also is provided at Effective selection in the kind, randomly to be used for this kind and/or the second kind (randomly, without additional selection the/screening). For example, in a kind, as selecting in the easy-to-operate kind (such as yeast cells etc.) or the assembly of screening O-tRNA/O-RS, and introduce the second eucaryote, for example, plant is (for example, higher plant, such as monocotyledon or dicotyledon), algae, protist, fungi, yeast, animal (for example, mammal, insect, arthropod etc.) etc., be used for and will mix the second kind in the alpha-non-natural amino acid body.
For example, saccharomyces cerevisiae (S.cerevisiae) can be elected to be the first eucaryote, because it is single celled, have fast generation time, and identified genetics characteristics relatively well. Referring to, for example, D.Burke, etc., (2000) " yeast genetics method " (Methods in Yeast Genetics), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. And, because Eukaryotic machine translator be high conservative (referring to, for example, (1996) " translation control " (Translational Control), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Y.Kwok and J.T.Wong, (1980), determine evolutionary relationship (Evolutionary relationship between Halobacterium cutirubrum and eukaryotes determined by use of aminoacyl-tRNA synthetases as phylogenetic probes) between red skin salt bacillus and the eucaryote, CanadianJournal of Biochemistry 58:213-218 with aminoacyl-tRNA synthetase as the systematic growth probe; (2001) " ribosomes " (The Ribosome), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), can introduce higher eucaryote for the aaRS gene that mixes alpha-non-natural amino acid with being found in saccharomyces cerevisiae, use with the cognate tRNA cooperation (referring to, for example, K.Sakamoto, Deng, (2002) the alpha-non-natural amino acid locus specificity is mixed in the protein of mammalian cell (Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells) Nucleic Acids Res. 30:4692-4699; And C.Kohrer, Deng, (2001), amber and ochre inhibition type tRNAs are inputted mammalian cell: with the universal method (Import of amber and ochre suppressor tRNAs into ma-malian cells:a general approach to site-specific insertion of amino acid analogues into proteins) in the amino acid analogue locus specificity ground insertion protein, Proc.Natl. Acad.Sci.U.S.A.98:1431)-14315) to mix alpha-non-natural amino acid.
In one embodiment, method of producing O-tRNA/O-RS in the first kind as herein described also comprises to be introduced the nucleic acid of the nucleic acid of coding O-tRNA and coding O-RS in the second kind (for example, mammal, insect, fungi, algae, plant etc.) eukaryotic. In another embodiment; by in eukaryotic preferably the quadrature tRNA of aminoacylation with alpha-non-natural amino acid method of producing quadrature aminoacyl-tRNA synthetase (O-RS) comprising: (a) in the presence of alpha-non-natural amino acid, the eukaryotic colony of the first kind (for example, yeast etc.) is just selected. Each eukaryotic comprises: the i) a member in aminoacyl-tRNA synthetase (RS) library, ii) quadrature tRNA (O-tRNA), iii) coding positive selected marker polynucleotides, and iv) coding negative selectable marker polynucleotides. The cell of survival is included in the active RS of aminoacylation quadrature tRNA (O-tRNA) in the presence of the alpha-non-natural amino acid under just selecting. The cell that to survive under just selecting is born selection in the situation that does not have alpha-non-natural amino acid, have the active RS of the O-tRNA of natural amino acid to remove aminoacylation. This provides aminoacylation preferably to have the O-RS of the O-tRNA of alpha-non-natural amino acid. The nucleic acid of coding O-tRNA and the nucleic acid (or assembly of O-tRNA and/or O-RS) of coding O-RS are introduced the second kind, for example, mammal, insect, fungi, algae, plant and/or analog) eukaryotic. Usually, select to obtain O-tRNA by the eukaryotic colony of the first kind is born, wherein eukaryotic comprises a member in tRNA library. Negative selection has been removed by the cell to a member in the tRNA library of eukaryotic endogenous aminoacyl-tRNA synthetase (RS) aminoacylation, and this provides the tRNA storehouse with the first kind and the second kind eukaryotic quadrature.
Select codon
Selection codon of the present invention has been expanded the genetic codon framework of protein biosynthesis machine. For example, select codon to comprise, for example, three unique base codons, nonsense codon such as terminator codon, for example, amber codon (UAG), opal codon (UGA), the non-natural codon, at least one four base codon, rare codon etc. Many selection passwords can be introduced required gene, for example, one or more, two or more, unnecessary three etc. In case gene can comprise a plurality of copies of given selection codon, just can comprise a plurality of different selection codons, or their any combination.
In one embodiment, method is included in the eukaryotic with mixing alpha-non-natural amino acid in the termination codon daughter of selecting in the codon. For example, produced the identification terminator codon, such as the O-tRNA of UAG, and by O-RS and required alpha-non-natural amino acid with the O-tRNA aminoacylation. This O-tRNA of the host's of natural generation aminoacyl-tRNA synthetase and nonrecognition. The site-directed mutagenesis of available routine is introduced terminator codon in the interested site of polypeptide of interest, for example, and TAG. Referring to, for example, Sayers, J.R., Deng (1988), based on 5 ', 3 ' exonuclease (5 ' in the oligonucleotides-directed mutagenesis of D2EHDTPA, 3 ' Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis), Nucleic Acids Res.791-802. When the nucleic acid of O-RS, O-tRNA and coding polypeptide of interest in vivo in conjunction with the time, mix alpha-non-natural amino acid in response to the UAG codon, be created in the polypeptide that assigned address contains alpha-non-natural amino acid.
Mix in the body of alpha-non-natural amino acid and finish and significantly do not upset eukaryotic host cell. For example, because the suppression efficiency of UAG codon depends on O-tRNA, such as amber inhibition type tRNA and eucaryon releasing factor (for example, eRF) competition between (it is attached to terminator codon and the initial peptide of growing discharges from ribosomes), so can pass through, the expression that for example increases O-tRNA such as inhibition type tRNA is regulated suppression efficiency.
Select codon also to comprise expansion cipher, for example, the codon of four or a plurality of bases is such as four, five, six or polybase base codon more. The example of four base codons comprises, for example, and AGGA, CUAG, UAGA, CCCU etc. The example of five base codons comprises, for example, and AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC etc. Feature of the present invention comprises that expansion cipher is used in inhibition according to frameshit. Four or a plurality of base codon can, for example, one or more alpha-non-natural amino acids insert in the same protein. For example, have anticodon loop, such as the sudden change O-tRNA of 8-10 nucleotides anticodon loop at least, under the existence such as special frameshit inhibition type tRNA, four or a plurality of base codon are read and are single amino acids. In other embodiments, anticodon loop can be decoded, for example, and at least four base codons, at least five base codons or hexabasic at least basic codon or more. Because 256 kinds of four possible base codons are arranged, so can be with four or a plurality of base codon a plurality of alpha-non-natural amino acids of encoding in same cell. Referring to, Anderson etc., (2002) explore the limit (Exploring the Limits of Codon and Anticodon Size) of codon and anticodon size, Chemistry and Biology, 9:237-244; Magliery, (2001) expansion genetic code: select the establishment agent of four base codons and identify " unsettled " four base codons (Expanding the Genetic Code:Selectiono of Efficient Suppressors of Four-base Codons and Identification of " Shifty " Four-base Codons with a Library Approach in Escherichia coli), J.Mol.Biol.307:755-769 with the library method in the Escherichia coli.
For example, be used for alpha-non-natural amino acid is mixed protein with external biological synthetic method four base codons. Referring to, for example, Ma etc., (1993) Biochemistry, 32:7939; With Hohsaka etc., (1999) J.Am. Chem.Soc., 121:34. The frameshit inhibition type tRNA that CGGG and AGGU are used for by two kinds of chemical acylation mixes streptavidin simultaneously with the NBD derivatives of 2-naphthyl alanine and lysine; referring to, for example, Hohsaka etc.; (1999) J.Am.Chem.Soc., 121:12194. In vivo in the research, Moore etc. have detected the ability of tRNALeu derivative and NCUA anticodon inhibition UAGN codon (N can be U, A, G or C), discovery is by tRNA Leu and the UCUA anticodon tetrad UAGA that can decode, efficient is 13 to 26%, and decoding is few in 0 or-1 frame. Referring to, Moore etc., (2000) J.Mol.Biol., 298:195. In one embodiment, the present invention can use expansion cipher based on rare codon or nonsense codon, and they can be reduced in other does not need missense liaison and frameshit on the site to suppress.
Give fixed system for one, select codon can comprise one of natural three base codons yet, wherein the endogenous system does not use (or seldom using) natural base codon. For example, this comprises that the system and/or the three base codons that lack the tRNA that identifies natural three base codons are systems of rare codon.
Select codon randomly to comprise the non-natural base-pair. These non-natural base-pairs have also been expanded existing genetic alphabet table. An extra base-pair can make the number of triplet codon be increased to 125 from 64. The character of the 3rd base-pair comprises that stable and selective base pairing, polymerase mix DNA with the effective enzymatic of high fidelity, and effectively continuous primer extended after newborn non-natural base-pair was synthetic. The description that goes for the non-natural base-pair of method and composition comprises, for example, and Hirao, etc., (2002) are used for amino acid analogue is mixed the non-natural base-pair of protein, Nature Biotechnology, 20:177-182. Other relevant publication is seen following listed.
For using in the body, non-natural nucleoside is that film is permeable, and phosphorylation forms corresponding triphosphate. In addition, the hereditary information of increase is stable, and can not destroyed by cellular enzymes. In the past Benner and effort that other people do utilized Watson-Crick from the model to different hydrogen bond patterns, and wherein the most noticeable example is different-C: different-G pair. Referring to, for example, Switzer etc., (1989) J.Am.Chem.Soc., 111:8322; With Piccirilli etc., (1990) Nature, 343:33; Kool, (2000) Curr.Opin.Chem.Biol., 4:602. Usually, these bases and natural base have mispairing to a certain degree, can not copy by enzymatic. Kool and colleagues prove that the hydrophobic accumulative facies mutual effect between base can replace hydrogen bond, form to order about base-pair. Referring to, Kool, (2000) Curr.Opin.Chem.Biol., 4:602; With Guckian and Kool, (1998) Angew.Chem. Int.Ed.Engl., 36,2825. On exploitation is satisfied in the effort of the non-natural base-pair of all requirements, Schultz, Romesberg and colleagues systematically synthesize and have studied the hydrophobic base of a series of non-naturals. Find that PICS:PICS self-natural base-pair of contrast is more stable, the crin promise fragment (KF) of e. coli dna polymerase I can effectively be mixed DNA with it. Referring to, for example, McMinn etc., (1999) J.Am.Chem.Soc., 121:11586; With Ogawa etc., (2000) J.Am.Chem.Soc., 122:3274. KF can be with the efficient that is sufficient to biological function and selective synthetic 3MN:3MN self-right. Referring to, for example, Ogawa etc., (2000) J.Am. Chem.Soc., 122:8803. Yet two kinds of bases all are used for further copying as chain terminating agent. It is right that recent findings, mutation DNA polymerase can be used for copying PICS self. In addition, it is right to copy 7AI self. Referring to, for example, Tae etc., (2001) J.Am.Chem.Soc., 123:7439. Also developed new metal base-pair Dipic:Py, in conjunction with Cu (II) time, form stablize right. Referring to, Meggers etc., (2000) J.Am.Chem. Soc., 122:10714. Because expansion cipher and non-natural codon in essence with natural codon quadrature, be their generation quadrature tRNA so the inventive method can be utilized this character.
The translation bypath system also is used in the required polypeptide mixes alpha-non-natural amino acid. In the translation bypath system, large sequence is inserted in the gene, but do not translated into albumen. This sequence comprises as inducing ribosomes to skip this sequence and the structure of the prompting of the downstream proceeding to insert translation.
Alpha-non-natural amino acid
Alpha-non-natural amino acid used herein refers to any amino acid, modified amino acid, or is not the amino acid analogue of selenocystein and/or pyrroles's lysine, and the below is the a-amino acid of 20 kinds of genetic codings: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. The general structure of formula I explanation a-amino acid:
Alpha-non-natural amino acid generally is any structure with formula I, and wherein the R group is any substituting group use in 20 kinds of natural amino acids a kind of. Referring to for example, " biochemistry " of L.Stryer, the third edition, 1988, Freeman and Company, the structure of 20 kinds of natural amino acids among the New York. It should be noted that alpha-non-natural amino acid of the present invention can be the compound of the natural generation except above-mentioned 20 kinds of a-amino acids.
Because alpha-non-natural amino acid of the present invention is general different from the natural amino acid in the side chain, alpha-non-natural amino acid and other amino acid, for example, the same way as formation amido link that forms in the natural or protein of alpha-non-natural amino acid with natural generation. Yet alpha-non-natural amino acid has makes its side-chain radical different from natural amino acid. For example; R among the formula I randomly comprise alkyl-, aryl-, acyl group-, ketone group-, azido-, hydroxyl-, hydrazine, cyano group-, halogen-, hydrazides, alkenyl, alkynyl, ether, mercaptan, selenium-, sulfonyl-, boric acid, borate, phosphoryl, phosphono, phosphine, heterocycle, ketenes, imines, aldehyde, ester, thio-acid, azanol, amine etc., or their any combination. Other interested alpha-non-natural amino acid includes but not limited to, but but but the amino acid, Fluorescent amino acid, metal that contain amino acid, spin labeling that can photoactivated crosslinking agent in conjunction with amino acid, metallic amino acid, radioactivity amino acid, have new functional group amino acid, with the covalently or non-covalently interactional amino acid of other molecules, the light cage covers and/or the amino acid of light isomery, contain biotin or biotin-analog amino acid, contain keto amino acid, contain the amino acid of the amino acid of polyethylene glycol or polyethers, amino acid chemical cleavage that heavy atom replaces or light cutting, compare with natural amino acid have prolong side chain amino acid (for example, polyethers or long chain hydrocarbon, as greater than about 5, greater than about 10 carbon etc.), contain carbon-connection sugar amino acid, have redox active amino acid, contain the amino acid of amino thio-acid and contain the amino acid of one or more toxic moieties. In some embodiments, alpha-non-natural amino acid has can photoactivated crosslinking agent, and it is used for, and for example, protein is connected on the solid support. In one embodiment, alpha-non-natural amino acid has sugar moieties (for example, glycosylation amino acid) and/or other carbohydrate modification that is attached to amino acid side chain.
Except the alpha-non-natural amino acid that contains new side chain, alpha-non-natural amino acid also randomly comprises the skeleton structure of modification, for example, and shown in the structure of formula II and III:
Wherein Z generally comprises OH, NH2, SH, NH-R ' or S-R '; X and Y can be identical or different, and they generally comprise S or O, and R and R ' are randomly identical or different, and they generally are selected from same list and the hydrogen of above-mentioned alpha-non-natural amino acid R group composition for having formula I. For example, alpha-non-natural amino acid of the present invention randomly is included in the replacement in amino or the carboxyl, shown in II and III. Such alpha-non-natural amino acid for example includes but not limited to, has alpha-hydroxy acid, α-thio-acid alpha-amido carbothioic acid ester with common 20 kinds of corresponding side chains of right amino acid or non-natural side chain. In addition, the replacement on α-carbon randomly comprises L, D or α-α-two substituted amino acids, such as D-Glu, D-alanine, D-methyl-O-tyrosine, aminobutyric acid etc. Other structure substitute comprises cyclic amino acids, such as proline analogs, and 3,4,6,7,8 and 9 yuan of loop proline analogs, β and γ amino acid are such as Beta-alanine and the GABA that replaces.
For example, a lot of alpha-non-natural amino acids are based on natural amino acid, such as tyrosine, glutamine, phenylalanine etc. Tyrosine Analogues comprises tyrosine, the tyrosine of ortho position replacement and the tyrosine that a position replaces that contraposition replaces; the tyrosine that wherein replaces comprises; for example, ketone group (for example acetyl group), benzoyl, amino, hydrazine, azanol, mercapto, carboxyl, isopropyl, methyl, C6-C 20Straight or branched hydrocarbon, saturated or unsaturated hydrocarbons, 0-methyl, polyethers, nitro, alkynyl etc. In addition, also consider polysubstituted aromatic ring. Glutamine analog of the present invention includes but not limited to, the Glutamine Derivatives that Alpha-hydroxy derivative, γ-substitutive derivative, cyclic derivatives and acid amides replace. The example of phenylalanine analogues includes but not limited to; the phenylalanine that the phenylalanine that the phenylalanine that contraposition replaces, ortho position replace and a position replace; wherein substituting group comprises; for example, hydroxyl, methoxyl group, methyl, pi-allyl, aldehyde, azido, iodine, bromine, ketone group (such as acetyl group), benzoyl, alkynyl etc. The object lesson of alpha-non-natural amino acid includes but not limited to, p-acetyl group-L-Phe, p-propargyloxy phenylalanine, 0-methyl-TYR, L-3-(2-naphthyl) alanine, 3-methylphenylalanine, 0-4-pi-allyl-TYR, 4-propyl group-TYR, three-O-acetyl group-GlcNAc β-serine, L-3,4 dihydroxyphenylalanine, fluoridize phenylalanine, isopropyl-L-Phe, p-azido-L-Phe, p-acyl group-L-Phe, p-benzoyl-L-Phe, L-phosphoserine, phosphono serine, phosphono tyrosine, p-iodo-phenylalanine, p-bromophenyl alanine, p-amino-L-Phe and isopropyl-L-Phe etc. The example of alpha-non-natural amino acid structure is seen Fig. 7, B group and Figure 11. For example, WO2002/085923 is entitled as the various alpha-non-natural amino acids that other structure is provided among Figure 16,17,18,19,26 and 29 of " mixing alpha-non-natural amino acid in the body ". Also can be from Kiick etc., (2002) connect azide mixed by Staudinger and are used for chemo-selective in the recombinant protein and modify, among Fig. 1 structure 2-5 of PNAS 99:19-24 referring to other methionine analogs.
In one embodiment, provide the composition that comprises alpha-non-natural amino acid (such as p-(propargyloxy)-phenylalanine). Also provide various contain p-(propargyloxy)-phenylalanine and, such as the composition of albumen and/or cell. In one aspect, the composition that comprises p-(propargyloxy)-phenylalanine alpha-non-natural amino acid also comprises quadrature tRNA. Alpha-non-natural amino acid can (such as covalency) be attached on the quadrature tRNA, for example, is covalently bound on the quadrature tRNA by amino-acyl bond, and 3 ' OH or the 2 ' OH of terminal ribose that is covalently bound to quadrature tRNA is first-class.
The chemical part of the alpha-non-natural amino acid by can mixing albumen provides the operation of various advantages and albumen. For example, unique reactive permission of ketone carried out the albumen selective modification in vitro and in vivo with many reagent that contain hydrazine or azanol. The heavy atom alpha-non-natural amino acid for example, can be used for orientation x-ray structure data. Introduce heavy atom with the alpha-non-natural amino acid locus specificity and also selecting to provide selective and flexibility aspect the heavy atom position. Photosensitive alpha-non-natural amino acid (for example, having the amino acid of benzophenone and aromatic yl azide (for example, the aziminobenzene compound) side chain) for example, allows albumen to carry out effective photo-crosslinking with external in vivo. The example of photosensitive alpha-non-natural amino acid includes but not limited to, for example, and p-azido-phenylalanine and p-benzoyl-phenylalanine. Then, can be by photosensitive group-provide temporary transient (and/or space) contrast, arbitrarily crosslinked protein with photosensitive alpha-non-natural amino acid be provided. In one embodiment, can be with isotope-labeled, the methyl of methyl substituted non-natural amino for example, methyl is as partial structurtes and dynamic (dynamical) probe in for example using nuclear magnetic resonance and vibrational spectroscopy. Alkynyl or azido functional group for example, allow by [3+2] cycloaddition reaction molecular selectivity modifying protein.
The chemical synthesis of alpha-non-natural amino acid
Many alpha-non-natural amino acids that provide above can from, for example, Sigma (USA) or Aldrich (Milwaukee, WI, USA) buy. The method that provides in the method that provides such as this paper or the various publication or with standard method well known by persons skilled in the art synthetic those alpha-non-natural amino acids that can not buy from the market randomly. Organic synthesis technology referring to, for example, " organic chemistry " of Fessendon and Fessendon, (1982, second edition, Willard Grant Press, Boston Mass.); " the senior organic chemistry " of March (third edition, 1985, Wiley and Sons, New York); " senior organic chemistry " (third edition, A and B part, 1990, Plenum Press, New York) with Carey and Sundberg. Describe other synthetic publication of alpha-non-natural amino acid and comprise that for example, WO2002/085923 is entitled as " mixing alpha-non-natural amino acid in the body " (In vivo incorporation of Unnatural Amino Acids); Matsoukas etc., (1995) J.Med.Chem., 38,4660-4669; King, F.E. and Kidd, D.A.A. (1949) newly synthesize glutamine and glutamic acid gamma-dipeptides (A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates) from the intermediate of phthalic acid, J.Chem.Soc., 3315-3319; Friedman, O.M. and Chatterrji, R. (1959) synthetic glutamine derivative is as the pattern substrate (Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents) of antitumor agent, J.Am.Chem.Soc.81,3750-3752; Craig, (1988) 7-chloro-4[[4-(the lignocaine)-1-methyl butyl such as J.C.] amino] absolute configuration (Absolute Configuration of the Enantiomers of 7-Chloro-4 [[4-(diethylamino)-1-methylbutyl] amino] quinoline (Chloroquine)) of enantiomer of quinoline (chloroquine), J.Org. Chem.53,1167-1170; Azoulay, M., Vilmont, M. and Frappier, F. (1991) is as the glutamine analog (Glutamin analogues as Potential Antimalarials) of potential antimalarial, Eur. J.Med.Chem.26,201-5; Koskinen, A.M.P. and Rapoport, H. (1989) synthesize the limited amino acid analogue 4-substituted prolines (Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues) of conformation, J.Org.Chem.54,1859-1866; Christie, B.D. and Rapoport, H. (1985) is from the pure 2-nipecotic acid of altheine synthesizing optical (Synthesis of Optically Pure Pipecolates from L-Asparagine). Be applied to entirely synthesize (+)-Apovincamine (Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization), J.Org.Chem.1989:1859-1866 by amino acid decarbonylation and imines ion cyclisation; Barton etc., (1987) with the synthetic new a-amino acid of free-radical chemistry and derivative: synthetic L-and D-alpha-amido-adipic acid, L-alpha-amido pimelic acid and suitable unsaturation derivative (Synthesis of Novel α-Amino-Acids and Derivatives Using Radical Chemistry:Synthesis of L-and D-α-Amino-Adipic Acids, L-α-aminopimelic Acid and Appropriate Unsaturated Derivatives), Tetrahedron Lett.43:4297-4308; With, Subasinghe etc., synthetic and the activity (Quisqualic acid analogues:synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site) on new quisqualic acid-sensitization site of (1992) quisqualic acid analog: β-heterocycle 2-alanine derivatives, J.Med.Chem.35:4602-7. Also the patent application referring to the attorney docket numbering P1001US00 that submitted on December 22nd, 2002 is entitled as " protein array " (Protein Arrays).
The method of synthetic p-(propargyloxy) phenylalanine compound is provided in one aspect of the invention. Method comprises, for example, and (a) with uncle N--butoxy carbonyl-tyrosine and K2CO 3Be suspended in the dry DMF; (b) propargyl bromide is added in the reactant mixture of (a), alkanisation hydroxyl and carboxyl produce the midbody compound of protection, and this compound has structure:
Figure A20048002115500501
(c) midbody compound with protection mixes in MeOH with anhydrous HCl, makes amine moiety go protection, thus synthetic p-(propargyloxy) phenylalanine compound. In one embodiment, the method comprises that also (d) dissolves p-(propargyloxy) phenylalanine HCl, stirring at room in the aqueous solution of NaOH and MeOH; (e) pH is adjusted to pH7; (f) precipitation p-(propargyloxy) phenylalanine compound. Referring to for example, the propargyloxy phenylalanine is synthetic among this paper embodiment 4.
The cellular uptake of alpha-non-natural amino acid
When design with when selecting alpha-non-natural amino acid, a problem generally can considering be eukaryotic to the picked-up of alpha-non-natural amino acid, for example, mix albumen. For example, to point out these compounds unlikely be that cell is permeable to the high charge density of a-amino acid. By the transportation system of collecting based on albumen natural amino acid is taken in eukaryotic. If any, can finish the alpha-non-natural amino acid that rapid screening is estimated cellular uptake. Referring to, for example, the application of the attorney docket numbering P1001US00 that submitted on December 22nd, 2002 for example is entitled as in " protein array " toxicity test; And Liu, D.R. and Schultz, P.G. (1999) have the progress of the biological evolution of expansion genetic code, PNAS United States 96:4780-4785. Although can easily analyze picked-up with various mensuration, the alternative route that design is fit to the alpha-non-natural amino acid of cellular uptake approach provides the amino acid whose biosynthesis pathway of generation in the body.
The biosynthesizing of alpha-non-natural amino acid
There have been a lot of biosynthesis pathways in the cell, have been used to produce amino acid and other compound.Yet at occurring in nature, for example in eukaryotic, may there be the biological synthesis method that is used for concrete alpha-non-natural amino acid, the invention provides this method.For example, in host cell by adding new enzyme or modifying the biosynthesis pathway that existing host cell approach randomly produces alpha-non-natural amino acid.Enzyme that additional new enzyme is randomly natural generation or the artificial enzyme that produces.For example, the combination of adding from other biological known enzyme depended in the biosynthesizing of right-aminobenzene alanine (being entitled as described in the embodiment in " mixing alpha-non-natural amino acid in the body " as WO2002/085923).Can be by the gene of these enzymes being introduced in the eukaryotic with the plasmid transformant that contains gene.When these genes were expressed in cell, they provided the enzymatic pathway of synthetic required compound.The example of the enzyme type of optional adding is provided in the following examples.The sequence of additional enzymes exists, and for example, finds among the Genbank.Also the enzyme that will manually produce randomly adds cell in the same manner.In this mode, the resource of manipulated cell machine and cell is to produce alpha-non-natural amino acid.
Be used for biosynthesis pathway or be used to develop the new enzyme of existing approach for production, can make in all sorts of ways.For example, with Maxygen, Inc exploitation as, circulation reorganization (can obtain from the www.maxygen.com of WWW) randomly is used to develop new enzyme and approach.Referring to, for example, Stemmer (1994), " reorganizing at external rapid evolution albumen " (Rapid evolution of a protein in vitro by DNA shuffling) by DNA, Nature 370 (4): 389-391; With, Stemmer, (1994), by random fragmentation and again assembling carry out DNA reorganization: the vitro recombination (DNA shuffling by random fragmentation andreassembly:In vitro recombination for molecular evolution) that is used for molecular evolution, Proc.Natl.Acad.Sci.USA., 91:10747-10751.Similarly, (can obtain) DesignPath that Genencor is developed from the genencor.com of WWW TMRandomly be used for metabolic pathway engineering, for example, design produces the approach of 0-methyl-L-tyrosine in cell.This technology for example identifies by functional genomics that with the combination of new gene the existing approach in host living beings has been rebuild in molecular evolution and design.Diversa Corporation (can obtain from WWW diversa.com) also provides the technology of rapid screening gene library and gene approach, for example, sets up new way.
Usually, producing alpha-non-natural amino acid with the biosynthesis pathway of the present invention's design produces in the enough biosynthetic concentration of effective protein proteins, for example, the amount of n cell, but be unlikely to reach the degree that influences other amino acid concentration or exhaust the cell resource.The typical concentration of producing in the body is that about 10mM is to about 0.05mM in this way.In case,, be used for the synthetic and cell growth of ribosomal protein just randomly with the production of selecting further to optimize alpha-non-natural amino acid in the body with containing the plasmid transformant that is useful on the gene of producing the required enzyme of concrete approach and producing alpha-non-natural amino acid.
Polypeptide with alpha-non-natural amino acid
Protein of interest or polypeptide with at least one alpha-non-natural amino acid are features of the present invention.The present invention also comprises having at least one polypeptide or albumen with the alpha-non-natural amino acid of the present composition and method production.Excipient (for example, pharmaceutically acceptable excipient) also can exist with this albumen.
By producing protein of interest or polypeptide with at least a alpha-non-natural amino acid in eukaryotic, albumen or polypeptide are modified after generally comprising eukaryotic translation.In some embodiments, albumen comprises at least one alpha-non-natural amino acid and at least one by the posttranslational modification that produces in the eukaryotic body, and wherein this posttranslational modification is not produced by prokaryotic.For example, this posttranslational modification comprises, for example, and acetylation, acidylate, lipid-modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid-connection modification, glycosylation etc.In one aspect, this posttranslational modification comprises with oligosaccharides (for example, (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc)) be attached on the asparagine by the connection of GlcNAc-asparagine.Also referring to, table 7, this tabular have gone out some examples of the oligosaccharides that the N-of eukaryotic protein (also can have additional residue, show) connects.On the other hand, posttranslational modification comprises oligosaccharides (for example, Gal-GaINAc, Gal-GlcNAc etc.) connected by GalNAc-serine or GaINAc-threonine, or GlcNAc-serine or GlcNAc-threonine connect and be attached on serine or the threonine.
Table 7: the example of the oligosaccharides that connects by GlcNAc-
Figure A20048002115500521
Aspect another, the protease hydrolytic processing that posttranslational modification comprises precursor (for example, peptide precursor, Pre Pro PTH, preproinsulin, proinsulin, the preceding opium casting skin element of calcitonin precursor, calcitonin gene-relevant is former, the opium casting skin is plain former etc.), be assembled into oligomeric protein or big molecule assembling, transfer to another site in the cell (organelle for example, as endoplasmic reticulum, golgiosome, nucleus, lysosome, peroxisome, mitochondria, chloroplast, vacuole etc., or pass through secretory pathway).In some embodiments, this albumen comprises secretion or positioning sequence, epi-position mark, FLAG mark, polyhistidine tag, GST and merges etc.
An advantage of alpha-non-natural amino acid is that it provides the additional chemical part, can be used for adding additional molecule.These modifications can generate in the body in eukaryotic, or external generation.Therefore, in some embodiments, posttranslational modification is by alpha-non-natural amino acid.For example, posttranslational modification can be passed through nucleophilic-electrophilic reaction.The reaction that major part is used for selective modification albumen now relates to covalent bond formation between nucleophilic and the electrophilic reaction gametophyte, for example has the reaction of histidine or cysteine side chain α-Lu Daitong.Selectivity in these situations is by the quantity and the accessibility decision of nucleophilic residues in the albumen.In protein of the present invention, can have more optionally reaction with other, as have the non-natural keto-acid-amino acid or the reaction in vitro and in vivo of aminooxy group compound of hydrazides.Referring to, for example, Cornish, etc., (1996) Am.Chem.Soc., 118:8150-8151; Mahal, etc., (1997) Science, 276:1125-1128; Wang, etc., (2001) Science 292:498-500; Chin, etc., (2002) Am.Chem.Soc.124:9026-9027; Chin, etc., (2002) Proc.Natl.Acad.Sci., 99:11020-11024; Wang, etc., (2003) Proc.Natl.Acad.Sci., 100:56-61; Zhang, etc., (2003) Biochemistry, 42:6735-6746; And Chin, etc., (2003) Science is in the printing.Many reagent are used in this permission, comprise that fluorophore, crosslinking chemical, sugar derivatives and cytotoxicity molecule carry out selected marker to any albumen basically.Also referring to the patented claim USSN10/686 that is entitled as " glycoprotein is synthetic " (Glycoprotein synthesis) that submits in, on October 15th, 2003,944.For example, the posttranslational modification of being undertaken by azido amino acid also can connect (for example, using triaryl phosphine reagent) by Staudinger and carries out.Referring to, for example, Kiick etc., (2002) triazo-compound is mixed be used in the recombinant protein connecting and carry out chemo-selective and modify (Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligtation), PNAS 99:19-24 by Staudinger.
The invention provides another high efficiency method of selective modification albumen, it comprises in response to selecting codon, with alpha-non-natural amino acid, for example, contain azide or alkynyl part alpha-non-natural amino acid (referring to, for example, Figure 11 2 and 1) heredity mixes in the protein.Can pass through then, for example, Huisgen[3+2] cycloaddition reaction (referring to, for example, Padwa, A. " comprehensive organic synthesis " (Comprehensive Organic Synthesis), the 4th volume, (1991) Trost, B.M. compile Pergamon, Oxford, 1069-1109 page or leaf; And Huisgen, R. " the bipolar also addition chemistry of 1.3-" (1,3-Dipolar Cycloaddition Chemistry), (1984) Padwa, A. compiles, Wiley, New York, the 1-176 page or leaf) for example to use respectively, alkynyl or azide derivatives are modified these amino acid side chains.Referring to, for example, Figure 16.Because this method comprises cycloaddition rather than nucleophilic displacement of fluorine, so can come modifying protein with high selectivity.This reaction can be at room temperature, join in the reaction mixture by Cu (I) salt with catalytic amount with fabulous regioselectivity (1,4＞1,5) in the aqueous conditions and carry out.Referring to, for example, Tornoe, etc., (2002) Org.Chem.67:3057-3064; And Rostovtsev, etc., (2002) Angew.Chem.Int.Ed.Eng.41:2596-2599.Operable other method is the ligand exchange that has on two arsenic compounds of four halfcystine motifs, referring to, for example, Griffin, etc., (1998) Science 281:269-272.
Can comprise in fact any molecule by the molecule of [3+2] cycloaddition adding albumen of the present invention with azido or alkynyl derivatives.Referring to, for example, this paper embodiment 3 and 5.This molecule includes but not limited to, dyestuff, fluorophore, crosslinking chemical, sugar derivatives, polymkeric substance are (for example, the derivant of polyglycol), the derivant of photocrosslinking agent, cytotoxic compound, affinity labeling, biotin, resin, pearl, second albumen or polypeptide (or more), polynucleotide (for example, DNA, RNA etc.), metal-chelator, co-factor, fatty acid, carbohydrates etc.Referring to, for example, Figure 13 A of this paper and embodiment 3 and 5.These molecules can be joined the alpha-non-natural amino acid with alkynyl respectively, as right-propargyloxy phenylalanine, or have the alpha-non-natural amino acid of azido, in right-azido-phenylalanine.For example, referring to Figure 13 B and Figure 17 A.
On the other hand, the invention provides the composition that comprises this molecule and produce these molecules, for example, the method for azido dyestuff (as shown in chemical constitution 4 and chemical constitution 6), alkynyl polyglycol (for example, shown in the chemical constitution 7), wherein n for example is, 50 and 10,000,75 and 5,000,100 and 2,000, the integer between 100 and 1,000 grade.In embodiments of the present invention, the molecular weight of alkynyl polyglycol is, for example, about 5,000 to about 100, and 000Da, about 20,000 is to about 50, and 000Da, about 20,000 is to about 10, and 000Da (for example, 20,000Da) etc.
Figure A20048002115500541
Figure A20048002115500551
Also provide to comprise these compounds, for example, had the various compositions of albumen and cell.In one aspect of the invention, (for example contain the azido dyestuff, chemical constitution 4 or chemical constitution 6) albumen also comprise at least a alpha-non-natural amino acid (for example, alkynyl amino acids), wherein the azido dyestuff is attached on the alpha-non-natural amino acid by [3+2] cycloaddition.
In one embodiment, albumen comprises the alkynyl polyglycol of chemical constitution 7.In another embodiment, said composition also comprises at least a alpha-non-natural amino acid (for example, azido amino acid), wherein by [3+2] cycloaddition the alkynyl polyglycol is attached on the alpha-non-natural amino acid.
The method that is used for synthetic azido dyestuff also is provided.For example, a kind of this method comprises: the dye composition that contains the sulfuryl halide part (a) is provided; (b) in the presence of 3-azido propylamine and triethylamine, dye composition is heated to the chamber, with the halogenide position coupling of the amine moiety and the dye composition of 3-azido propylamine, thus synthetic azido dyestuff.In one embodiment, this dye composition comprises dansyl Cl, and this azido dyestuff comprises the composition of chemical constitution 4.In one aspect, this method also comprises purifying azido dyestuff from reaction mixture.Referring to, for example, this paper embodiment 5.
In another embodiment, the method for synthetic azido dyestuff comprises that (a) provides the dye composition that contains amine; (b) dye composition that will contain amine in suitable solvent mixes with carbodiimide and 4-(3-azido propyl group carbamyl)-butyric acid, the carbonyl that this is sour and the amine moiety coupling of dye composition, thereby synthetic azido dyestuff.In one embodiment, carbodiimide comprises 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCI).In one aspect, the dyestuff that contains amine comprises fluorescein amine, and suitable solvent comprises pyridine.For example, the dyestuff that contains amine randomly comprises fluorescein amine, and the azido dyestuff randomly comprises the composition of chemical constitution 6.In one embodiment, this method also comprises (c) precipitation azido dyestuff; (d) use the HCl washing precipitation; (e) precipitation of dissolving washing in EtOAc; (f) precipitation azido dyestuff in hexane.Referring to, for example, this paper embodiment 5.
The method of synthetic propargyl acid amides polyglycol also is provided.For example, this method comprises propargyl amine and polyglycol (PEG)-hydroxysuccinimide eater at organic solvent (for example, CH 2Cl 2) in react under the room temperature, produce the propargyl acid amides polyglycol of chemical constitution 7.In one embodiment, this method also comprises with ethyl acetate precipitation propargyl acid amides polyglycol.In one aspect, this method also is included in the methyl alcohol crystallization propargyl acid amides polyglycol again; With desciccate under the vacuum.Referring to, for example, the embodiment 5 of this paper.
Eukaryotic of the present invention provides the synthetic ability that comprises the albumen of the alpha-non-natural amino acid that consumption is arranged greatly.In one aspect, said composition randomly comprises, for example, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams or the albumen that contains alpha-non-natural amino acid more, or with the obtainable amount of protein production method (this paper provides recombinant protein production and purifying in detail) in the body.On the other hand, this albumen is randomly to be present in the composition, promptly for example, cell lysate, damping fluid, medicine damping fluid or other suspending liquid are (for example, volume is, for example, receive from about 1 and to rise to about 100 liters) concentration for for example, whenever rise to few 10 microgram albumen, whenever rise to few 50 microgram albumen, whenever rise to few 75 microgram albumen, whenever rise to few 100 microgram albumen, whenever rise to few 200 microgram albumen, whenever rise to few 250 microgram albumen, whenever rise to few 500 microgram albumen, whenever rise to few 1 milligram of albumen or whenever rise to few 10 milligrams of albumen or more.The a large amount of productions (for example, than using other method, for example, but external translation generality energy is bigger) that comprise the albumen of at least a alpha-non-natural amino acid in eukaryotic are features of the present invention.
Can finish alpha-non-natural amino acid mix with, for example, revise the variation in protein structure and/or the function, for example, change the accessibility of size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, proteinase target site, part of target (for example, protein arrays) etc.That the albumen that comprises alpha-non-natural amino acid can have an increase or even new catalysis or physical property.For example, by comprising the character of alpha-non-natural amino acid below randomly having modified in the albumen: toxicity, bio distribution, structural property, spectral quality, chemistry and/or spectrochemical property, catalytic capability, half life period (for example serum half-life), with the ability of other molecular reaction, for example covalently or non-covalently wait.The composition that contains the albumen that comprises at least a alpha-non-natural amino acid can be used for, for example, and new acology, diagnostics, catalyzing enzyme, industrial enzyme, and for example, the research of protein structure and function in conjunction with albumen (for example, antibody).Referring to, for example, Dougherty, (2000) alpha-non-natural amino acid is as the probe (Unnatural Amino Acids as Probesof Protein Structure and Function) of protein structure and function, Current 0pinion in Chemical Biology, 4:645-652.
In one aspect of the invention, composition comprises at least aly having at least one, the albumen of for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or more alpha-non-natural amino acid.Alpha-non-natural amino acid can be identical or different, for example, in albumen, can have 1,2,3,4,5,6,7,8,9 10 or more different loci comprise 1,2,3,4,5,6,7,8,9 or 10 or how different alpha-non-natural amino acid.On the other hand, composition comprises exist in the albumen at least a, but is less than the albumen that whole concrete amino acid is replaced by alpha-non-natural amino acid.For the given albumen that has more than an alpha-non-natural amino acid, alpha-non-natural amino acid can be identical or different (for example, this albumen can comprise the alpha-non-natural amino acid that two or more are dissimilar, maybe can comprise two kinds of identical alpha-non-natural amino acids).For the given albumen that has more than two alpha-non-natural amino acids, alpha-non-natural amino acid can be a plurality of alpha-non-natural amino acids identical, different or of the same race and the combination of at least a different alpha-non-natural amino acid.
Can produce any basically alpha-non-natural amino acid albumen (or its part) of (with any respective coding nucleic acid, for example, this nucleic acid comprises one or more selection codons) that comprises with composition and the method for this paper.The not trial that the known protein of hundreds of thousands is identified, any one all can be modified to and comprise one or more alpha-non-natural amino acids in these albumen, for example, in relevant translation system, comprise the selection codon that one or more are suitable by revising any available mutation method.The common sequence library of known protein comprises GenBank EMBL, DDBJ and NCBI.Can easily discern other storehouse by the search the Internet.
Usually, albumen and any available albumen are (for example, treatment albumen, diagnosis albumen, industrial enzyme or their part etc.), for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or at least 99% or more how identical, they comprise one or more alpha-non-natural amino acids.Can modify the treatment that comprises one or more alpha-non-natural amino acids, the example of diagnosis and other albumen includes but not limited to, for example, α-1 antitrypsin, angiostatin, AHF, antibody (being described in further detail as follows of antibody), apolipoprotein, apoprotein, atrial natriuretic peptide, atrium natriuresis polypeptide, atrial natriuretic peptide, the C-X-C chemotactic factor (CF) (for example, T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), calcitonin, the CC chemotactic factor (CF) (for example, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, MCP-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-1 β, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262), the CD40 part, the C-kit part, collagen, colony stimulating factor (CSF), complement factor 5 α, complement inhibitor, complement receptor 1, cell factor, (for example, epithelium neutrophil cell activating peptide-78, GRO α/MGSA, GRO β, GRO γ, MIP-1 α, MIP-1 δ, MCP-1), epidermal growth factor (EGF), hematopoietin (" EPO ", representative is by mixing the preferred target that one or more alpha-non-natural amino acids are modified), exfoliative toxin A and B, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropic hormone, growth factor, Hedgehog albumen (for example, Sonic, Indian, Desert), haemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumins, insulin, insulin-like growth factor (IGF), interferon (for example, IFN-α, IFN-β, IFN-Y), interleukin (for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 etc.), keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, Neurturin, neutrophil cell inhibiting factor (NIF), oncostatin M, BMP, parathyroid hormone, PD-ECSF, PDGF, peptide hormone (for example, human growth hormone (HGH)), the multi-effect nutrient factor, albumin A, Protein G, the thermal source exotoxin A, B and C, relaxain, feritin, SCF, soluble complement acceptor I, solubility I-CAM1, soluble interleukin-6 receptor (IL-1,2,3,4,5,6,7,9,10,11,12,13,14,15), soluble TNF acceptor, somatomedin, somatostatin, somatotropin, streptokinase, super antigen is staphylococcal enterotoxin (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), superoxide dismutase (SOD), toxic shock syndrome toxin (TSST-1), thymosin, tissue plasminogen activator, tumor necrosis factor (TNF β), Tumor Necrosis Factor Receptors (TNFR), tumor necrosis factor-alpha (TNF α), vascular endothelial growth factor (VEGEF), urokinase and many other materials.
One class can be used for mixing in the body composition of alpha-non-natural amino acid and albumen that method is made comprises transcriptional regulatory agent or its part with described herein.The example of transcriptional regulatory agent comprises gene and the transcript regutation protein of regulating cell growth, differentiation, adjusting etc.Comprise prokaryotes, virus and eucaryote that fungi, plant, yeast, insect and animal comprise and found the transcriptional regulatory agent in the mammal, this provides a large amount of treatment targets.Should understand expression and transcriptional activator by a lot of mechanism, for example, by with the reaction of receptors bind, stimulus signal transductory cascade, regulate transcription factor expression, combine with promoter and enhancer, and be attached to the protein combination of promoter and enhancer, the DNA that untwists, montage premessenger RNA, gather adenosine RNA and degradation of rna and regulate and transcribe.For example, the composition of the GAL4 albumen in the eukaryotic or its part also is a feature of the present invention.Usually, GAL4 albumen or its part contain at least one alpha-non-natural amino acid.Also referring to the part that is entitled as " quadrature aminoacyl-tRNA synthetase " herein.
One class albumen of the present invention (for example, albumen with one or more alpha-non-natural amino acids) comprise the expression activator, as cell factor, inflammation molecule, growth factor, their acceptor and oncoprotein, for example, interleukin (for example, IL-1, IL-2, IL-8 etc.), interferon, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1 and hyaluronic acid glycosides/CD44; Signal transducers and corresponding oncoprotein, for example, Mos, Ras, Raf and Met; And transcriptional activator and mortifier, for example, p53, Tat, Fos, Myc, Jun, Myb, Rel and steroid hormone acceptor such as estrogen, progesterone, testosterone, aldosterone, ldl receptor part and cortisone acceptor.
The present invention also provides enzyme (for example, industrial enzyme) or its part with at least one alpha-non-natural amino acid.The example of enzyme includes but not limited to; for example, amidase, amino acid racemase, acylase, dehalogenase, dioxygenase, diaryl propane peroxidase, epimerase, EH, esterase, isomerase, kinases, glucose isomerase, glycosidase, glycosyl transferase, haloperoxidase, monooxygenase (as p450), lipase, lignin peroxidase, nitrile hydratase, nitrilase, proteinase, phosphatase, subtilopeptidase A, transaminase and nuclease.
A lot of these albumen be commercially available (referring to, for example, Sigma Bio Sciences 2002 catalogues and price list), corresponding proteins sequence and gene, a lot of variants that generally also have them be know (referring to, for example, Genbank).Can for example, change albumen according to the present invention by inserting one or more alpha-non-natural amino acids to arbitrary modification the in them according to one or more treatments, diagnosis or interested enzymatic property.The example of treatment relevant nature comprises serum half-life, stores half life period, stability, immunogenicity, therapeutic activity, detectability (for example, comprising reporter group (for example, mark or mark binding site) in alpha-non-natural amino acid), LD 50Reduction or other spinoff, enter ability (for example oral availability) of health etc. by alimentary canal.The example of diagnostic properties comprises storage half life period, stability, diagnosis activity, detectability etc.The example of relevant enzyme character comprises storage half life period, stability, enzymatic activity, productive capacity etc.
Also can modify various other albumen, to comprise one or more alpha-non-natural amino acid of the present invention.For example, the present invention for example can comprise, from infectious fungi, for example, aspergillus, Candida kind; Bacterium specifically is as the Escherichia coli of pathogen model and medically important bacterium such as staphylococcus (for example, golden yellow (staphylococcus)) or streptococcus (for example, pneumonia (streptococcus)); Protozoan such as sporozoa (for example plasmodium), rhizopodea (for example Endamoeba) and Flagellata (Trypanosomonas, Leishmania, Trichomonas, Giardia etc.); (example comprises poxvirus to virus, as vaccinia virus as (+) RNA viruses; Pico+ribonucleic acid+virus, for example poliovirus; Togavirus, for example rubella virus; Flavivirus, for example HCV; And coronavirus), (-) RNA viruses (for example, rhabdovirus, for example VSV; Paramyxovirus, for example RSV; Orthomyxovirus, for example influenza virus; Bunyavirus and arenavirus), dsDNA virus (for example arc reovirus virus), RNA is to dna virus, it is retrovirus, as HIV and HTLV, and some DNA in the albumen as hepatitis B, in one or more vaccine proteins replaces one or more natural amino acids with alpha-non-natural amino acid to RNA viruses.
The agricultural associated protein, as insect-resistant albumen (for example, Cry albumen), starch and lipid production enzyme, plant and insect toxins, toxin resistance protein, mycotoxin (are for example separated toxalbumin, plant growth enzyme, ribulose 1,5-diphosphonic acid carboxylase/oxygenase " RUBlSCO "), lipoxygenase (LOX) and phosphoenolpyruvate (PEP) carboxylase also are the suitable targets that alpha-non-natural amino acid is modified.
The present invention also is provided at and produces at least a method (with the albumen of this method production) that contains the albumen of at least one alpha-non-natural amino acid in the eukaryotic.For example, method comprises: cultivate the eukaryotic that contains nucleic acid in proper culture medium, this nucleic acid comprises at least one and selects the codon and this albumen of encoding.This eukaryotic also comprises: the also quadrature tRNA (O-tRNA) of identification selection codon works in cell; Preferably aminoacylation have alpha-non-natural amino acid O-tRNA quadrature aminoacyl tRNA synthetase (O-RS) and contain the nutrient culture media of alpha-non-natural amino acid.
In one embodiment, this method also comprises mixes alpha-non-natural amino acid in the albumen, and wherein alpha-non-natural amino acid comprises first reactive group; Then (for example with this albumen and the molecule that contains second reactive group, the derivant of the derivant of dyestuff, polymkeric substance such as polyglycol, photocrosslinking agent, cytotoxic compound, affinity labeling, biotin, resin, second albumen or polypeptide, metal-chelator, co-factor, fatty acid, carbohydrates, polynucleotide (for example, DNA, RNA etc.) etc.) contact.First reactive group and the reaction of second reactive group make this molecule be attached on the alpha-non-natural amino acid by [3+2] cycloaddition.In one embodiment, first reactive group is alkynyl or azido part, and second reactive group is azido or alkynyl part.For example, first reactive group be alkynyl part (for example, alpha-non-natural amino acid right-the propargyloxy phenylalanine), second reactive group is the azido part.In another embodiment, first reactive group be azido part (for example, alpha-non-natural amino acid right-azido-L-phenylalanine), second reactive group is the alkynyl part.
In one embodiment, the efficient that the O-RS aminoacylation has the O-tRNA of alpha-non-natural amino acid for example is equivalent to have, among the SEQ ID NO.:86 or 45 at least 50% of the efficient of the O-RS of listed amino acid sequence.In another embodiment, O-tRNA comprises by SEQ ID NO.:65 or 64 processing or coding, or their complementary polynucleotide sequence.In another embodiment, O-RS comprises any one listed amino acid in SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86.
This encoding proteins can comprise, for example, treats albumen, diagnosis albumen, industrial enzyme or their part.Randomly, further modify the albumen that this method is produced by alpha-non-natural amino acid.For example, the albumen produced of the method for randomly modifying in vivo by at least one posttranslational modification.
The method of producing screening or selecting transcript regutation protein (with screening of producing in this way or selection transcript regutation protein) also is provided.For example, method comprises: select first polynucleotide sequence, polynucleotide sequence coding nucleic acid binding domain wherein; And the sudden change of first polynucleotide sequence selected codon to comprise at least one.This provides screening or selects polynucleotide sequence.This method also comprises: select second polynucleotide sequence, wherein second polynucleotide sequence coding transcription activating domain; Provide and contain screening that is operably connected to second polynucleotide sequence or the construction of selecting polynucleotide sequence; With this construction, alpha-non-natural amino acid, quadrature tRNA synzyme (O-RS) and quadrature tRNA (O-tRNA) are introduced cell.In response to the selection codon in screening or the selection polynucleotide sequence; O-RS rely on these assemblies preferably aminoacylation have the O-tRNA of alpha-non-natural amino acid; O-tRNA identification selection codon also mixes alpha-non-natural amino acid in the nucleic acid binding domain, thereby screening is provided or selects transcript regutation protein.
In some embodiments, in this method protein of interest or polypeptide (or its part) and/or the present composition by nucleic acid coding.Usually, this nucleic acid comprises at least one and selects codon, at least two selection codons, at least three selection codons, at least four selection codons, at least five selection codons, at least six selection codons, at least seven selection codons, at least eight selection codons, at least nine selection codons, ten or more options codon more.
Can be with the gene of method well known to those skilled in the art and " mutagenesis and other Protocols in Molecular Biology " described herein mutagenesis coding protein of interest or polypeptide, to comprise, for example, one or more selection codons that are used to mix alpha-non-natural amino acid.For example, will be used for the nucleic acid mutation of protein of interest,, provide the insertion of one or more alpha-non-natural amino acids to comprise one or more selection codons.The present invention includes any described variant, for example, mutant, the form of arbitrary protein for example, comprises at least one alpha-non-natural amino acid.Similarly, the present invention also comprises corresponding nucleic acids, promptly any nucleic acid with one or more selection codons, one or more alpha-non-natural amino acids of this nucleic acid coding.
In an illustrative embodiments, the invention provides composition (with the composition of the inventive method production), comprise the Arg110TAG mutant of Thr44, GAL4, wherein GAL4 albumen comprises at least one alpha-non-natural amino acid.In another embodiment, the invention provides the composition of the Trp33TAG mutant that comprises human superoxide dismutase (hSOD), wherein hSOD albumen comprises at least one non-natural amino.
Purifying contains the recombinant protein of alpha-non-natural amino acid
The standard step purifying albumen of the present invention that can be known according to those skilled in the art and uses for example, contains the albumen of alpha-non-natural amino acid, contains the antibody etc. of the albumen of alpha-non-natural amino acid, reaches part or basic homogeney.Therefore, can be by any one reclaims and purifying polypeptide of the present invention in many methods well known in the art, comprise, for example, ammonium sulfate or precipitation with alcohol, acid or alkali extracting, column chromatography, affinity column chromatography, negative ion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, agglutinin chromatography, gel electrophoresis etc.In generating correct folding maturation protein, can use albumen folding step more as required.In the end need in the highly purified purification step, can use high performance liquid chroma-tography (HPLC), affinity chromatography or other appropriate method.In one embodiment, the antibody that will resist the alpha-non-natural amino acid albumen of alpha-non-natural amino acid (or contain) for example, is used for the protein purification based on affinity as purified reagent, and this albumen contains one or more alpha-non-natural amino acids.In case purifying reaches part homogeney or homogeney as required, then polypeptide randomly is used as, for example measure assembly, therapeutic agent or be used as the immunogene of producing antibody.
Except other list of references of quoting herein, various purifying/protein folding method all is well known in the art, and these methods comprise, for example, R.Scopes, " protein purification " (Protein Purification), Springer-Verlag, N.Y. (1982); Deutscher, " Enzymology method " (Methods in Enzymology) the 182nd volume: " protein purification guide " (Guide to Protein Purification), Academic Press, Inc.N.Y. (1990); Sandana (1997) " bio-separation of albumen " (Bioseparation of Proteins), Academic Press, Inc.; Bollag etc. (1996) " protein process " (Protein Methods) the 2nd edition, Wiley-Liss, NY; Walker (1996) " albumen Handbook Of Operating Procedures " (The Protein ProtocolsHandbook) Humana Press, NJ, the IRL Press of Harris and Angal (1990) " protein purification is used: practical approach " (Protein Purification Applications:A Practical Approach) Oxford, Oxford, England; The IRL Press of Harris and Angal " method for purifying proteins: practical approach " (Protein PurificationMethods:A Practical Approach) Oxford, Oxford, England; Scopes (1993) " protein purification: principle and put into practice " (Protein Purification:Principles and Practice) the 3rd edition SpringerVerlag, NY; Janson and Ryden (1998) " protein purification: principle, high resolution method and application " (Protein Purification:Principles, High Resolution Methods andApplications), second edition, Wiley-VCH, NY; And Walker (1998) " the albumen running program on the CD-ROM " (Protein Protocols on CD-ROM) Humana Press, NJ; Listed method in the list of references of wherein quoting.
An advantage with alpha-non-natural amino acid production protein of interest or polypeptide in eukaryotic is that this albumen or polypeptide are generally folding with their original conformation.Yet, In some embodiments of the present invention, those skilled in the art will recognize that behind synthetic, expression and/or the purifying, albumen can have and the different conformation of the required conformation of related polypeptide.In one aspect of the invention, expressing protein is sex change randomly, then renaturation.This is by for example, chaperone is added protein of interest or polypeptide and/or by protein dissolution etc. is finished.
Usually, need occasionally to make polypeptide be folded into preferred conformation more then express polypeptide sex change and reduction.For example, guanidine, urea, DTT, DTE and/or chaperone can be added interested translation product.The method of reduction, sex change and recombinant protein is well known to those skilled in the artly (referring to above-mentioned list of references, and Debinski, to wait (1993) J.Biol.Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4:581-585; And Buchner, etc., (1992) Anal.Biochem., 205:263-270).For example, Debinski waits and has described sex change and reduction occlusion body albumen in guanidine-DTE.Albumen can contain, and is for example folding again in the arginic potential buffer solution of oxidized glutathione and L-.Folding again reagent can flow or move to one or more polypeptide or other expression product and contact, and vice versa.
Antibody
In one aspect, the invention provides molecule of the present invention, for example synzyme, tRNA and comprise the antibody of the albumen of alpha-non-natural amino acid.The antibody of molecule of the present invention is used as purified reagent, for example, is used for purifying molecule of the present invention.In addition, antibody can be used as the existence that indicator is indicated synzyme, tRNA or comprised the albumen of alpha-non-natural amino acid, for example, and to follow the trail of existing or locate (for example, body is interior or original position) of molecule.
Antibody of the present invention can be to comprise the albumen of one or more basic or parts by the fragment coding polypeptide of immunoglobulin gene or immunoglobulin gene.The immunoglobulin gene of generally acknowledging comprises κ, λ, α, γ, δ, ε and υ constant region gene, and countless immune globulin variable region genes.Light chain is categorized as κ or λ.Heavy chain is categorized as γ, υ, α, δ or ε, and they define immunoglobulin class IgG, IgM, IgA, IgD and IgE respectively successively.The structural units of a kind of typical immunoglobulin (Ig) (as antibody) comprises the tetramer.To forming, each is to having one " light chain " (about 25kD) and one " heavy chain " (about 50-70kD) by two identical polypeptied chains for each tetramer.The N of each chain is terminal to determine the about 100-110 or the variable region of amino acids more, mainly responsible antigen recognizing.Variable light chain of term (VL) and variable heavy chain (VH) refer to these light chains and heavy chain respectively.
Antibody exists with complete immunoglobulin (Ig) or with the fragment that produces many well-characterized with different peptide enzymic digestions.Therefore, for example, digest antibody below the disulfide bond of pepsin in hinge area, produce F (ab ') 2, the dimer of Fab itself is to be connected in V by disulfide bond H-C H1 light chain.Can under temperate condition, reduce F (ab ') 2Interrupting the disulfide bond in the hinge area, thereby with F (ab ') 2Dimer is converted into Fab ' monomer.Fab ' monomer comes down to have the Fab (referring to, " basic immunology " (Fundermental Immunology), the 4th edition, W.E.Paul compiles, Raven Press, N.Y. (1999) to the more detailed description of other antibody fragment) of part hinge area.Though the digestion according to complete antibody has defined the different antibodies fragment, it will be understood by those skilled in the art that also available chemical method or from the beginning synthesize described Fab ' fragment etc. by the method for utilizing recombinant DNA.Therefore, used herein term antibody also randomly comprises by the whole antibody modification or with recombinant DNA method and from the beginning synthesizes the antibody fragment that is produced.Antibody comprises single-chain antibody, comprises strand Fv (sFv or scFv) antibody, wherein by variable heavy chain and variable light chain link together (directly or via peptide linker) form continuous polypeptide.Antibody of the present invention can be, for example, and polyclone, monoclonal, chimeric, humanization, strand, Fab fragment, the fragment that produces by the Fab expression library etc.
Usually, antibody of the present invention in various molecular biosciences or method of pharmacy, be used as general reagent and the treatment reagent be valuable.It is available producing polyclone and monoclonal antibody method, can be applied to production antibody of the present invention.Many basic readers have been described the antibody production method of standard, comprise, for example, Borrebaeck (volume) (1995) " antibody engineering " (Antibody Engineering), second edition, Freeman and Company, NY (Borrebaeck); McCafferty etc. (1996) " antibody engineering, practical approach " (Antibody Engineering, A Practical Approach) IRL of Oxford Press, Oxford, England (McCafferty), and Paul (1995) " antibody engineering scheme " (Antibody Engineering Protocols) Humana press, Towata, NJ (Paul); Paul (volume), (1999) " basic immunology " (Fundamental Immunology), the 5th edition Raven Press, N.Y.; Coligan (1991) " newly organized immunological experiment guide " (CurrentProtocols in Immunology) Wiley/Greene, NY; Harlow and Lane (1989) " antibody: laboratory manual " (Antibodies:A Laboratory Manual) Cold Harbor Press, NY; Stites etc. (volume) " basis and clinical immunology " (Basic and Clinical Immunology) (the 4th edition) LangeMedical Publications, Los Altos, CA and the list of references of wherein quoting; Goding (1986) " monoclonal antibody: principle and put into practice " (Monoclonal Antibodies:Principles andPractice) (second edition) Academic Press, New York, NY; And Kohler and Milstein (1975) Nature 256:495-497.
Developed the various recombinant techniques that are used to not rely on as to the Antibody Preparation of animal injections of antigens, they can be used for content of the present invention.For example, may in bacteriophage or similar substrates, produce and select the recombinant antibodies library.Referring to, for example, Winter etc. (1994) produce antibody (Making Antibodiesby Phage Display Technology) by display technique of bacteriophage, Annu.Rev.Immunol.12:433-455 and quote the list of references of making summary.Also referring to, Griffiths and Duncan (1998) select the strategy (Strategies for selection of antibodies by phage display) of antibody, Curr OpinBiotechnol 9:102-108 by phage display; Hoogenboom etc. (1998) antibody phage display technique and application thereof (Antibody phage display technology and its applications), Immunotechnology4:1-20; Gram etc. (1992) are external selection and affine ripe antibody (in vitro selection and affinity maturation of antibodies from a naivecombinatorial immunoglobulin library) PNAS 89:3576-3580 from the combination immunoglobulin (Ig) library of naivety; Huse etc. (1989) Science 246:1275-1281; With (1989) Nature 341:544-546 such as Ward.
In one embodiment, antibody library can comprise the V gene repertoire (as, from lymphocyte populations, collect or assembled in vitro), with its clone, be used on the surface of filobactivirus, showing relevant heavy chain and light chain variable territory.Select bacteriophage by combining with antigen.By the bacterial expression soluble antibody that infects bacteriophage, for example improve this antibody by mutagenesis.Referring to, antibody engineering (Antibody Engineering by Parsimonious Mutagenesis) the Gene 137:109-118 that is undertaken by brief mutagenesis as Balint and Larrick (1993); Stemmer etc. (1993) prepare selection (Selection of an Active Single Chain Fv Antibody From a Protein Linker LibraryPrepared by Enzymatic Inverse PCR) Biotechniques 14 (2): the 256-65 of active single-chain Fv antibody from albumen joint library by the enzyme inverse PCR; Crameri etc. (1996) make up by DNA reorganization and development antibody-phage library (Construction and evolutionof antibody-phage libraries by DNA shuffling) Nature Medicine 2:100-103; Set up all conversion (Combinatorial multiple cassette mutagenesis creates all the permutationsof mutant and wildtype cassettes) BioTechniques 18:194-195 of saltant and wild flask with many cassette mutagenesiss of Crameri and Stemmer (1995) combination.
The kit that is used to clone with expressing recombinant antibody bacteriophage system also is known and available, for example, originate from Amersham-Pharmacia Biotechnology (Uppsala, Sweden) " recombinant phages antibody system; mouse ScFv module " (recombinant phage antibody system, mouse ScFv module).Phage antibody library also be used for by chain reorganization produce high affine human antibody (referring to, for example, (1992) by-passing Immunization such as Marks: make up high affine people's antibody (By-Passing Immunization:BuildingHigh Affinity Human Antibodies by Chain Shuffling) Biotechniques 10:779-782 by chain reorganization.Also be recognized that, any one preparation antibody that can be by many commerce services (as Bethyl Laboratories (Montgomery, TX), Anawa (Switzerland), Eurogentec (Belgium and Philadelphia, PA, USA etc.) and many other companies.
In some embodiments, the present invention " humanization " antibody is useful, as, antibody is used for the treatment of when using.The use of humanized antibody be tending towards reducing to the unwanted immunoreactive incidence of treatment antibody (as, when the patient is a man-hour).Above-mentioned antibody list of references has been described the humanization strategy.Except humanized antibody, people's antibody also is feature of the present invention.People's antibody is made up of distinctive human immunoglobulin(HIg) sequence.People's antibody can pass through produced in several ways (referring to, for example, Larrick etc., United States Patent (USP) 5,001,065 as the summary).The conventional method of producing people's antibody by trisome hybridoma (trioma) technology is by Ostberg etc., and (1983), Hybridoma 2:361-367, Ostberg, United States Patent (USP) 4,634,664 and Engelman etc., United States Patent (USP) 4,634,666 describe.The known the whole bag of tricks that uses antibody in purifying and detection albumen, these methods can be applied to detect and the purifying protein that contains alpha-non-natural amino acid as described herein.Usually, antibody is useful reagent to enzyme linked immunological adsorption reaction, Western trace, immunochemistry, affinity chromatography, SPR and a lot of additive method.Above-mentioned list of references provides the details of how to carry out enzyme linked immunological adsorption reaction, Western trace, surface plasmon resonance (SPR) etc.
In one aspect of the invention, antibody of the present invention itself comprises alpha-non-natural amino acid, and the antibody with interested character (for example improved half life period, stability, toxicity etc.) is provided.Also referring to, be entitled as " polypeptide " part herein with alpha-non-natural amino acid.Antibody account for all compounds in the present clinical testing (Wittrup, (1999) phage display Tibtech 17:423-424, antibody are generally as diagnostic reagent near 50%.Therefore, the ability with the alpha-non-natural amino acid modified antibodies provides important instrument for modifying these valuable reagent.
For example, Mab has a lot of application in diagnostic field.The assay method wider from simple spot test to involvement aspect is as the radiolabeled NR-LU-10 Mab from DuPont Merck Co., and it is used for tumor imaging (Rusch etc. (1993) NR-LU-10 monoclonal anti swept-volume.The useful new tool (NR-LU-10monoclonal antibody scanning.A helpful new adjunct to computedtomography in evaluating non-small-cell lung cancer) of non-small cell lung cancer, J Thorac CardiovascSurg 106:200-4 are estimated in the computed tomography video picture).As mentioned above, Mab is the center reagent of ELISA, Westerm trace, immunochemistry, affinity chromatography etc.Can modify any described diagnosis antibody, comprise one or more alpha-non-natural amino acids, change that for example Ab is to the specificity or the affinity of target, or for example, change one or more detectable character by in alpha-non-natural amino acid, comprising detectable label (as spectrum, fluorescence, luminous etc.).
The valuable antibody reagent of one class is a treatment antibody.For example, antibody can be the Mab of tumour-specific, it can suppress tumor growth by the target tumor cell, and the cell-mediated cytotoxicity (ADCC) that relies on by antibody or the cracking (CML) of complement-mediated destroy tumor growth (these universal Ab are sometimes referred to as " magic bullet ").An example is a sharp appropriate glycosides (Rituxan), a kind of anti-CD20 Mab, be used for the treatment of non-its golden lymphomas suddenly (the sharp appropriate glycosides of Scott (1998): a kind of new monoclonal antibody (Rituximab:a newtherapeutic monoclonal antibody for non-Hodgkin ' s lymphom) for the treatment of non-its golden lymphomas suddenly, Cancer Pract6:195-7).Second example is involved in the antibody of the key component of disturbing tumor growth.He Saiting (Herceptin) is a kind of anti-HER-2 monoclonal antibody, be used for the treatment of metastatic breast cancer, and provide the example (Baselga etc. of antibody with this kind mechanism of action, (1998) recombinant humanized Anti-HER 2 (He Saiting) enhancing taxol and adriamycin are to crossing the antitumor activity (Recombinanthumanized anti-HER2antibody (Herceptin) enhances the antitumor activity ofpaclitaxel and doxorubicin against HER2/neu overexpressing human breastcancer xenografts) [misarrangement is published in Cancer Res (1999) 59 (8): 2020] of the human breast carcinoma heterograft knurl of expressing HER2/neu, Cancer Res 58:2825-31).The 3rd example relates to the antibody that directly cytotoxic compound (toxin, radioactive nuclide etc.) is passed to tumour or other interested positions.For example, a kind of application Mab is CYT-356, the antibody that 90Y connects, it directly will radiate the target prostate tumor cells, and (Deb etc. (1996) are with the anti-prostate cancer of controlling of 90Y-CYT-356 mab treatment hormone (Treatment of hormone-refractory prostate cancer with90Y-CYT-356 monoclonal antibody) Clin Cancer Res 2:1289-97.The 4th application is antibody targeted enzyme prodrug therapy, and the enzyme that wherein is positioned to tumour altogether activates the prodrug that whole body gives near tumour.For example, developed the anti-Ep-CAM1 antibody that is connected in Carboxypeptidase A, be used for the treatment of colorectal cancer (Wolfe etc., (1999) the antibody targeted enzyme prodrug therapy that carries out with the T268G mutant of human carboxypeptidase A 1: the inside and outside research (Antibody-directed enzymeprodrug therapy with the T268G mutant of human carboxypeptidase Al:in vitroand in vivo studies with prodrugs of methotrexate and the thymidylate synthaseinhibitors GW1031 and GW1843) of prodrug amethopterin and thymidylate synthase inhibitor GW1031 and GW1843, Bioconjug Chem 10:38-48).Other Ab (as antagonist) is designed to specificity suppresses the normal cell function, to obtain curative effect.An example is just to clone OKT3, a kind of anti-CD3Mab that provides by Johnson and Johnson, (Strate etc. (1990) are just cloning OKT3 and are being used for acute kidney allograft rejection reaction (Orthoclone OKT3 as first-line therapy in acute renal allograft rejection), Transplant Proc 22:219-20 as first-line treatment to be used to reduce the acute organ graft rejection.Another kind of antibody preparation is an activator.These monoclonal antibodies are designed to specificity strengthen the normal cell function, to obtain curative effect.For example, (Xie etc. (1997) were tested and appraised activator ScFv and prove that directly MuSK participates in acetylcholinergic receptor bunch collection (Direct demonstration of MuSK involvement inacetylcholine receptor clustering through ident ification of agonist ScFv), Nat.Biotechnol.15:768-71 among the acetyl choline receptor agonists based on monoclonal antibody that is used for psychiatric treatment was being developed.Can be with any one is modified into and comprises one or more alpha-non-natural amino acids in these antibody, to strengthen one or more therapeutic properties (specificity, affinity, serum half-life etc.).
Another kind of antibody product provides new function.Main antibody is catalytic antibody in this group, as engineered Ig sequence with the mimetic enzyme catalysis ability (Wentworth and Janda (1998) catalytic antibody (Catalyticantibodies) Curr Opin Chem Biol 2:138-44).For example, interesting application be in vivo with catalytic antibody mAb-15A10 hydrolysis cocaine with the treatment habituation (catalytic antibody of a kind of anti-cocaine of Mets etc. (1998) prevents that cocaine from strengthening and poisonous effect (A catalytic antibody against cocaineprevents cocaine ' s reinforcing and toxic effects in rats) in rat, Proc Natl AcadSci U S A 95:10176-81).Also can modify catalytic antibody, make it comprise one or more alpha-non-natural amino acids, to improve one or more interested character.
By immunoreactivity definition polypeptide
Because polypeptide of the present invention provides various novel polypeptide sequences (to comprise non-natural amino acid under the situation as synthetic proteins in this paper translation system, or as under the situation of the new synzyme of this paper, the new sequence of standard amino acid), these polypeptide also provide for example discernible new construction characteristic in immunoassays.Antibody or specificity are in conjunction with the production of antibodies of this paper invention polypeptide, and the polypeptide of antibody or antiserum combination is feature of the present invention.
For example, the present invention includes the synthetase albumen that combines with antibody or antiserum specificity or they and antibody or antiserum immune response specifically, generation comprises the immunogene that is selected from amino acid sequences one or more in (SEQ ID NO:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86).In order to eliminate the cross reactivity with other homologues, with available contrast synzyme homologue, for example wild-type e. coli tyrosyl synzyme (TyrRS) (as, SEQ ID NO.2) is subdued antibody or antiserum.
In a kind of canonical form, polyclonal antiserum is used in immunoassays, produce the antiserum of anti-one or more polypeptide, described polypeptide comprises corresponding to SEQ ID NO:36-63 (as any other subgroup of 36-47,48-63 or 36-63) and/or 86, or their substantive subsequence (as, the full length sequence at least about 30% is provided) in one or more one or more sequences.This group is referred to as hereinafter from the potential polypeptide immunogen of SEQ ID NO:36-63 and 86 " immunogenic polypeptide ".Randomly select the antiserum of gained, to have low cross reactivity with contrast synzyme homologue, before polyclonal antiserum is used for immunoassays, for example, by removing any this cross reactivity with one or more synzyme homologue immunoadsorption.
In order to produce the antiserum that is used for immunoassays, one or more immunogenic polypeptides of production as described herein and purifying.For example, can in recombinant cell, produce recombinant protein.With with standard adjuvant, as the immunogenic protein of Freund combination and the immunity of standard mouse immune scheme (the relevant antibody that can be used for determining specific immune response produces, the standard to describe of immunoassays form and condition referring to, for example, Harlow and Lane (1988) " antibody, laboratory manual " (Antibodies, A Laboratory Manual), Cold Spring HarborPublications, New York.This paper has also described the additional reference and the discussion of antibody, and this paper can be applicable to produce antibody by immunoreactivity definition/detection polypeptide) mouse (because the actual genetic identity of mouse, the result more can repeat, so this mouse of this mensurations use) of inbred strais.Perhaps, will be from one or more of the open sequence of this paper synthetic or recombinant polypeptide conjugation to carrier protein, and as immunogene.
In immunoassays, collect polyclonal antiserum, and the anti-immunogenic polypeptide of titration, for example, carry out solid-phase immunoassay with one or more fixing on solid support immunogenic proteins.Selecting, concentrate and subduing titre with contrast synzyme polypeptide is 10 6Or bigger polyclonal antiserum, polyclonal antiserum that subdue with generation, that concentrate, titration.
Test is that subdue, that concentrate, the polyclonal antiserum of titration in relative immunity is measured with the cross reaction of contrast homologue.In this comparative measurements, measure difference in conjunction with condition for polyclonal antiserum that subdue, that concentrate, titration, make signal to noise ratio (S/N ratio) that the polyclonal antiserum of titration is attached to the immunogenicity synzyme be attached to contrast synzyme homologue compare high at least about 5-10 doubly.That is to say, by adding nonspecific competitor such as albumin or skimmed milk power, and/or by regulating salt condition, temperature, and/or the severity of combination/washing reaction is regulated in other aspects.In subsequent measurements with these combination/wash conditions in order to determine that whether test polypeptide (comparing the polypeptide of immunogenic polypeptide and/or contrast polypeptide) is by the polyclonal antiserum specificity combination of concentrating, subdue.Particularly, the test polypeptide shows in conjunction with under the condition in difference, comparison is according to the high at least 2-5 of signal to noise ratio (S/N ratio) times of the synzyme homologue, and compare its signal to noise ratio (S/N ratio) with immunogenic polypeptide at least about 1/2, compare with known synzyme, therefore the total basic structure similarity of this test polypeptide and immunogenic polypeptide is polypeptide of the present invention.
In another embodiment, the immunoassays of competition convolution are used to test the detection of polypeptide.For example, as mentioned above, by from the anti-serum mixture of concentrating, removing cross reacting antibody with the absorption of contrast polypeptide immune.Then, immunogenic polypeptide is fixed on the solid support that contacts with the antiserum of subduing of concentrating.Add in the mensuration and tried albumen, with the concentrated antiserum of subduing of competitive combination.Compare with ankyrin, tried albumen and the competitive ability that combines of the antiserum of concentrating of subduing, compare with the ability (immunogenic polypeptide and fixing immunogenic polypeptide effective competition are with the concentrated antiserum of combination) that adding is measured with competitive binding immunoassay originality polypeptide.Calculate the cross reactivity percentage that is tried albumen with the criterion calculation method.
In replicate determination, by combining sero-fast ability with immunogenic polypeptide competitiveness relatively, it is competitive in conjunction with the sero-fast ability of concentrating of subduing randomly to measure reference protein.In addition, calculate the cross reaction percentage of contrast polypeptide with the criterion calculation method.When the cross reactivity percentage comparison of test polypeptide according to the polypeptide height at least 5-10 times the time, or the combination of test polypeptide thinks that the test polypeptide is specifically in conjunction with the antiserum of concentrating of subduing greatly in the incorporation range of immunogenic polypeptide the time.
Usually, antiserum immunoadsorption and that concentrate can be used for competitive binding immunoassay described herein to be measured, with more any test polypeptide and immunogenicity and/or contrast polypeptide.In order to carry out this relatively, in wide concentration range, measure each immunogenicity, test and contrast polypeptide, the amount of each polypeptide need can to suppress to subdue antiserum with, for example 50% combination of fixing contrast is with measured by standard techniques test or immunogenic protein.If the aequum of test polypeptide combination is less than the twice of the amount of required immunogenic polypeptide in the competitive assay, think that the test polypeptide combines specifically with the antibody of the anti-immunogenic protein of generation, the amount that provides be the contrast polypeptide at least about 5-10 times.
As specific additional determination, with the antiserum that the randomly complete immunoadsorption of immunogenic polypeptide (but not contrast polypeptide) is concentrated, the antiserum of subduing up to the immunogenic polypeptide that does not almost have or do not have gained of concentrating can be detected with not combining of the immunogenic polypeptide that is used for immunoadsorption.Then, test the reactivity of antiserum with the test polypeptide of panimmunity absorption.If almost do not have or do not observe reactivity (that is, the antiserum of observing fully absorption is not higher than 2 times with the signal to noise ratio (S/N ratio) that combines of immunogenic polypeptide), the antiserum that is brought out by immunogenic protein is specifically in conjunction with testing polypeptide so.
Pharmaceutical composition
Randomly with polypeptide of the present invention or albumen (as synzyme, comprise the albumen of one or more alpha-non-natural amino acids etc.) being used for the treatment of property purposes, as combining with suitable pharmaceutical carrier.This composition for example, comprises compound and the pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.Described carrier or excipient include but not limited to, salt solution, buffer saline, glucose, water, glycerine, ethanol and/or their combination.Make formulation adapt to administering mode.Usually, the protein medicine-feeding mode is well known in the art, can be applied to the administration of polypeptide of the present invention.
At one or more in the external and/or body in the animal model of disease randomly test comprise the therapeutic combination of one or more polypeptide of the present invention, according to method conclusive evidence usefulness, tissue metabolism and estimation dosage as known in the art.Particularly, at first can by active, stability or herein alpha-non-natural amino acid to other suitable measuring method of natural amino acid homologue (as, modification is compared with natural amino acid EPO with the EPO that comprises one or more alpha-non-natural amino acids), promptly in a relevant mensuration, determine dosage.
Administration is to be undertaken by being generally used for introducing any approach that molecule makes it finally to contact with blood or histocyte.Randomly use one or more pharmaceutically acceptable carriers, give non-natural amino acid polypeptides of the present invention with any desired manner.The suitable medication that gives the described polypeptide of patient in the content of the present invention is available, and though availablely give concrete composition more than a kind of approach, concrete approach can often provide than quicker and more effective effect of another approach or reaction.
By the concrete composition that gives, and the concrete grammar that is used to give said composition partly determines pharmaceutically acceptable carrier.Therefore, pharmaceutical composition of the present invention has various suitable formulations.
Can include but not limited to by many approach: per os, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, local, hypogloeeis or the rectal peptide composition that gives.Also can give the non-natural amino acid polypeptides composition by liposome.This method of administration and dosage forms are normally well known by persons skilled in the art.
Also non-natural amino acid polypeptides can be united separately or with other suitable components and make aerosol (being that they can " atomize ") to pass through inhalation.Aerosol can be inserted the propellant accepted of pressurization, as dichlorodifluoromethane, propane, nitrogen etc.
The formulation that is fit to the intestines and stomach external administration, for example (in the joint), intravenous, intramuscular, intracutaneous, intraperitoneal and subcutaneous route comprise and can contain antioxidant, damping fluid, bacteriostatic agent and make formulation and the water-based and the non-aqueous isotonic sterile injection liquid of the solute that receptor's blood of plan etc. oozes and can comprise the water and the nonaqueous phase sterile suspension of suspending agent, solubilizer, thickening agent, stabilizing agent and antiseptic in the joint.The formulation of packing nucleic acid can unit dose or multiple dose airtight container, presents as the form of ampoule bottle and bottle.
Intestines and stomach external administration and intravenous administration are preferred administering modes.Particularly, be used for natural amino acid homologue treatment (as, generally be used for EPO, GCSF, GMCSF, IFN, interleukin, antibody and/or any albumen that pharmaceutically transmits) method of administration, provide preferred method of administration and formulation (as the variant of the adding polyglycol of current treatment albumen etc.) with the formulation of current use for the albumen that comprises alpha-non-natural amino acid of the present invention.
In content of the present invention, to pass in time, the dosage that gives patient is enough to patient is produced useful therapeutic response, or for example, according to the application, suppresses pathogenic infection, or other suitable activity.Dosage is by the usefulness of concrete composition/formulation and activity, stability or the serum half-life of used non-natural amino acid polypeptides, patient's state of an illness, and patient's to be treated body weight or body surface area are determined.The size of dosage is also by existence, character among the concrete patient and the decisions such as degree of following any adverse side effect that gives concrete composition/formulation.
In determining treatment or prevent disease (as cancer, hereditary disease, diabetes, acquired immune deficiency syndrome (AIDS) etc.), use the composition/formulation of effective dose, the generation aspect of the polypeptide antibody of doctor's evaluation cycle plasma concentration, formulation toxicity, progression of disease and/or relevant alpha-non-natural amino acid.
Give as 70 kilograms of patients' dosage in the scope suitable, and change according to compositions related activity or serum half-life and to make adjustment with the treatment albumen dosage of current use.The present composition/formulation can comprise antibody administration by any known routine treatment, vaccine administration, and the administration of cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleic acid analog, biological response modifier etc. comes the supplementary therapy illness.
For administration, give formulation of the present invention with the LD-50 of relevant formulation and/or the speed that the observation of alpha-non-natural amino acid any spinoff under variable concentrations is determined, for example according to patient body weight and general health.Can finish administration by single dose and equal divided dose.
If carry out that the patient of a kind of formulation of infusion generates heat, shiver with cold or myalgia, he should accept aspirin, brufen, paracetamol or other pain/heating control medicine of suitable dose so.For experience infusion reaction,, should give aspirin, paracetamol in preceding 30 minutes in advance or as diphenhydramine at infusion as the patient of heating, myalgia and shiver with cold.To alexipyretic and the antihistamine not more serious shiver with cold and the myalgia of reaction rapidly, then use Sauteralgyl.The order of severity according to reaction slows down or TD.
Nucleic acid and peptide sequence and variant
As above hereinafter described, the invention provides nucleic acid polynucleotide sequence and polypeptid acid sequence, as O-tRNA and O-RS and, as the composition and the method for sequence as described in comprising.The example of described sequence is disclosed herein, as O-tRNA and O-RS (referring to table 5, as the SEQ ID NO.3-65,86 except SEQ ID NO.:1 and 2).Yet, it will be appreciated by those skilled in the art that the present invention is not limited to sequence disclosed herein, for example, embodiment and table 5.It will be understood by those skilled in the art that the present invention also provide many relevant and even incoherent sequence with function described herein, as coding O-tRNA or O-RS.
The present invention also provides polypeptide (O-RS) and polynucleotide, and as O-tRNA, the polynucleotide of coding O-RS or its part (as the avtive spot of synzyme) are used to make up the oligonucleotides of aminoacyl tRNA synthetase mutant etc.For example, polypeptide of the present invention comprises and comprises SEQ ID NO.:36-63 (as 36-47, any other subgroup of 48-63 or 36-63) polypeptide of arbitrary listed amino acid sequence and/or in 86, comprise by SEQ ID NO.:3-35 (as 3-19, any other subgroup of 20-35 or 3-35) polypeptide of the amino acid sequence of arbitrary listed polynucleotide sequence coding in, with with to the immunoreactive specifically polypeptide of the specific antibody of polypeptide, this polypeptide comprises SEQ ID NO.:36-63, and/or in 86 any listed amino acid sequence polypeptide or (for example comprise SEQ ID NO.:3-35,3-19,20-35, or any other subgroup of sequence 3-35) in the polypeptide of amino acid sequence of listed any polynucleotide sequence coding.
Polypeptide of the present invention comprises that also the tyrosyl aminoacyl-tRNA synthetase (TyrRS) (for example, SEQ ID NO.:2) with natural generation has the polypeptide of at least 90% same acid sequence and comprises two or more amino acid whose polypeptide in the A-E family.For example, A family comprises valine, isoleucine, leucine, glycocoll, serine, alanine or the threonine on the Tyr37 opposite position with Escherichia coli TyrRS.B family comprises the aspartic acid on the Asn126 opposite position with Escherichia coli TyrRS; C family comprises threonine, serine, arginine, asparagine or the glycocoll on the Asp182 opposite position with Escherichia coli TyrRS; D family comprises methionine, alanine, valine or the tyrosine on the Phel83 opposite position with Escherichia coli TyrRS; E family comprises serine, methionine, valine, halfcystine, threonine or the alanine on the Leul86 opposite position with Escherichia coli TyrRS.The subgroup of any of these family combination also is a feature of the present invention.For example, in one embodiment, O-RS has two or more and is selected from valine, isoleucine, leucine or the threonine that occurs on the Tyr37 opposite position with Escherichia coli TyrRS; With threonine, serine, arginine or the glycocoll on the Asp182 opposite position of Escherichia coli TyrRS; With methionine or the tyrosine on the Phel83 opposite position of Escherichia coli TyrRS; With with the Leul86 opposite position of Escherichia coli TyrRS on serine or the amino acid of alanine.In another embodiment, O-RS comprises that two or more are selected from glycocoll, serine or alanine on the Tyr37 opposite position with Escherichia coli TyrRS, with the aspartic acid on the Asnl26 opposite position of Escherichia coli TyrRS, with the asparagine on the Aspl82 opposite position of Escherichia coli TyrRS, with alanine on the Phel83 opposite position of Escherichia coli TyrRS or valine and/or and with the Leul86 opposite position of Escherichia coli TyrRS on methionine, valine, halfcystine or threonine.
Similarly, polypeptide of the present invention also comprise the polypeptide that contains at least 20 continuous amino acids in SEQ ID NO.:36-63 (for example, any other subgroup of 36-47,48-63 or 36-63) and/or 86 and as above-mentioned A-E family in two or more aminoacid replacement.Also referring to this paper table 4,6 and/or table 8.Polypeptide of the present invention also comprises the amino acid sequence of the conservative variant that comprises arbitrary aforementioned polypeptides.
In one embodiment, composition comprises polypeptide of the present invention and excipient (for example, damping fluid, water, pharmaceutically acceptable excipient etc.).The present invention also provides and immunoreactive specifically antibody of polypeptide of the present invention or antiserum.
The present invention also provides polynucleotide.Polynucleotide of the present invention comprise code book invention protein of interest or polypeptide or comprise one or more selection codons, or the two.For example, polynucleotide of the present invention comprise, for example, contain the polynucleotide of any one listed nucleotide sequence among SEQ ID NO.:3-35 (for example, any other subgroup of 3-19,20-35 or sequence 3-35), the 64-85; Polynucleotide with this polynucleotide sequence complementation or its polynucleotide sequence of encoding; And/or coding contains the polynucleotide of the polypeptide of any one listed amino acid sequence in SEQ ID NO.:36-63 and/or 86 or its conservative variant.Polynucleotide of the present invention also comprise the polynucleotide of code book invention polypeptide.Similarly, surpassing basically with the nucleic acid of above-mentioned multi-nucleotide hybrid under the rigorous condition of height, the nucleic acid of total length is polynucleotide of the present invention.
Polynucleotide of the present invention also comprise the polynucleotide of coded polypeptide; this polypeptide comprise with the tyrosyl aminoacyl-tRNA synthetase (TyrRS) of natural generation (for example; SEQ ID NO.:2) at least 90% identical amino acid sequence and comprise the two or more sudden changes described in the A-E family (above-mentioned).With above-mentioned polynucleotide and/or contain the identical polynucleotide of the polynucleotide at least 70% (or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% or more) of the conservative variant of arbitrary above-mentioned polynucleotide and be also included within the polynucleotide of the present invention.Also referring to table 4, table 6 and/or the table 8 of this paper.
In some embodiments, carrier (for example, plasmid, clay, bacteriophage, virus etc.) comprises polynucleotide of the present invention.In one embodiment, carrier is an expression vector.In another embodiment, expression vector comprises the promoter that is operably connected to one or more polynucleotide of the present invention.In another embodiment, cell contains the carrier that comprises polynucleotide of the present invention.
Those skilled in the art also will understand, and the present invention includes a lot of variants of open sequence.For example, the present invention includes the conservative variant of the open sequence that produces the function identical sequence.Think and the present invention includes and at least a openly variant of the nucleic acid polynucleotide sequence of sequence hybridization.Unique subsequence of the open sequence of this paper, as by for example, the subsequence that the standard sequence correlation technique is determined is also included among the present invention.
Conservative variant
Because the degeneracy of genetic code, " the reticent replacement " (promptly not causing the replacement in the nucleotide sequence that coded polypeptide changes) be each coded amino acid nucleotide sequence infer feature.Similarly, amino acid whose with in the similar different aminoacids substituted-amino acid sequence of character height one or more " conserved amino acid replaces ", also easily be accredited as with to disclose the construction height similar.This conservative variant of each open sequence is a feature of the present invention.
Concrete nucleotide sequence " conservative variant " nucleic acid of refer to encode identical or essentially identical amino acid sequence, or this nucleic acid does not become amino acid sequence encode essentially identical sequence.Those skilled in the art will recognize that, change in coded sequence, adding or removal single amino acids or little number percent (are generally less than 5%, more be generally less than 4%, 2% or 1%) amino acid independent replacement, disappearance or the adding carried out be " conservative modification variation ", wherein change and cause amino acid whose disappearance, amino acid whose adding or with chemically similar aminoacid replacement amino acid.Therefore, the listed peptide sequence of the present invention " conservative variant " comprise with identical and guard substituent conservative selection amino acid with little number percent, be generally less than 5%, more be generally less than 2% or 1% and replace peptide sequence amino acid.At last, adding the sequence of the coding activity that does not change nucleic acid molecules, as the adding of NOT-function sequence, is the conservative variant of basic nucleic acid.
It is well known in the art that functionally similar amino acid whose conservative replacement table is provided.Listed the example group of the natural amino acid that comprises mutually " the conservative replacement " below.
Conservative replacement group
1 Alanine (A) serine (S) threonine (T)
2 Aspartic acid (D) glutamic acid (E)
3 Asparagine (N) glutamine (Q)
4 Arginine (R) lysine (K)
5 Isoleucine (I) leucine (L) methionine (M) valine (V)
6 Phenylalanine (F) tyrosine (Y) tryptophane (W)
Nucleic acid hybridization
Can identify nucleic acid of the present invention with relatively hybridizing, comprise the conservative variant of nucleic acid of the present invention, this comparison hybrid method is the method for optimizing of difference nucleic acid of the present invention.In addition, the target nucleic acid of the nucleic acid hybridization of representing with SEQ ID NO:3-35 (for example, any other subgroup of 3-19,20-35 or sequence 3-35), 64-85 under height, superelevation and the rigorous condition of superelevation is a feature of the present invention.Compare with given nucleotide sequence, the example of described nucleic acid comprises having the nucleic acid that one or several reticent or conservative nucleic acid replaces.
Be equivalent to when the hybridization of test nucleic acid and probe Perfect Matchings complementary target at least 1/2, be probe and the target signal to noise ratio (S/N ratio) of hybridizing under the following conditions up at least 1/2 the time, think and test nucleic acid and probe nucleic acid specific hybrid, under the described conditions, the signal to noise ratio (S/N ratio) ratio that combines with the Perfect Matchings complementary target of Perfect Matchings probe hybridizes to and does not match during target nucleic acid the high at least about 5-10 of observed signal to noise ratio (S/N ratio) doubly arbitrarily.
When nucleic acid generally in solution in conjunction with the time, their " hybridization ".Nucleic acid is because of the physical-chemical power of various well-characterized, as hybridization such as hydrogen bond, solvent repulsion, base stackings.In Tijssen (1993) " laboratory technique in biological chemistry and the molecular biology--use nucleic acid probe hybridization " (Laboratory Techniques inBiochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes) the 2nd chapter part i, " the tactful summary of hybridization principle and nucleic acid probe determining " (Overview of principlesof hybridization and the strategy of nucleic acid probe assays) (Elsevier, and found the extensive guide of nucleic acid hybridization among the Ausubel (above-mentioned) New York).Hames and Higgins (1995) " gene probe 1 " (Gene Probes 1) IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) " gene probe 2 " (GeneProbes 2) IRL Press at Oxford University Press, Oxford, that England (Hames and Higgins 2) provides is synthetic, mark, detection and quantitatively DNA and RNA, comprises the details of oligonucleotides.
The example that is used for having the rigorous hybridization conditions of hybridizing more than the complementary nucleic acid of 100 complementary residues in Southern or Northern trace on filter membrane is to spend the night in the 42 ℃ of hybridization of 50% formalin that contain 1 milligram of heparin.The example of rigorous wash conditions is 0.2xSSC in 65 ℃ of washings 15 minutes (description of SSC damping fluid referring to, Sambrook, above-mentioned).Before the high rigorous washing low rigorous washing, to remove the background probe signals.The example of low rigorous washing is that 2xSSC was 40 ℃ of washings 15 minutes.In the usually concrete hybridization assays, signal to noise ratio (S/N ratio) shows to have detected specific hybrid than observed high 5 times (or higher) in the uncorrelated probe.
The nucleic acid hybridization experiment is sequence dependent as " rigorous hybridization wash conditions " in the content of Southern and Northern hybridization, and is different under the varying environment parameter.At Tijssen (1993) (above-mentioned) and Hames and Higgins, found the extensive guide of nucleic acid hybridization in 1 and 2.Can easily empirical rigorous hybridization and the wash conditions of determining any test nucleic acid.For example, when definite high rigorous hybridization and wash conditions, increase hybridization and wash conditions gradually (for example, by improving temperature, reduce salinity, improve scaling agent concentration and/or improving organic solvent, concentration as the formalin in hybridization or the washing), up to meeting a group selection standard.For example, hybridization and wash conditions increase gradually, and viewed signal to noise ratio (S/N ratio) was high at least 5 times when the signal to noise ratio (S/N ratio) that combines up to probe and perfection pairing complementary target was hybridized with the target that do not match than probe.
" very rigorous " condition of selection is with the heat fusion joint (Tm) that equals concrete probe.Tm is the temperature (under ionic strength that limits and pH) of 50% cycle tests and Perfect Matchings probe hybridization.Be the object of the invention, usually will " high rigorous " hybridization and wash conditions be chosen as and be lower than concrete sequence about 5 ℃ of the ionic strength of qualification and the Tm under the pH.
" superelevation is rigorous " hybridization and wash conditions are the preciseness that has increased hybridization and wash conditions, up to probe and Perfect Matchings complementary target nucleic acid combine signal to noise ratio (S/N ratio) be hybridize with the target nucleic acid that do not match arbitrarily in viewed signal to noise ratio (S/N ratio) up at least 10 times.With the target nucleic acid of probe hybridization, its signal to noise ratio (S/N ratio) is at least 1/2 of a Perfect Matchings complementary target nucleic acid, thinks to combine with probe under the rigorous condition of superelevation under this condition.
Similarly, even can determine higher levels of preciseness by the hybridization and/or the wash conditions that increase relevant hybridization assays gradually.For example, in those conditions, increased the preciseness of hybridization and wash conditions, up to the signal to noise ratio (S/N ratio) of probe and the combination of Perfect Matchings complementary target nucleic acid be with the target nucleic acid hybridization that do not match arbitrarily in viewed signal to noise ratio (S/N ratio) up at least 10 times, 20 times, 50 times, 100 times or 500 times or higher.With the target nucleic acid of probe hybridization, its signal to noise ratio (S/N ratio) is at least 1/2 of a Perfect Matchings complementary target nucleic acid, thinks to combine with probe under the rigorous condition of superelevation under the described conditions.
If their encoded polypeptides are basic identical, the nucleic acid of not hybridizing mutually under rigorous condition is still essentially identical so.This occurs in, for example, and when the maximum codon degeneracy that allows with genetic code produces the copy of nucleic acid.
Unique subsequence
In one aspect, the invention provides nucleic acid, this nucleic acid comprises subsequence unique in the nucleic acid that is selected from 0-tRNA disclosed herein and O-RS sequence.Compare with any known 0-tRNA or the corresponding nucleic acid of O-RS nucleotide sequence, this unique subsequence is unique.Can use, for example, the BLAST that is set to default parameters compares.Any unique subsequence all is useful, for example, identifies nucleic acid of the present invention as probe.
Similarly, the present invention includes polypeptide, this polypeptide comprises the subsequence that is selected from uniqueness in the O-RS polypeptide of sequence disclosed herein.Here, compare with the corresponding polypeptide of any known peptide sequence, unique subsequence is unique.
The present invention also is provided at the target nucleic acid of hybridizing with the oligonucleotides coding of uniqueness under the rigorous condition, this oligonucleotides coding is selected from subsequence unique in the O-RS polypeptide of sequence, wherein with any contrast polypeptide (for example, from it for example, parental generation sequence by sudden change acquisition synzyme of the present invention) corresponding polypeptide is compared, and unique subsequence is unique.Determine unique sequence as described above.
Sequence compares, homogeneity and homology
When adopting as one of following sequence comparison algorithm (or those skilled in the art can use other algorithm) or measure with relatively and when contrasting maximum consistance by visual inspection, the term " identical " in two or more nucleic acid or peptide sequence content or " homogeneity " percentage refer to that two or more are identical or have specified amino acid residues or the sequence of nucleotide same percentage or subsequence.
When measuring with relatively and when contrasting maximum consistance with sequence comparison algorithm or by visual inspection, at two kinds of nucleic acid or polypeptide (for example, the DNA of coding O-tRNA or O-RS, or the amino acid sequence of O-RS) term " basic identical " in the content refers to that two or more have at least about 60%, preferred 80%, the most preferably sequence or the subsequence of 90-95% nucleotide or amino acid residue homogeneity.Do not having to it is generally acknowledged that with reference under actual ancestors' the situation sequence of " basic identical " is " homology ".Preferably, on the sequence area of length, more preferably on zone, there is " basic homogeneity ", most preferably at least about 100 residues at least about 50 residues, surpassing at least about 150 residues, or basic identical above sequence on two full length sequences to be compared.
Determine for sequence comparison and homology, generally a sequence is used as reference sequence, compare with cycle tests.When using sequence comparison algorithm, will test and reference sequence input computing machine, specify the subsequence coordinate if necessary, specified sequence algorithm routine parameter.This sequence comparison algorithm calculates the sequence homogeneity percentage of cycle tests with respect to reference sequence according to the program parameter of appointment then.
Can pass through, for example, the local clustalw algorithm of Smith and Waterman, Adv.Appl.Math.2:482 (1981), the homology contrast algorithm of Needleman and Wunsch, J.Mol.Biol.48:443 (1970), the search similarity method of Pearson and Lipman, Proc.Nat ' l.Acad.Sci.USA 85:2444 (1988), (the GAP in the Wisconsin science of heredity software package is carried out in the computerize of these algorithms, BESTFIT, FASTA and TFASTA, science of heredity computer set (Genetics Computer Group), 575Science Dr., Madison, WI) or visual inspection (usually referring to, Ausubel etc., following) for relatively carrying out optimized sequence contrast.
One is suitable for determining that the examples of algorithms of sequence homogeneity percentage and sequence similarity is the BLAST algorithm, Altschul etc., and J.Mol.Biol.215:403-410 has described this algorithm in (1990).Can obtain carrying out the software that BLAST analyzes publicly by NCBI (www.ncbi.nlm.nih.gov/).This algorithm comprises, at first is tested and appraised sequence that the short word of length W in the search sequence identifies high score to (HSP), when with database sequence in during the word contrast of equal length, search sequence coupling or satisfy the threshold score T that some are just being estimated.T is called neighborhood word score threshold (Altschul etc., above-mentioned).These initial neighborhood word are hit (word hits) seeks the longer HSP that contains them as initial search seed.Word hits along the both direction of each sequence and extends then, up to increasing accumulation contrast mark.For nucleotide sequence, with parameter M (the right award mark of coupling residue; Always＞0) and N (the punishment mark of mispairing residue; Always＜0) calculate running summary of the points scored.For amino acid sequence, the sub matrix of using tricks calculates running summary of the points scored.When: when accumulation contrast mark descends by the parameter X from its maximum acquisition value; Because one or more negative score residue contrasts reach below zero or zero running summary of the points scored; Or when reaching each sequence terminal, the extension that word hits on all directions stops.BLAST algorithm parameter W, T and X determine the sensitivity and the speed of contrast.The default setting that BLASTN program (being used for nucleotide sequence) is used is the comparison of word length (W) 11, expectation value (E) 10, cutoff value 100, M=5, N=-4 and two chains.For amino acid sequence, the default setting that the BLASTP program is used is word length (W) 3, expectation value (E) 10 and BLOSUM62 score matrix (referring to Henikoff and Henikoff (1989) Proc.Natl.Acad.Sci.USA 89:10915).
Except sequence of calculation homogeneity percentage, the BLAST algorithm also to the similarity between two kinds of sequences carry out statistical study (referring to, for example, Karlin and Altschul, Proc.Nat ' 1.Acad.Sci.USA 90:5873-5787 (1993)).A kind of method of measuring similarity that the BLAST algorithm provides is minimum summation probability (P (N)), and it provides the probability explanation, the accidental coupling between following two kinds of nucleotide of this probability or the amino acid sequence.For example, if in the comparison of test nucleic acid and reference nucleic acid minimum summation probability less than about 0.1, be more preferably less than about 0.01, most preferably less than about 0.001, think that then nucleic acid is similar to reference sequence.
Mutagenesis and other Protocols in Molecular Biologies
The common textbook of describing Protocols in Molecular Biology comprises Berger and Kimmel, the molecule clone technology guide, " Enzymology method " the 152nd volume (Guide to Molecular Cloning Techniques, Methods inEnzymology) Academic Press, Inc., San Diego, CA (Berger); Sambrook etc., " molecular cloning-laboratory manual " (Molecular Cloning-A Laboratory Manual) (second edition), the 1-3 volume, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") and " newly organized molecular biology experiment guide " (Current Protocols in MolecularBiology), volumes such as F.M.Ausubel, Current Protocols, it is Greene PublishingAssociates, Inc. and John Wiley﹠amp; Sons, the co-partnership company of Inc., (1999 rise augment) (" Ausubel ")).These textbooks described the purposes, promoter of mutagenesis, carrier and much other with, produce relevant problem as gene, comprise be used to produce comprise alpha-non-natural amino acid, quadrature tRNA, quadrature synzyme and they to the selection codon of albumen.
The present invention uses various types of mutagenesis, for example, and to produce the tRNA library, to produce the synzyme library, with the selection codon insertion protein of interest or the polypeptide of the alpha-non-natural amino acid of will encoding.They include but not limited to site-directed mutagenesis, random point mutagenesis, homologous recombination, DNA reorganization or other recurrence method of mutagenesis, chimeric construct, with the mutagenesis that contains the uracil template, the mutagenesis of oligonucleotides-guiding, gapped duplex DNA mutagenesis etc. is used in the DNA mutagenesis of D2EHDTPA-modification, or their combination in any.Other suitable method comprises a mispairing reparation, usefulness repair-deficiency type host strain mutagenesis, restricted selection and restricted purifying, deletion mutagenesis, synthesizes mutagenesis, double-strand break reparation etc. by full gene.The present invention for example also comprises, comprises the mutagenesis of chimeric construct thing.In one embodiment, can instruct mutagenesis according to the Given information of natural generation molecule natural generation molecule or change or sudden change, for example, sequence, sequence comparison, physical property, crystal structure etc.
The foregoing of this paper and example have been described these steps.Can find additional information: Ling etc. in publication below and the incorporated by reference document, the DNA method of mutagenesis: summary (Approaches to DNA mutagenesis:an overview), Anal Biochem.254 (2): 157-178 (1997); Dale etc., carry out the random mutagenesis (Oligonucleotide-directed random mutagenesisusing the phosphorothioate method) of oligonucleotides-guiding, Methods Mol.Biol.57:369-374 (1996) with the D2EHDTPA method; Smith, mutagenesis in vitro (In vitro mutagenesis), Ann.Rev.Genet.19:423-462 (1985); Botstein and Shortle, the strategy of mutagenesis in vitro and application (Strategies and applications of invitro mutagenesis), Science 229:1193-1201 (1985); Carter, site-directed mutagenesis (Site-directed mutagenesis), Biochem.J.237:1-7 (1986); Kunkel, the efficiency of inducing mutation (The efficiency of oligonucleotide directed mutagenesis) of oligonucleotides guiding is published in " nucleic acid and molecular biology " (Nucleic Acids﹠amp; Molecular Biology) (D.M.J. compiles for Eckstein, F. and Lilley, Springer Verlag, Berlin)) (1987); Kunkel, need not the quick and effective site-directed mutagenesis (Rapid and efficient site-specific mutagenesiswithout phenotypic selection) of Phenotypic Selection, Proc.Natl.Acad.Sci.USA 82:488-492 (1985); Kunkel etc. need not the quick and effective site-directed mutagenesis (Rapid and efficientsite-specific mutagenesis without phenotypic selection) of Phenotypic Selection, Methods in Enzymol.154,367-382 (1987); Bass etc. have the mutation T rp mortifier (Mutant Trprepressors with new DNA-binding specificities) of new DNA-binding specificity, Science 242:240-245 (1988); Methods in Enzymo.100:468-500 (1983); Methods in Enzymol.154:329-350 (1987); Zoller and Smith, the carrier of deriving with M13 carries out the mutagenesis of oligonucleotides-guiding: produce the effective and universal method (Oligonucleotide-directed mutagenesisusing M13-derived vectors:an efficient and general procedure for theproduction of point mutations in any DNA fragment) of point mutation in any dna fragmentation, Nucleic Acids Res.10:6487-6500 (1982); Zoller and Smith, be cloned into the mutagenesis (Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors) of the oligonucleotides-guiding of dna fragmentation in the M13 carrier, Methods in Enzymol.100:468-500 (1983); Zoller and Smith, the mutagenesis of oligonucleotides-guiding: use the straightforward procedure (Oligonucleotide-directed mutagenesis:a simple method using twooligonucleotide primers and a single-stranded DNA template) of two kinds of Oligonucleolide primers and single stranded DNA template, Methods inEnzymol.154:329-350 (1987); Taylor etc., the purposes (The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA) of DNA in Restriction Enzyme prepared in reaction breach DNA that D2EHDTPA is modified, Nucl.Acids Res.13:8749-8764 (1985); Taylor etc., the sudden change (The rapidgeneration of oligonucleotide-directed mutations at high frequency usingphosphorothioate-modified DNA) that the DNA high-frequency of modifying with D2EHDTPA produces oligonucleotides-guiding fast, Nucl.Acids Res.13:8765-8787 (1985); Nakamaye and Eckstein, the D2EHDTPA group suppresses restriction endonuclease Nci I cutting and the application in the mutagenesis of oligonucleotides-guiding (Inhibition of restriction endonuclease Nci Icleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis) thereof, Nucl.Acids Res.14:9679-9698 (1986); Sayers etc., based on the Y-T exonuclease (Y-TExonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis) in the mutagenesis of the oligonucleotides-guiding of D2EHDTPA, Nucl.Acids Res.16:791-802 (1988); Sayers etc., by in the presence of ethidium bromide, containing the DNA (Strandspecific cleavage of phosphorothioate-containing DNA by reaction withrestriction endonucleases in the presence of ethidium bromide) of D2EHDTPA, (1988) Nucl.Acids Res.16:803-814 with the cutting of restriction endonuclease reaction chain specificity; Kramer etc., make up the gapped duplex DNA method (The gapped duplex DNA approach to oligonucleotide-directed mutationconstruction) of the sudden change of oligonucleotides-guiding, Nucl.Acids Res.12:9441-9456 (1984); Kramer and Fritz are by the structure sudden change (Oligonucleotide-directedconstruction of mutations via gapped duplex DNA) of gapped duplex DNA oligonucleotides-guiding, Methods in Enzymol.154:350-367 (1987); Kramer etc., the improved enzymatic vitro reactions of gapped duplex DNA method (Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction of mutations) that is used for the structure sudden change of oligonucleotides-guiding, Nucl.Acids Res.16:7207 (1988); Fritz etc., the structure sudden change of oligonucleotides-guiding: need not the gapped duplex DNA method (Oligonucleotide-directed construction ofmutations:a gapped duplex DNA procedure without ezymatic reactions invitro) of enzymatic vitro reactions, Nucl.Acids Res.16:6987-6999 (1988); Kramer etc., some mispairing reparation (PointMismatch Repair), Cell 38:879-887 (1984); Carter etc. improve oligonucleotides site-directed mutagenesis (Improved oligonucleotide site-directed mutagenesis using M13vectors), Nucl.Acids Res.13:4431-4443 (1985) with the M13 carrier; Carter is with the mutagenesis (Improved oligonucleotide-directed mutagenesis using M13vectors) of M13 carrier improvement oligonucleotides-guiding, Methods in Enzymol.154:382-403 (1987); Eghtedarzadeh and Henikoff, oligonucleotides are used to produce big disappearance (Use of oligonucleotides to generate largedeletions), Nucl.Acids Res.14:5115 (1986); Wells etc., the importance (Importance of hydrogen-bond formation instabilizing the transition state of subtilisin) that hydrogen bond forms in stablizing the transition state of subtilopeptidase A, Phil.Trans.R.Soc.Lond.A 317:415-423 (1986); Nambiar etc., the synthetic entirely and clone (Total synthesis and cloning of a gene coding for the ribonuclease S protein) of the gene of coding ribonuclease S albumen, Science 223:1299-1301 (1984); Sakamar and Khorana, ox bacillus outer segments guanylic acid-in conjunction with the gene of the α-subunit of albumen (transducin) is synthetic entirely and express (Total synthesis andexpression of a gene for the α-subunit of bovine rod outer segment guaninenucleotide-binding protein (transducin)), Nucl.Acids Res.14:6361-6372 (1988); Wells etc., cassette mutagenesis: limiting the effective ways (Cassettemutagenesis:an efficient method for generation of multiple mutations atdefined sites) that the site produces multimutation, Gene 34:315-323 (1985); Grundstr  m etc., by the synthetic mutagenesis (Oligonucleotide-directed mutagenesis bymicroscale ' shot-gun ' gene synthesis) of oligonucleotides-guiding, the Nucl.Acids Res.13:3305-3316 (1985) of carrying out of ' shotgun ' gene of trace; Mandecki, the double-strand break reparation of oligonucleotides-guiding in the escherichia coli plasmid: site-directed mutagenesis method (Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli:a method for site-specific mutagenesis), Proc.Natl.Acad.Sci.USA, 83:7177-7181 (1986); Arnold is used for the protein engineering (Proteinengineering for unusual environments) of unusual environment, Current Opinion in Biotechnology 4:450-455 (1993); Sieber etc., Nature Biotechnology, 19:456-460 (2001) .W.P.C.Stemmer, Nature 370,389-91 (1994); And I.A.Lorimer, I.Pastan, Nucleic AcidsRes.23,3067-8 (1995).Other details of above-mentioned a lot of methods can be referring to Methods in Enzymology the 154th volume, and it has also described useful measure, to solve the failure problems that occurs in the various method of mutagenesis.
The present invention also relates to eukaryotic host cell and biology, be used for by quadrature tRNA/RS mixing alpha-non-natural amino acid in the body.With polynucleotide of the present invention or comprise the construction of polynucleotide of the present invention, carrier for example of the present invention can be, as (for example, conversion, transduction or transfection) host cell of cloning vector or expression vector genetic modification.Carrier can be, as plasmid, bacterium, virus, exposed polynucleotide or the form of conjugation polynucleotide.By standard method, comprise electroporation (From etc., Proc.Natl.Acad.Sci.USA 82,5824 (1985)), viral vector infection, in the matrix of globule or particle or on the surface by having the granule high speed trajectory infiltration (Klein etc. of nucleic acid, Nature 327,70-73 (1987)) carrier is introduced cell and/or microorganism.
Can cultivate the host cell of transforming in the conventional nutrient medium of revising as screening step, activate promoter or selecting the activity of transformant for being applicable to.These cells can randomly be cultivated into genetically modified organism.Other is used for, the useful list of references of for example cell separation and cultivation (as follow-up separate nucleic acid) comprises Freshney (1994) " animal cell culture, basic fundamental handbook (Culture of Animal Cells, a Manual of BasicTechnique), the third edition, Wiley-Liss, New York and the list of references of quoting thereof; Payne etc. (1992) " in liquid system, cultivating vegetable cell and tissue " (Plant Cell and Tissue Culture in LiquidSystems) John Wiley﹠amp; Sons, Inc.New York, NY; Gamborg and Phillips (volume) (1995) " vegetable cell, tissue and organ culture " (Plant Cell, Tissue and Organ Culture); " basic skills Springer Verlag laboratory manual " (Fundamental Methods Springer Lab Manual), Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (volume) " microbiological culture media handbook (The Handbook of Microbiological Media) (1993) CRC Press, BocaRaton, FL.
Several well-known process of target nucleic acid being introduced cell are available, and wherein any one all can be used for the present invention.These methods comprise: will contain the bacterium bioplast of DNA and recipient cell fusion, electroporation, projectile bombardment and with viral vector infection (following further discussion) etc.The number of the plasmid that contains DNA construction of the present invention can be used to bacterial cell to increase.Bacterial growth is to logarithmic phase, can by the plasmid in the whole bag of tricks separation of bacterial known in the art (referring to, for example, Sambrook).In addition, can buy from market the kit that much is used for from the bacterium plasmid purification (referring to, for example, the EasyPrep of Pharmacia Biotech TM, FlexiPrep TMThe StrataClean of Stratagene TMQIAprep with Qiagen TM).Further handle the plasmid of separation and purifying then,, be used for transfectional cell or mix relevant carriers with the infection biological body to produce other plasmid.Typical carriers comprises and is used to regulate transcribing with translation termination, transcribing and translation initiation sequence of concrete target nucleic acid expression, and promoter.Carrier randomly comprises and contains at least one independent terminator sequence, allows this box in eucaryote or prokaryotes or the sequence (as shuttle vector) of duplicating in the two be used for protokaryon and the common expression cassette of the selected marker of eukaryotic system.Carrier is suitable for prokaryotes, eucaryote or preferably duplicates and integrate in the two.Referring to, Giliman and Smith, Gene8:81 (1979); Roberts, etc., Nature, 328:731 (1987); Schneider, B., etc., ProteinExpr.Purif.6435:10 (1995); Ausubel, Sambrook, Berger (above-mentioned).For example ATCC publishes as (volume) ATCC such as " ATCC bacterium and bacteriophage catalogue " (The ATCC Catalogue of Bacteria and Bacteriophage) (1992) Gherna, and the bacterium and the bacteriophage catalogue that are used to clone are provided.Be used to check order, the additional basic skills of clone and molecular biology others and basic theory be according to also referring to (1992) " recombinant DNA " (Recombinant DNA) second edition Scientific American Books such as Watson, NY.In addition, basically customization of can be from various commercial source any family or standard are ordered any nucleic acid (nucleic acid of mark in fact arbitrarily, standard or non-standard no matter), as Midland Certified Reagent Company (Midland, TXmcrc.com), The Great American Gene Company (Ramona, CA can login WWW genco.com), ExpressGen Inc. (Chicago, IL, can login WWW expressgen.com), OperonTechnologies Inc. (Alameda, CA) and a lot of other companies.
Kit
Kit also is a feature of the present invention.For example, the kit of producing the albumen that contains at least one alpha-non-natural amino acid in cell is provided, wherein this kit comprises polynucleotide sequence and/or the O-tRNA that contains the O-tRNA that encodes, and/or the polynucleotide sequence of coding O-RS and/or the container of O-RS.In one embodiment, this kit also comprises at least a alpha-non-natural amino acid.In another embodiment, this kit also comprises the illustrative material of producing albumen.
Embodiment
It is in order to illustrate that the following examples are provided, but not the present invention of requirement for restriction protection.Those skilled in the art will recognize that, can under the situation that does not deviate from the claimed scope of the invention, change various non-important parameters.
Embodiment 1: in eukaryotic, mix alpha-non-natural amino acid aminoacyl-tRNA synthetase production method and Composition
The expansion the eukaryotic genetic code provides strong instrument to comprise the alpha-non-natural amino acid with new physics, chemistry or biological property for analyze and control protein function in these cells.For this purpose, described and in saccharomyces cerevisiae (S.cerevisiae), be used for separating the aminoacyl-tRNA synthetase that with high fidelity alpha-non-natural amino acid is mixed albumen in response to amber codon.This method activates the reactive reporter HIS3 of GAL4, URA3 or LacZ based on by suppress amber codon between the DNA of GAL4 is in conjunction with territory and transcription activating domain.The optimization of the GAL4 reporter that is used for just selecting active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS) variant has been described.Also developed the negative selection of carrying out inactivation EcTyrRS variant with the URA3 reporter, used to add the micromolecule (5-fluororotic acid (5-FOA)) of growth medium as ' poisonous allele '.Importantly, can on unicellular and carry out positive and negative with the severity of certain limit and select.This can help to separate aminoacyl-tRNA synthetase (aaRS) activity of certain limit from big mutant synzyme library.Model Selection has proved that this method is used to separate the effect of required aaRS phenotype.
Recently, alpha-non-natural amino acid is added provide in the genetic code of Escherichia coli (E.coli) and analyze in vitro and in vivo and the effective new tool of operon protein 26S Proteasome Structure and Function.With efficient and the fidelity that is equal to mutually with common 20 seed amino acids, will have the amino acid of photoaffinity labeling, heavy atom, ketone and alkylene and chromophore and mix protein in the Escherichia coli.Referring to, for example, Chin, Deng, (2002), photocrosslinking agent is joined (Addition of a Photocrosslinker to the Genetic Code ofEscherichia coli) in the colibacillary genetic code, Proc.Natl.Acad.Sci.U.S.A.99:11020-11024; Chin and Schultz, (2002) carry out photo-crosslinking (In vivo Photocrosslinkingwith Unnatural Amino Acid Mutagenesis), ChemBioChem 11:1135-1137 with alpha-non-natural amino acid mutagenesis in the body; Chin etc., (2002), right-azido-L-phenylalanine is added (Addition ofp-Azido-L-phenylalanine to the Genetic code of Escherichia coli) in the colibacillary genetic code, J.Am.Chem.Soc.124:9026-9027; Zhang etc., (2002), the alkene selectivity is mixed albumen (Theselective incorporation of alkenes into proteins in Escherichia coli) in the Escherichia coli, Angew.Chem.Int.Ed.Engl.41:2840-2842; And Wang and Schultz, (2002), expansion genetic code (Expanding the Genetic Code), Chem.Comm.1-10.
In the past; the tetrahymena thermophila tRNA by the wrong acidylate of microinjection chemistry (for example; M.E.Saks; Deng (1996); be used for suppressing and to mix the engineering tetrahymena tRNAGln (An engineered Tetrahymena tRNAGln in vivo incorporation ofunnatural amino acids into proteins by nonsense suppression) of protein in the alpha-non-natural amino acid body by nonsense; J.Biol.Chem.271:23169-23175) with relevant mRNA; alpha-non-natural amino acid (is for example introduced in the nicotinic acetylcholine receptor in the xenopus leavis oocytes; M.W.Nowak; Deng (1998); Method Enzymol.293:504-529 will be mixed in the ion channel of xenopus leavis oocytes expression system (In vivo incorporation of unnatural amino acids into ionchannels in Xenopus oocyte expression system)) in the alpha-non-natural amino acid body.This amino acid that has allowed to contain the side chain of tool unique physical or chemical property by introducing carries out detailed biophysical studies to the acceptor in the egg mother cell.Referring to, for example, D.A.Dougherty (2000) is as the alpha-non-natural amino acid (Unnatural amino acids as probes of protein structureand function) of protein structure and function probe, Curr.Opin.Chem.Biol.4:645-652.Unfortunately, this method only limits to the protein in can the cell of microinjection because tRNA external by chemical acylation, can not be by acidylate again, so the albumen productive rate is very low.This needs to measure the sensitive technology of protein function conversely.
In eukaryotic,, alpha-non-natural amino acid heredity is mixed the interest that has caused everybody in the protein in response to amber codon.Also referring to, H.J.Drabkin etc., (1996), amber in the mammalian cell suppresses to depend on Escherichia coli aminoacyl-tRNA synthetase expression of gene (Amber suppression in mammalian cellsdependent upon expression of an Escherichia coli aminoacyl-tRNA synthetasegene), Molecular﹠amp; Cellular Biology 16:907-913; A.K.Kowal, Deng, (2001), the 21 aminoacyl-tRNA synthetase-inhibition type tRNA is to mixing the amino acid analogue locus specificity the possible purposes (Twenty-first aminoacyl-tRNAsynthetase-suppressor tRNA pairs for possible use in site-specificincorporation of amino acid analogues into proteins in eukaryotes and ineubacteria) in the albumen in eucaryote and eubacteria, [comment], Proc.Natl.Acad.Sci.U.S.A.98:2268-2273; And K.Sakamoto, Deng, (2002), in mammalian cell, the alpha-non-natural amino acid locus specificity mixed (Site-specific incorporation of an unnatural amino acid into proteins inmammalian cells) in the protein, Nucleic Acids Res.30:4692-4699.This will have significant technology and real advantage, because the related synzyme by tRNA will be to its acidylate-cause producing mass mutation albumen again.And the aminoacyl-tRNA synthetase of genetic coding and tRNA are heritable in principle, allow alpha-non-natural amino acid to mix in the albumen by a lot of cell divisions, and do not have the index dilution.
Described with amino acid join essential step in the colibacillary genetic code (referring to, for example, D.R.Liu and P.G.Schultz, (1999), progress (Progresstoward the evolution of an organism with an expanded genetic code) with biological evolution of expansion genetic code, Proc.Natl.Acad.Sci.U.S.A.96:4780-4785; Similar principles can be used for expanding Eukaryotic genetic code.The first step is identified quadrature aminoacyl-tRNA synthetase (aaRS)/tRNA CUARight.This works to needs and host cell machine translator one, but aaRS should not make any endogenous tRNAs have amino acid, tRNA CUAShould be by any endogenous synzyme aminoacylation.Referring to; for example; D.R.Liu; Deng; be designed for the tRNA and the aminoacyl-tRNA synthetase (Engineering atRNA and aminoacyl-tRNA synthetase for the site-specific incorporation ofunnatural amino acids into proteins in vivo) that in vivo the alpha-non-natural amino acid locus specificity are mixed in the protein, Proc.Natl.Acad.Sci.U.S.A.94:10092-10097.In second step, only the aaRS/tRNA with alpha-non-natural amino acid is right to select those from mutant aaRS library.In Escherichia coli, utilize the variant of MjTyrRS to select alpha-non-natural amino acid to select to carry out by adopting for two steps ' two sieve '.Referring to, for example, D.R.Liu and P.G.Schultz, (1999), progress (Progress toward the evolution of an organism withan expanded genetic code) with biological evolution of expansion genetic code, Proc.Natl.Acad.Sci.U.S.A.96:4780-4785.In eukaryotic, use the system of selection of modifying.
(S.cerevisiae) is elected to be the eucaryon host biology with saccharomyces cerevisiae, because it is unicellular, has generation time fast, and characterized the science of heredity feature relatively well.Referring to, for example, D.Burke, etc., (2000) " yeast genetics method " (Methods in Yeast Genetics), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY.And, because Eukaryotic machine translator be high conservative (referring to, for example, (1996) " translation control " (Translational Control), Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY; Y.Kwok and J.T.Wong, (1980), determine evolutionary relationship (Evolutionary relationship between Halobacterium cutirubrum and eukaryotesdetermined by use of aminoacyl-tRNA synthetases as phylogenetic probes) between red skin salt bacillus and the eucaryote, Canadian Journal of Biochemistry 58:213-218 with aminoacyl-tRNA synthetase as the systematic growth probe; (2001) " ribosomes " (TheRibosome), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), probably, being found in the aaRS gene that saccharomyces cerevisiae is used for mixing alpha-non-natural amino acid can be by ' cutting and paste ' to senior eucaryote, use with cognate tRNA s cooperation (referring to, for example, K.Sakamoto, Deng, (2002) in mammalian cell, the alpha-non-natural amino acid locus specificity mixed (Site-specific incorporation of anunnatural amino acid into proteins in mammalian cells) in the protein, Nucleic Acids Res.30:4692-4699; And C.Kohrer, Deng, (2001), amber and ochre inhibition type tRNAs are imported mammalian cell: with the universal method (Import of amber andochre suppressor tRNAs into mammalian cells:a general approach tosite-specific insertion of amino acid analogues into proteins) in the amino acid analogue locus specificity insertion protein, Proc.Natl.Acad.Sci.U.S.A.98:1431 (14315) is to mix alpha-non-natural amino acid.Therefore, the expansion of saccharomyces cerevisiae genetic code is the approach of the Eukaryotic genetic code of the complicated many cells of expansion.Referring to, for example, M.Buvoli, Deng, (2000), inhibition type tRNA gene inhibition nonsense mutation (Suppression of nonsense mutations in cell culture and mice by multimerizedsuppressor tRNA genes) by polymerization in cellular incubation and mouse, Molecular﹠amp; Cellular Biology 20:3116-3124.Methanococcus jannaschii TyrRS (the MjTyrRS)/tRNA that is used to expand the Escherichia coli genetic code before deriving from is (referring to for example, L.Wang and P.G.Schultz, (2002), expansion genetic code (Expanding the Genetic Code), Chem.Comm.1-10) tyrosyl in eucaryote be not quadrature (for example, P.Fechter, Deng, (2001), Methanococcus jannaschii and main tyrosine homogeneity determinant among the saccharomyces cerevisiae tRNA (Tyr) be guard but express different (Major tyrosine identity deterninants in Methanococcus jannaschiiand Saccharomyces cerevisiae tRNA (Tyr) are conserved but expresseddifferently), Eur.J.Biochem.268:761-767), need new quadrature to the expansion the eukaryotic genetic code.Schimmel and colleague point out, in saccharomyces cerevisiae, Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA CUATo suppressing amber codon; And in yeast cells colloidal sol, the endogenous aminoacyl tRNA synthetase is not loaded with Escherichia coli tRNA CUA(Fig. 2).Also referring to, for example, H.Edwards, Deng, (1991), Escherichia coli tyrosine transfer RNA is leucine-transspecific RNA (An Escherichia colityrosine transfer RNA is a leucine-specific transfer RNA in the yeastSaccharomyces cerevisiae) in saccharomyces cerevisiae, Proc.Natl.Acad.Sci.U.S.A.88:1153-1156; And H.Edwards and P.Schimmel (1990), bacterium amber in the bacterium aminoacyl-tRNA synthetase selectivity identification saccharomyces cerevisiae suppresses son (A bacterial amber suppressor in Saccharomyces cerevisiaeis selectively recognized by a bacterial aminoacyl-tRNA synthetase), Molecular﹠amp; Cellular Biology 10:1633-1641.In addition, EcTyrRS has shown not at the external yeast tRNA that is loaded with.Referring to, for example, Y.Kwok and J.T.Wong, (1980), determine evolutionary relationship (Evolutionaryrelationship between Halobacterium cutirubrum and eukaryotes determined byuse of aminoacyl-tRNA synthetases as phylogenetic probes) between red skin salt bacillus and the eucaryote, Canadian Journalof Biochemistry 58:213-218 with aminoacyl-tRNA synthetase as the systematic growth probe; B.P.Doctor, Deng, (1966), the species specific research of yeast and Escherichia coli tyrosine tRNA (Studies on the species specificity of yeast and E.colityrosine tRNAs), Cold Spring HarborSymp.Quant.Biol.31:543-548; And K.Wakasugi; Deng; (1998); genetic code in the evolution: with species specificity aminoacylation and peptide graft exchange (Genetic codein evolution:switching species-specific aminoacylation with a peptidetransplant), EMBO Journal 17:297-305.Therefore, EcTyrRS/tRNA CUATo being that the right material standed for of quadrature is (for example in saccharomyces cerevisiae and the senior eucaryote; A.K.Kowal; Deng; (2001); the 21 aminoacyl-tRNA synthetase-inhibition type tRNA is to mixing the amino acid analogue locus specificity the possible purposes (Twenty-first aminoacyl-tRNA synthetase-suppressor tRNApairs for possible use in site-specific incorporation of amino acid analoguesinto proteins in eukaryotes and in eubacteria) in the albumen in eucaryote and eubacteria; [comment], Proc.Natl.Acad.Sci.U.S.A.98 (2001) 2268-2273).
In order to expand the substrate specificity of EcTyrRS in the Escherichia coli, Nishimura and colleagues have screened the EcTyrRS mutant library of the PCR generation that easily makes mistakes, the mutant of having found to have the improved 3-of mixing nitrogen tyrosine ability.Referring to, for example, F.Hamano-Takaku, Deng, (2000), the mutant Escherichia coli tyrosyl-t RNA synthetase utilizes alpha-non-natural amino acid nitrogen tyrosine Billy with tyrosine more effective (A mutant Escherichia colityrosyl tRNA synthetase utilizes the unnatural amino acid azatyrosine moreefficiently than tyrosine), J.Biol.Chem.275:40324-40328.Yet this amino acid mixes in the whole colibacillary protein group, and the still preferred tyrosine of the enzyme of generation is as substrate.Yokoyama and colleagues have screened the EcTyrRS avtive spot variant of sub-fraction design in the wheat germ translation system, found to utilize 3-iodotyrosine Billy to use the more effective EcTyrRS variant of tyrosine.Referring to, D.Kiga, Deng, (2002), in eukaryotic translation, the alpha-non-natural amino acid locus specificity mixed engineering colon bacillus tyrosyl-tRNA synthetase and the application in wheat germ cell-free system (An engineered Escherichia colityrosyl-tRNA synthetase for site-specific incorporation of an unnaturalamino acid into proteins in eukaryotic translation and its application ina wheat germ cell-free system) thereof in the albumen, Proc.Natl.Sci.U.S.A.99:9715-9720.The enzyme of developing in Escherichia coli with us is opposite (for example, J.W.Chin, Deng, (2002), photocrosslinking agent is joined (Addition of a Photocrosslinker to the Genetic Code ofEscherichia coli) in the colibacillary genetic code, Proc.Natl.Acad.Sci.U.S.99:11020-11024; J.W.Chin, Deng, (2002), right-azido-L-phenylalanine is added (Addition ofp-Azido-L-phenylalanine to the Genetic code of Escherichia coli) in the colibacillary genetic code, J.Am.Chem.Soc.124:9026-9027; L.Wang, etc., (2001) expand colibacillary genetic code (Expanding theGenetic Code of Escherichia coli), Science 292:498-500; And L.Wang, Deng, (2002), L-3-(2-naphthyl) alanine is added (AddingL-3-(2-naphthyl) alanine to the genetic code of E-coli) in the colibacillary genetic code, J.Am.Chem.Soc.124:1836-1837), this enzyme is not having still to mix tyrosine under the situation of alpha-non-natural amino acid.Referring to, for example, D.Kiga etc., (2002), in eukaryotic translation, the alpha-non-natural amino acid locus specificity mixed engineering colon bacillus tyrosyl-tRNA synthetase and the application in wheat germ cell-free system (An engineeredEscherichia coli tyrosyl-tRNA synthetase for site-specific incorporation ofan unnatural amino acid into proteins in eukaryotic translation and itsapplication in a wheat germ cell free system) thereof in the albumen, Proc.Natl.Acad.Sci.U.S.A.99:9715-9720.Recently, Yokoyama and colleagues also prove in mammalian cell this EcTyrRS mutant and tRNA from bacillus stearothermophilus CUAOne works gives to suppress amber codon.Referring to, K.Sakamoto, etc., mix the alpha-non-natural amino acid locus specificity in the albumen in mammalian cell (2002), Nucleic AcidsRes.30:4692-4699.
Require the amino acid of any adding eucaryon genetic code to mix with the fidelity that is similar to 20 common seed amino acids.In order to finish this purpose, in saccharomyces cerevisiae, work in response to amber codon TAG with discovery with system of selection in the common body, mix alpha-non-natural amino acid but not the EcTyrRS/tRNA of common amino acid CUAVariant.The major advantage of selecting is can be from 10 8Select rapidly in the EcTyrRS avtive spot variant library and the enrichment selectivity is mixed the enzyme of alpha-non-natural amino acid, this diversity than in-vitro screening manys 6-7 the order of magnitude.Referring to, for example, D.Kiga, Deng, (2002), in eukaryotic translation, the alpha-non-natural amino acid locus specificity mixed engineering colon bacillus tyrosyl-tRNA synthetase and the application in wheat germ cell-free system (An engineered Escherichia colityrosyl-tRNA synthetase for site-specific incorporation of an unnaturalamino acid into proteins in eukaryotic translation and its application ina wheat germ cell-free system) thereof in the albumen, Proc.Natl.Acad.Sci.U.S.A.99:9715-9720.This multifarious increase has improved the possibility of separating the EcTyrRS variant greatly, and variant is used for mixing with very high fidelity the useful functionality of different range.Referring to, for example, L.Wang and P.G.Schultz, (2002), expansion genetic code (Expanding the Genetic Code), Chem.Comm.1-10.
In order to promote the system of selection of saccharomyces cerevisiae, transcription activating protein, GAL4 (referring to Fig. 1) have been used.Referring to, for example, A.Laughon, Deng, (1984), identify two kinds of albumen (Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4gene), Molecular﹠amp by saccharomyces cerevisiae GAL4 gene code; Cellular Biology 4:268-275; A.Laughon and R.F.Gesteland, (1984), the primary structure of saccharomyces cerevisiae GAL4 gene (Primary structure of the Saccharomycescerevisiae GAL4 gene), Molecular﹠amp; Cellular Biology 4:260-267; L.Keegan, Deng, (1986), regulate the transcribing of albumen-mobilizing function DNA isolation in conjunction with (Separation of DNAbinding from the transcription-activating function of a eukaryoticregulatory protein), Science 231:699-704 from eucaryon; And M.Ptashne, (1988), the eukaryotic transcription activator is (the How eukaryotic transcriptional activators work) how to work, Nature335:683-689.These 881 terminal 147 amino acid of amino acid whose albumen N-form DNA in conjunction with territory (DBD), and it combines specifically with dna sequence dna.Referring to, for example, M.Carey, etc., (1989), the amino-terminal fragment of GAL4 and DNA are combined into dimer (An amino-terminal fragment of GAL4binds DNA as adimer), J.Mol.Biol.209:423-432; And E.Giniger, etc., (1985), GAL4, the positive specific DNA of albumen of regulating of a primary yeast is in conjunction with (Specific DNA binding of GAL4, a positiveregulatory protein of yeast), Cell 40:767-774.By interleaving protein sequence DBD is connected to terminal 113 the amino acid activation territories (AD) of C-, but when this activation domain combines with DNA activated transcription.Referring to, for example, J.Ma and M.Ptashne, (1987), the deletion analysis of GAL4 defines two kinds of transcriptional activation sections (Deletion analysis of GAL4 defines two transcriptional activating segments), Cell 48:847-853: and J.Ma and M.Ptashne, (1987), 30 amino acid (The carboxy-terminal 30amino acids of GAL4are recognized byGAL80) of GAL80 identification GAL4 carboxyl-end, Cell 50:137-142.We imagine, by amber codon being placed the N-end DBD place near the single polypeptide of terminal DBD of the N-that contains GAL4 and the terminal AD of its C-, pass through EcTyrRS/tRNA CUARight amber suppresses to be connected with the transcriptional activation by GAL4 (Fig. 1, A group).By the reporter of selecting suitable GAL4 to activate, positive and negative selects to carry out with this gene (Fig. 1, B group).Though much can be used for just selecting (as URA3 based on the amino acid nutrient deficiency reporter that replenishes cell, LEU2, HISS, LYS2), but the HISS gene is attracting reporter, because can regulate the activity (imidzoleglycerol phosphate dehydrogenase) of its encoded protein in dosage dependence mode by adding 3-aminotriazole(ATA) (3-AT).Referring to, for example, G.M.Kishore and D.M.Shah, (1988), as the amino acid bio synthetic inhibitor (Amino acid biosynthesisinhibitors as herbicides) of herbicide, Annual Review of Biochemistry 57:627-663.In saccharomyces cerevisiae, less gene has been used for negative the selection.One of several negative selection strategies that success is used (referring to, for example, A.J.DeMaggio, etc., (2000), yeast division-crossing system (The yeast split-hybridsystem), Method Enzymol.328:128-137; H.M.Shih, Deng, (1996), positive heredity selects to destroy protein-protein interaction: identify to stop CREB sudden change (the A positivegenetic selection for disrupting protein-protein interactions: identification of CREB mutations that combines with coactivator CBP, that prevent association with thecoactivator CBP), Proc.Natl.Acad.Sci.U.S.A.93:13896-13901; M.Vidal, Deng, (1996), characterize the hereditary feature (Genetic characterization of a mammalian protein-protein interactiondomain by using a yeast reverse two-hybrid system) in mammalian proteins-protein-interacting territory with the reverse two-hybrid system heredity of yeast, [comment], Proc.Natl.Acad.Sci.U.S.A.93:10321-10326; And M.Vidal, Deng, (1996), oppositely double cross and single crosses system detection protein-protein dissociates and DNA-protein-interacting (Reverse two-hybrid and one-hybridsystems to detect dissociation of protein-protein and DNA-proteininteractions), [comment], Proc.Natl.Acad.Sci.U.S.A.93:10315-10320) develop negative (for example the selection of URA3/5-fluororotic acid (5-FOA) of describing in ' oppositely double cross ' system at Vidal and colleagues, J.D.Boeke, Deng, (1984), in yeast, just selecting to lack Orotidine-5 '-'-mutant of phosphate decarboxylase activity: 5-fluororotic acid resistance (A positive selection for mutants lackingorotidine-5 '-phosphate decarboxylase activity in yeast:5-fluoroorotic acidresistance), Molecular﹠amp; General Genetics 197:345-346) system.Referring to, M.Vidal, Deng, (1996), characterize mammalian proteins-protein-interacting territory (Genetic characterizationof a mammalian protein-protein interaction domainby using a yeast reverse two-hybrid system) with the reverse two-hybrid system heredity of yeast, [comment], Proc.Natl.Acad.Sci.U.S.A.93:10321-10326; And M.Vidal, Deng, (1996), oppositely double cross and single crosses system detection protein-protein dissociates and DNA-protein-interacting (Reverse two-hybrid and one-hybrid systems todetect dissociation of protein-protein and DNA-protein interactions), [comment], Proc.Natl.Acad.Sci.U.S.93:10315-10320).In reverse two-hybrid system, the URA3 reporter of genome conformity is placed under the promoter of strict control, this promoter contains the GAL4DNA binding site.When interactional two kinds of albumen and GAL4DBD and GAL4AD generation fusion, they have just rebuild the activity of GAL4, and activate transcribing of URA3.In the presence of 5-FOA, the URA3 gene outcome changes into toxic products with 5-FOA, cell killing.Referring to, J.D.Boeke, etc., above-mentioned.This selection is used to select to destroy the albumen of protein-protein interaction and the sudden change that selection destroys protein-protein interaction.The variant of the micromolecular inhibitor that is used to screen protein-protein interaction has also been described.Referring to, for example, J.Huang and S.L.Schreiber, (1997) be used for selecting the yeast genetic system (A yeastgenetic system for selecting small molecule inhibitors of protein-proteininteractions in nanodroplets) of the micromolecular inhibitor of protein-protein interaction, Proc.Natl.Acad.Sci.U.S.A.94:13396-13401. at nano-liquid droplet
The suitable selection of amber codon allows effectively just selecting active EcTyrRS variant with the reporter that HIS3 or URA3GAL4 activate in total length GAL4, with supplementation group propylhomoserin or uracil auxotrophy in yeast cells.And the URA3 reporter can be used for the negative inactivation EcTyrRS variant of selecting in the presence of 5-FOA.In addition, can will use the colorimetric estimation of lacZ to be used for reading yeast cells aminoacyl-tRNA synthetase activity.
Result and discussion
Under the control of composing type ADH1 promoter, express the EcTyrRS gene, from identical height copy yeast plasmid (pEcTyrRStRNA CUA, Fig. 1, C group) and the middle tRNA that expresses CUAGene.At pEcTyrRStRNA CUADuring with the cotransformation of low copy reporter, this report gene contains single amber mutation between the DNA of the GAL4 construction that embeds MaV203 is in conjunction with territory and activation domain, cell is lacking histidine and containing growth (Fig. 2) on the selective medium of 10-20mM 3-AT.When the MaV203 cell transfecting identical GAL4 construction and inactivation synthase mutant (A5) or when lacking the construction of EctRNA gene, on 10mM 3-AT, do not observe growth (Fig. 2).These experiments determine that EcTyrRS can be from the ADH1 promoter with the functional form constitutive expression, in MaV203, have minimum endogenous amber to suppress and in this system the yeast synzyme almost be not loaded with EctRNA CUAReferring to, for example, H.Edwards, Deng, (1991), Escherichia coli tyrosine transfer RNA is leucine-transspecific RNA (AnEscherichia coli tyrosine transfer RNA is a leucine-specific transfer RNAin the yeast Saccharomyces cerevisiae) in saccharomyces cerevisiae, Proc.Natl.Acad.Sci.U.S.A.88:1153-1156; And H.Edwards and P.Schimmel, (1990), bacterium amber in the bacterium aminoacyl-tRNA synthetase selectivity identification saccharomyces cerevisiae suppresses son (A bacterial amber suppressor inSaccharomyces cerevisiae is selectively recognized by a bacterialaminoacyl-tRNA synthetase), Molecular﹠amp; Cellular Biology 10:1633-1641.Because EcTyrRS (for example is not loaded with saccharomyces cerevisiae tRNA, Y.Kwok and J.T.Wong, (1980), determine evolutionary relationship (Evolutionary relationship between Halobacterium cutirubrum and eukaryotesdetermined by use of aminoacyl-tRNA synthetases as phylogenetic probes) between red skin salt bacillus and the eucaryote, Canadian Journal of Biochemistry 58:213-218 with aminoacyl-tRNA synthetase as the systematic growth probe; B.P.Doctor, Deng, (1966), the species specific research of yeast and Escherichia coli tyrosine tRNA (Studies on the species specificityof yeast and E.coli tyrosine tRNAs), Cold Spring HarborSymp.Quant.Biol.31:543-548; And K.Wakasugi; Deng; (1998); genetic code in the evolution: with species specificity aminoacylation and peptide graft exchange (Genetic code in evolution:switching species-specificaminoacylation with a peptide transplant); EMBO Journal 17:297-305), these experiment confirms EcTyrRS/EctRNA CUABe that quadrature is right in saccharomyces cerevisiae.
Though first generation GAL4 chimera can activate transcribing of weak HIS3 reporter, it can not activate in MaV203 that transcribing of URA3 reporter is enough on greater than the 3-AT concentration of 20mM or obviously grow (Fig. 2) on-URA flat board.In order to select the purpose of EcTyrRS, made variant second generation GAL4 construction.This GAL4 reporter is designed to be more activity, has bigger dynamic range, assembles to avoid revertant.In order to improve the activity of GAL4 reporter, under the control of strong ADH1 promoter, use total length GAL4 (its transcriptional activation activity be the DBD-AD fusion twice (referring to, for example, J.Ma and M.Ptashne, (1987), the deletion analysis of GAL4 defines two kinds of transcriptional activation sections (Deletion analysis of GAL4 defines twotranscriptional activating segments), Cell 48:847-853), and used high copy 2-micron plasmid (copy number be the chimeric centromere of initial GAL4 plasmid 10-30 doubly).The dynamic range that the increase of plasmid copy number and its encoded protein activity should be extended reporter.Amber mutation is the GAL4 gene region (Fig. 3) of target coded amino acid residue 2 and 147.The enough sequence specific DNA combinations in this zone (referring to, for example, M.Carey, Deng, (1989), amino-the terminal fragment of GAL4 and DNA are combined into dimer (An amino-terminalfragment of GAL4 binds DNA as a dimer), J.Mol.Biol.209:423-432), be arranged in the GAL4 gene first hidden activation domain 5 ' side (referring to, for example, J.Ma and M.Ptashne, (1987) deletion analysis of GAL4 defines two kinds of transcriptional activation sections (Deletion analysis of GAL4 defines twotranscriptional activating segments), Cell 48:847-853), Cell 48:847-853), so that do not estimate that the brachymemma product that suppresses to produce under the non-existent situation at amber can activated transcription.The selection of amino acid code sudden change by in the past to the saturation mutagenesis of GAL4 select to instruct (referring to, for example, M.Johnston and J.Dover, (1988), the mutation analysis (Mutational analysis ofthe GAL4-encoded transcriptional activator protein of Saccharomycescerevisiae) of the transcription activating protein of saccharomyces cerevisiae GAL4-coding, Genetics 120:63-74), and the terminal DNA of the N-of GAL4 in conjunction with the x-ray structure in territory (referring to, for example, R.Marmorstein, Deng, (1992), carry out DNA identification by GAL4: the structure of protein-dna compound (DNA recognition by GAL4:structure of a protein-DNA complex), [comment], Nature 356:408-414; And J.D.Baleja, Deng, (1992), the DNA-of saccharomyces cerevisiae Cd2-GAL4 is in conjunction with the solution structure (Solution structure of the DNA-binding domain ofCd2-GAL4from S.cerevisiae) in territory, [comment], Nature 356:450-453) and the NMR structure in its dimerization zone.Referring to, for example, P.Hidalgo, Deng, (2001), raise transcriptional machinery by GAL11P: the structure in GAL4 dimerization territory and interaction (Recruitment of the transcriptional machinerythrough GALllP:structure and interactions of the GAL4 dimerization domain), Genes﹠amp; Development15:1007-1020.
Total length GAL4 is cloned in the carrier based on little pUC, to make up 10 single amber mutants (at the codon place of amino acid L3, I13, T44, F68, R110, V114, T121, I127, S131, T145) rapidly by site-directed mutagenesis.Then, under the control of total length ADH1 promoter with the amber mutant subclone of GAL4 and generation in the yeast vector of 2-micron, to set up amber mutant (Fig. 1 of pGADGAL4 and a series of pGADGAL4 of being called (xxTAG), the C group), wherein xx refers to amino acid code that sports amber codon in the GAL4 gene.Use ECTyrRS/tRNA CUAOr A5/tRNA CUAEach GAL4 mutant cotransformation in the MaV203 cell, is converted into leucine and tryptophane prototrophy (protrophy) with transformant.PGADGAL4 itself transforms (10 of＜GAL4 amber mutant with low-down efficient -3Doubly), probably poisonous to the MaV203 cell under so high copy; Amber mutant with GAL4 is not observed this effect.
In the presence of active or dead synzyme, measure the phenotype (Fig. 3, A organizes) of GAL4 reporter on the dull and stereotyped and 0.1%5-FOA flat board at-URA.In the presence of wild type or inactivation EcTyrRS, five GAL4 mutant (L3TAG, I13TAG, T44TAG, F68TAG, S131TAG) are grown on-URA flat board, can not grow on 0.1%5-FOA.In these amber mutants, endogenous suppresses obviously enough with EcTyrRS/tRNA CUAThe inhibition of mediation is advanced to beyond the dynamic range of URA3 reporter among the MaV203.The single amber mutant of five GAL4 (R110TAG, V114TAG, T121TAG, I127TAG T145TAG) is not having uracil and is having EcTyrRS/tRNA CUASituation under the growth (but be not A5/tRNA CUA), on 5-FOA, shown reverse phenotype.These mutant have shown EcTyrRS dependence phenotype, belong in the dynamic range of URA3 reporter among the MaV203.Observe the cleanest EcTyrRS dependence phenotype on-URA and 0.1%5-FOA with the R110TAG mutant of GAL4.Yet when with the A5 cotransformation, this mutant shows some bluenesss in X-GAL measures.In order further to improve dynamic range, make two amber mutants of a series of six GAL4 contain R110TAG (Fig. 3, B group), (L3TAG, R110TAG; I13TAG, R110TAG; T44TAG, R110TAG; R110TAG, T121TAG; R110TAG, I127TAG; R110TAG, T145TAG).Four (I13TAG, R110TAG in these double-mutants; R110TAG, T121TAG; R110TAG, I127TAG and T145TAG, R110TAG) can not grow under the condition of uracil not having, and can on 0.1%5-FOA, grow.These double-mutants have the activity beyond the dull and stereotyped dynamic range of measuring.Two (L3TAG, R110TAG and T44TAG, R110TAG) in the double-mutant can be at wild type EcTyrRS/tRNA CUABut not A5/tRNA CUAOn-URA flat board, grow under the condition that exists; These mutant also show the mutual phenotype of expection on 5-FOA.Select pGADGAL4 (T44TAG, R110TAG), more activated characterize in more detail (Fig. 4) in these two GAL4 mutant.Contain pGADGAL4 (T44TAG, R110TAG)/pEcTyrRS-tRNA CUAMaV203 be blue on X-GAL, but contain pA5/tRNA accordingly CUAStrain then be not.Similarly, contain pGADGAL4 (T44TAG, R110TAG)/pEcTyrRS/tRNA CUAMaV203 have concentration up to the flat board of the 3-AT of 75mM and-grow vigorously on the URA flat board, but contain pA5/tRNA accordingly CUAStrain can or have at 10mM 3AT to grow under the situation of uracil.In a word, (T44TAG R110TAG) can cross over URA3 among the MaV203 to the EcTyrRS dependence phenotype of pGADGAL4, the dynamic range of HIS3 and lacZ reporter.
Interested is the activity of determining the GAL4 mutant; wherein T44 or R110 be by except the aminoacid replacement of tyrosine, may be useful to the sudden change aminoacyl-tRNA synthetase of selecting alpha-non-natural amino acid can be mixed albumen because replace the ability of different aminoacids under the situation that does not change the GAL4 activity.Referring to; for example; M.Pasternak; Deng; (2000); the new quadrature inhibition type tRNA/ aminoacyl-tRNA synthetase that is used to form the biology with expansion genetic code is to (A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair forevolving an organism with an expanded genetic code), Helvetica Chemica Acta83:2277.With a series of five mutant of residue T44 among the GAL4 (T44Y, T44W, T44F, T44D T44K) is building up among the pGADGAL4 (R110TAG), because pGADGAL4 itself is poisonous.With the mutant of the similar series of R110 position among the GAL4 (R110Y, R110W, R110F, R110D R110K) is building up among the pGADGAL4 (T44TAG).These mutant and we interested big hydrophobic amino acid side chain in mixing albumen has deflection, but also comprises positively charged and residue negative charge, as the strictness test that allows.Use pEcTyrRS/tRNA CUAIn the MaV203 cell, the lacZ that measures the leu+trp+ separator with o-nitrophenyl-β-D-galactose pyranoside (0NPG) hydrolysis produces (Fig. 5) with each mutant cotransformation.In all situations, the iuntercellular activity difference is less than 3 times, and this cell contains the GAL4 of the different aminoacids that has replaced T44 or R110.The changeability of this minimum has proved that these sites allow to carry out aminoacid replacement under the situation of the transcriptional activity that does not change GAL4.As by the single amber mutant of measuring on the selectivity flat board active expect that the T44 mutant of making causes the hydrolysis of ONPG slower than the R110 mutant of making in GAL4 (T44TAG) background in GAL4 (R110TAG) background.
Carry out model enrichment research with detect this system from big excessive inactivation synzyme, select the ability of active synzyme (table 1, table 2, Fig. 6).The ability of selecting active synzyme in the presence of alpha-non-natural amino acid from the variant library has been imitated in this selection.To contain GAL4 (T44, R110) and EcTyrRS/tRNA CUAThe MaV203 cell with determine excessive 10 to 10 by 0D660 6GAL4 doubly (T44TAG, R110TAG) and A5/tRNA CUA, and when being plated on non-selective-leu, partially mixed on the-trp nutrient culture media by the blue bacterium colony of X-Gal overlay measurement change.Select those can be at 50mM 3-AT or do not having a cell of surviving under the situation of uracil.3-AT or-cell of surviving on the URA measures the ratio of Smalt and white at X-Gal, and do not exist when selecting same ratio down relatively, clearly proof just selecting can be from dead synzyme enrichment activity synzyme (table 1), coefficient＞10 5Initial ratio was greater than 1: 10 5, it generally is impossible measuring accurate enrichment, because no more than 10 6Individual cell is bed board and do not have remarkable iuntercellular cross-talk to cause unreliable phenotype easily.
Table 1. model is just being selected functional EcTyrRS.
Figure A20048002115500941
A) pass through OD 660Measure
B) on X-Gal
The negative no function EcTyrRS (A5) that selects of table 2. model.
A) pass through OD 660Measure
B) on X-Gal
After just selecting in the presence of the alpha-non-natural amino acid, the cell of selection will contain the synzyme that can use natural amino acid and can use the alpha-non-natural amino acid of adding.For separating the synzyme that only can use alpha-non-natural amino acid, must from the clone who selects, remove the cell that coding uses the synzyme of natural amino acid.This can finish with negative the selection, and in negative the selection, alpha-non-natural amino acid is retained, and those are removed with the synzyme that natural amino acid plays a role.Just to select similar mode to carry out the negative selection of model with model.With EcTyrRS/tRNA CUAWith excessive 10 to 10 5A5/tRNA doubly CUAMix, on 0.1%5-FOA, select.To be that white and blue ratio compare (referring to table 2) with the same ratio under the non-selective condition in X-GAL measures at survivaling cell on the 0.1%5-FOA, be clear that bear selection can be from active synzyme the dead synzyme of enrichment, coefficient is 0.6x10 at least 4In initial ratio greater than 1: 10 4, it generally is impossible measuring accurate enrichment, because no more than 10 5Individual cell is bed board and do not have remarkable iuntercellular cross-talk to cause unreliable phenotype easily.
Developed a kind of universal method, the aaRS that discerns alpha-non-natural amino acid is just selecting and is discerning negative selection of aaRS of natural amino acid.By changing the severity of selecting, can separate various synthase activities.With this method be applied to be presented in the Model Selection with the EcTyrRS variant single-wheel just selecting in enrichment greater than 10 5, bear in the selection greater than 0.6x10 in single-wheel 4These observe these methods of prompting can provide quick arrival quadrature aminoacyl-tRNA synthetase, its function be with have various side chains the alpha-non-natural amino acid site-specific mix in the albumen of saccharomyces cerevisiae.And the enzyme that produces in the saccharomyces cerevisiae can be used for higher eucaryote.
Material and method
Vector construction
With primer tRNA 5 ': GGGGGGACCGGTGGGGGGACCGGTAAGCTTCCCGATAAGGGAGCAGGCCAGTAAAA AGCATTACCCCGTGGTGGGTTCCCGA (SEQ ID NO:89) and tRNA3 ': GGCGGCGCTAGCAAGCTTCCCGATAAGGGAGCAGGCCAGTAAAAAGGGAAGTTCAG GGACTTTTGAAAAAAATGGTGGTGGGGGAAGGAT (SEQ ID NO:90) pcr amplification tRNA from pESCSU3URA CUAGene.The ExpandPCR kit that Roche is all used in this and other all PCR reactions carries out according to manufacturer's instructions.After restriction endonuclease NheI and the AgeI digestion, between the same loci among these tRNA gene 2 microns carrier pESCTrp of insertion (Stratagene), produce ptRNA CUAThe pcr amplification total length ADH1 promoter from pDBLeu (Invitrogen) with primer PADHf:IGGGGGGACCGGTIGGGGGGACCGGTCGGGATCGAAGAAATGATGGTAAA TGAAATAGGAAATCAAGG (SEQ ID NO:91) and pADHR:GGGGGGGAATTCAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGC AGAAAAAGAAAC (SEQ ID NO:92) is with AgeI and EcoRI digestion.With primer pESCTrp1:TCATAACGAGAATTCCGGGATCGAAGAAATGATGGTAAATGAAATAG GAAATCTCATAACGAGAATTCATGGCAAGCAGTAACTTG (SEQ ID NO:93) and pESCTrp2:TTACTACGTGCGGCCGCATGG CAAGCA GTAACTTGTTACTACGTGCGGCCGCTTATTTCCAGCAAATCAGAC (SEQ ID NO:94) amplification EcTyrRS.With EcoRI and NotI digestion EcTyrRS PCR product.Then with AgeI and NotI digestion ptRNA CUAThese three DNA three are connected generation pEcTyrRS-tRNA CUAWith oligonucleotides F37Afwd:CCGATCGCGCTCGCTTGCGGCTTCGATC (SEQ ID NO:95), N126Afwd:ATCGCGGCGAACGCCTATGAC TGGTTC (SEQ ID NO:96), 182,183,186A, GTTGCAGGGTTATGCCGCCGCCTGTGCGAACAAACAG TAC (SEQ ID NO:97) and their reverse complement, and the oligonucleotides 4783:GCCGCTTTGCTATCAAGTATAAATAG of flank (SEQID NO:98), 3256:CAAGCCGACAACCTTGATTGG (SEQ ID NO:99) and as the pEcTyrRS-tRNA of template CUACarry out overlapping PCR, set up plasmid pA5-tRNA with amino acid residue (37,126,182,183 and 186 sport alanine in the avtive spot) CUAWith EcoRI and NotI digestion PCR product, be connected to the pEcTyrRS-tRNA of release when digest with same enzyme CUABig fragment in.Be to make up first generation DB-AD intelligencer, with forward primer pADfwd:GGGGACAAGTTTGTACAAAAAAGCAGGCTACGCCAATTTTAATCAAAGT GGGAATATTGC (SEQ ID NO:100) or pADfwd (TAG) GGGGACAAGTTTGTACAAAAAAGCAGGCTAGGCCAATTTTAATCAAAGTGGGAATA TTGC (SEQ ID NO:101) and ADrev:GGGGACCACTTTGTACAAGAAAGCTGGGTTACTCTTTTTTTGGGTTTGGT GGGGTATC (SEQ ID NO:102) from pGADT7 (Clontech) pcr amplification GAL4DNA in conjunction with the territory.Use the Clonase step, according to manufacturer's instructions with these PCR product cloning in carrier pDEST3-2 (invitrogen), produce pDB-AD and pDB-(TAG)-AD.For making up PGADGAL4 and variant, with primer ADH1428-1429AAGCTATACCAAGCATACAATC (SEQID NO:103) and GAL4C:ACAAGGCCTTGCTAGCTTACTCTTTTTTTGGGTTTGGTGGGGTATCTTC (SEQ ID NO:104) pcr amplification GAL4 gene from pCL1 (Clontech).According to manufacturer's instructions with this fragment cloning in carrier pCR2.1 TOPO (Invitrogen).Contain the clone (pCR2.1TOPOGAL4) of GAL4 gene with HindIII digestion, with 2.7kb GAL4 fragment gel-purified and be connected on the big fragment with the pGADT7 of HindIII digestion, handle gel-purified with the calf intestinal phosphatase enzyme.Carry out Quikchange reaction (Stratagene) according to manufacturer's instructions, the variant of GAL4 gene is based upon on the pCR2.1 with the primer that is listed in the side information.With the mode identical with wild type GAL4 gene with the GAL4 mutant clone in pGADT7.All final constructions all pass through dna sequencing and confirm.
Yeast Cultivation base and operation
Saccharomyces cerevisiae strain MaV203 (Invitrogen) is MAT α; Leu2-3,112; Trp1109; His3 Δ 200; Ade2-101; Cyh2 RCyh1 RThe GAL4 Δ; The gal80 Δ; GAL1::lacZ; HIS3UASGAL1::HIS3@LYS2; SPALlOUASGALl::URA3.The Yeast Cultivation base is available from Clontech, and 5-FOA and X-GAL are available from Invitrogen, and 3-AT is available from BIO 101.YPER (Yeast protein extraction agent) and ONPG are available from Pierce Chemicals.By PEG/ lithium acetate method (referring to, for example, D.Burke, Deng, (2000) " yeast genetics method " (Methods in YeastGenetics), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) carry out plasmid and transform, select transformant suitable synthetic removing fully on the composition nutrient culture media.Phenotype for the various plasmid combinations of test on MaV203 give will be resuspended in the 15 microlitre sterilized waters from the synthetic yeast colony of removing the composition flat board fully of each conversion, rules on interested selective medium then.Each phenotype is confirmed with five independent bacterium colonies at least.
Carrying out X-GAL by the gel cladding process measures.Referring to, I.G.Serebriiskii and E.A.Golemis, (2000), the purposes of lacZ in the research gene function: be used for the evaluation (Uses of lacZ to study gene function:evaluation of beta-galactosidaseassays employed in the yeast two-hybrid system) of the beta galactose glycosides mensuration of yeast two-hybrid system, Analytical Biochemistry 285:1-15.Briefly say, on agar plate, pass through to add pure several times chloroform cracking bacterium colony or cell fritter.After the chloroform evaporated, will contain 1% agarose of 0.25 grams per liter XGAL and contain 0.1M Na 2PO 4Damping fluid be added on planar surface.Agarose one solidifies, and just flat board is placed 37 ℃ to hatch 12 hours.By with 1 milliliter of SD-leu ,-trp is seeded in 96 orifice plates with single bacterium colony, and hatch 30 ℃ of vibrations and to carry out ONPG and measure.The OD of parallel record 100 microlitre cells and several cell diluents in 96 hole microtiter plates 660Cell (100 microlitre) is mixed with 100 microlitre YPER:ONPG (1xPBS, 50%v/v YPER, 20mM MgCl2,0.25%v/v beta-mercaptoethanol and 3mM ONPG), hatch 37 ℃ of vibrations.When colour developing, centrifugation cell is transferred to clean 96 hole microtiter plates (Nunclon, catalog number (Cat.No.) 167008) with supernatant, record A420.The mean value of at least 4 the independent cloning tests of data show, the error bars display standard is poor.Use equation: beta galactosidase unit=1000.A420/ (V.t.OD 660) calculate the ONPG hydrolysis, wherein V is the cell volume of representing with microlitre, t is with minute incubation time of expression.Referring to, for example, I.G.Serebriiskii and E.A.Golemis, (2000), the purposes of lacZ in the research gene function: be used for the evaluation (Uses of lacZ to study genefunction:evaluation of beta-galactosidase assays employed in the yeasttwo-hybrid system) of the beta galactose glycosides mensuration of yeast two-hybrid system, Analytical Biochemistry 285:1-15.A beta galactosidase unit is equivalent to every cell per minute hydrolysis 1 micromole ONPG.Referring to, Serebriiskii and Golemis, above-mentioned.On SPECTRAmaxl90 plate reader, carry out the spectrophotometric reading.
Model Selection
Just select: with two overnight culture cultivate SD-Leu ,-Trp in.One comprises and is loaded with pEcTyrRS-tRNA CUAThe MaV203 of/pGADGAL4 (T44, R110TAG), another is loaded with pA5-tRNASU3/pGADGAL4 (T44, R110TAG).These cells of centrifugal collection are resuspended in 0.9%NaCl by vortex.Then two cell solutions are diluted to identical OD 660To be loaded with pEcTyrRS-tRNA CUAThe MaV203 serial dilution of/pGADGAL4 (T44, R110TAG) is 7 orders of magnitude, then with each dilution and the undiluted pA5-tRNA that is loaded with CUAThe MaV2031 of/pGADGAL4 (T44, R110TAG): 1 volume: volume mixture, so that the cell that contains active and inactivation tyrosyl-tRNA synthetase of determining ratio to be provided.Each is carried out the serial dilution second time than row dilution, and wherein cell quantity reduces, but keeps being loaded with pEcTyrRS-tRNA CUA/ pGADGAL4 (T44, R110TAG) and pA5-tRNA CUA/ pGADGAL4 (T44.R110TAG) cell proportion.With these dilutions pave plate in SD-Leu ,-trp, SD-Leu ,-Trp ,-URA and SD-Leu ,-Trp ,-His+50mM 3-AT on.After 60 hours, to the colony counting on each flat board, measure the phenotype of confirming survivaling cell with the X-GAL beta galactosidase with Eagle Eye CCD camera (Stratagene).Separation from the cell of several independent bluenesss or white colony and SD-leu ,-grow among the trp saturatedly, use the standard method isolated plasmid dna.Confirm the identity of EcTyrRS variant with dna sequencing.
The negative selection: to carry out the negative selection of model, except being loaded with pA5-tRNA with just selecting similar mode CUAThe MaV203 serial dilution of/pGADGAL4 (T44, R110TAG) and with the pEcTyrRS-tRNA that is loaded with of constant density CUAThe MaV203 of/pGADGAL4 (T44, R110TAG) mixes.With cell pave plate in SD-leu ,-trp+0.1%5-FOA on, calculate colony counts after 48 hours, the dull and stereotyped processing as mentioned above.
Following oligonucleotides (table 3) and their reverse complement coupling are to make up the positional mutation body by Quikchange mutagenesis.Sudden change position runic textual representation.
Table 3: the oligonucleotides that is used to make up the positional mutation body.
Amber
The mutant oligonucleotide sequence
L3TAG5’-ATGAAGTAGCTGTCTTCTATCGAACAAGCATGCG-3’(SEQ ID NO：66)
I13TAG5’-CGAACAAGCATGCGATTAGTGCCGACTTAAAAAG-3’(SEQ ID NO：67)
T44TAG5’-CGCTACTCTCCCAAATAGAAAAGGTCTCCGCTG-3’(SEQ ID NO：68)
F68TAG5’-CTGGAACAGCTATAGCTACTGATTTTTCCTCG-3’(SEQ ID NO：69)
R110TAG5’-GCCGTCACAGATTAGTTGGCTTCAGTGGAGACTG-3’(SEQDNO：70)
V114TAG5’-GATTGGCTTCATAGGAGACTGATATGCTCTAAC-3’(SEQ ID NO：71)
T121TAG5’-GCCTCTATAGTTGAGACAGCATAGAATAATGCG-3’(SEQ ID NO：72)
I127TAG5’-GAGACAGCATAGATAGAGTGCGACATCATCATCGG-3’(SEQ ID NO：73)
S131TAG5’-GAATAAGTGCGACATAGTCATCGGAAGAGAGTAGTAG-3’(SEQ ID NO：74)
T145TAG5’-GGTCAAAGACAGTTGTAGGTATCGATTGACTCGGC-3’(SEQ ID NO：75)
Allow
Site mutation body oligonucleotide sequence
T44F5’-CGCTACTCTCCCCAAATTTAAAAGGTCTCCGCTG-3’(SEQ ID NO：76)
T44Y5’-CGCTACTCTCCCCAAATATAAAAGGTCTCCGCTG-3’(SEQ ID NO：77)
T44W5’-CGCTACTCTCCCCAAATGGAAAAGGTCTCCGCTG-3’(SEQ ID NO：78)
T44D5’-CGCTACTCTCCCCAAAGATAAAAGGTCTCCGCTG-3’(SEQ ID NO：79)
T44K5’-CGCTACTCTCCCCAAAAAAAAAAGGTCTCCGCTG-3’(SEQ ID NO：80)
R110F5’-GCCGTCACAGATTTTTTGGCTTCAGTGGAGACTG-3’(SEQ ID NO：81)
R110Y5’-GCCGTCACAGATTATTTGGCTTCAGTGGAGACTG-3’(SEQ ID NO：82)
R110W5’-GCCGTCACAGATTGGTTGGCTTCAGTGGAGACTG-3’(SEQ ID NO：83)
R110D5’-GCCGTCACAGATGATTTGGCTTCAGTGGAGACTG-3’(SEQ ID NO：84)
R110K5’-GCCGTCACAGATAAATTGGCTTCAGTGGAGACTG-3’(SEQ ID NO：85)
Embodiment 2: the eukaryotic genetic code of expansion
Described alpha-non-natural amino acid has been joined approach usually and fast in the saccharomyces cerevisiae genetic code.In response to nonsense codon TAG, five amino acid is effectively mixed in the protein with high fidelity.These amino acid whose side chains contain ketone group, can it be modified chemical probe and the reagent with wide scope uniquely in external or body; The amino acid that contains heavy atom is used for structural research; And photocrosslinking agent is used for the cell research of protein interaction.This method is not only removed the restriction of forcing of genetic code to our operon protein 26S Proteasome Structure and Function in yeast, and it provides the approach of the Eukaryotic genetic code of systemic expansion many cells.
Though the chemist developed synthetic and handle the effective ways of small molecule structure and strategy (referring to, for example, E..J.Corey and X.-M.Cheng, " logic of chemosynthesis " (The Logic of ChemicalSynthesis) (Wiley-Interscience, New York, 1995)), the ability of still reasonable control protein structure and function still is in the embryonic stage.Though may competitively in whole protein group mix the analogue approaching with common amino acid under many circumstances, method of mutagenesis is limited to 20 common amino acid members.Referring to, for example, K.Kirshenbaum, etc., (2002), ChemBioChem 3:235-7; With V.Doring etc., (2001), Science 292:501-4.Complete synthesizing (referring to, for example, B.Merrifield, (1986), Science232:341-7 (1986)) and semisynthesis (referring to, for example, D.Y.Jackson etc., (1994) Science 266:243-7; With P.E.Dawson and S.B.Kent, (2000), Annual Review of Biochemistry 69:923-60 made synthetic peptide and small protein become possibility, but for the albumen that surpasses 10 kilodaltons (kDa), purposes is more limited.Comprise the quadrature tRNA of chemical acylation biological synthesis method (referring to, for example, D.Mendel, etc., (1995), Annual Review of Biophysics and Biomolecular Structure 24:435-462; And V.W.Cornish, Deng (March 31 nineteen ninety-five), Angewandte Chemie-International Editionin English 34:621-633 allowed external (referring to, for example, J.A.Ellman, Deng, (1992), Science 255:197-200) or in the cell of microinjection (referring to, for example, D.A.Dougherty, (2000), Current Opinion in Chemical Biology4:645-52) alpha-non-natural amino acid is mixed in the bigger protein.Yet the stoichiometry characteristic of chemical acylation has seriously limited the amount of the protein that can produce.Therefore, although made very big effort, in whole evolution, the amino acid of 20 genetic codings (remove pyrroles's lysine and selenocystein (referring to, for example, A.Bock etc., (1991), Molecular Microbiology 5:515-20; And G.Srinivasan, etc., (2002), Science 296:1459-62) in addition) and limit protein, may be the character of whole biology.
In order to overcome this restriction, New Parent is joined (for example, L.Wang in the protein biosynthesizing machines of prokaryotes Escherichia coli (E.coli), Deng, (2001), Science 292:498-500), this makes, and the genetic coding alpha-non-natural amino acid becomes possibility in the body.In response to amber codon TAG, some amino acids with new chemistry, physics or biological property effectively and are optionally mixed in the protein.Referring to, for example, J.W.Chin etc., (2002), Journal of the American Chemical Society 124:9026-9027; J.W.Chin and P.G.Schultz, (2002), ChemBioChem 11:1135-1137; J.W.Chin, etc., (2002), PNASUnited States of America 99:11020-11024: and L.Wang and P.G.Schultz, (2002), Chem.Comm., 1:1-10.Yet, because machine translator is not very conservative between prokaryotes and eucaryote, the assembly that adds colibacillary biosynthesizing machine can not be used for usually eukaryotic with the alpha-non-natural amino acid site-specific mix in the protein, with research or manipulated cell process.
Therefore, be based upon the amino acid no purpose translation component that to expand genetic coding in the eukaryotic.Select saccharomyces cerevisiae as initial eucaryon host biology, because it is useful pattern eucaryote, carry out easily genetic manipulation (referring to, for example, D.Burke, Deng, (2000), " yeast genetics method " (Methods in YeastGenetics) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), the machine translator height homology of its machine translator and higher eucaryote (referring to, for example, T.R.Hughes, (2002), Funct.Integr.Genomics 2:199-211).New structural member add the saccharomyces cerevisiae genetic code need not with codon, tRNA and the aminoacyl-tRNA synthetase (' aaRS ') of the uniqueness of any assembly cross reaction of yeast machine translator (referring to, for example, Noren etc., (1989) Science 244:182; Furter (1998) ProteinSci.7:419; With Liu etc., (1999) PNAS USA 96:4780).Candidate's quadrature is to being to suppress tyrosyl-tRNA synthetase-tRNA from colibacillary amber CUATo (referring to, for example, H.M.Goodman, etc., (1968), Nature 217:1019-24; And D.G.Barker, etc., (1982), FEBS Letters 150:419-23).As Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and Escherichia coli tRNA CUAGenetic coding in saccharomyces cerevisiae but not during aminoacylation saccharomyces cerevisiae kytoplasm tRNA, Escherichia coli tyrosyl-tRNA synthetase (TyrRS) is aminoacylation Escherichia coli tRNA effectively CUAReferring to, for example, H.Edwards and P.Schimmel, (1990), Molecular﹠amp; CellularBiology 10:1633-41; And H.Edwards, etc., (1991), PNAS United States of America88:1153-6.In addition, for the saccharomyces cerevisiae aminoacyl-tRNA synthetase, Escherichia coli tyrosyl tRNA CUABe the difference substrate (referring to, for example, V.Trezeguet, etc., (1991), Molecular﹠amp; Cellular Biology11:2744-51), but it is processed in saccharomyces cerevisiae, from nuclear output to kytoplasm (referring to, for example, S.L.Wolin and A.G.Matera, (1999) Gene﹠amp; Development13:1-10) and useful effect in protein translation.Referring to, for example, H.Edwards and P.Schimmel, (1990) Molecular﹠amp; Cellular Biology 10:1633-41; H.Edwards, etc., (1991), PNAS United States of America 88:1153-6; And V.Trezeguet, etc., (1991), Molecular﹠amp; Cellular Biology 11:2744-51.And Escherichia coli TyrRS does not have editor's mechanism, therefore should not proofread and correct the alpha-non-natural amino acid that is connected with tRNA.
For the amino acid specificity that changes quadrature TyrRS so that it is with required alpha-non-natural amino acid with without any endogenous amino acid aminoacylation tRNA CUA, produce a big library of TyrRS mutant, and carry out heredity and select.According to from the crystal structure of the homology TyrRS of bacillus stearothermophilus (referring to, for example, P.Brick, Deng, (1989), Journal of Molecular Biology 208:83), will be arranged in five residue (bacillus stearothermophiluses in conjunction with the Escherichia coli TyrRS avtive spot of contraposition 6.5  of tyrosine aromatic ring, Fig. 7, the A group) sudden change.For example, for setting up mutant EcTyrRS library, the position that five targets are suddenlyd change at first is converted into the alanine codon to produce the A5RS gene.This in gene between two plasmids on the unique PstI site separately.Basically according to technology known in the art (referring to, for example, Stemmer etc., (1993) Biotechniques 14:256-265) description set up this library.A plasmid contains 5 ' half of A5RS gene, and another plasmid contains 3 ' half of A5RS gene.Carry out PCR by Oligonucleolide primers, each segment is carried out mutagenesis with the whole plasmid of amplification.The primer that mixes contains NNK, and (N=A+G+T+C is K=G+T) with BsaI restriction endonuclease recognition site.With BsaI digestion, connect two kinds of circular plasmids that produce, respectively contain half sudden change copy of EcTyrRS gene.Digest this two kinds of plasmids with PstI then, and, cause the assembling of total length mutator by assembling into single plasmid.The EcTyrRS gene that will suddenly change downcuts and connects into pA5RS/tRNA from this plasmid CUAIn EcoRI and the NotI site between.With PEG-lithium acetate method this library is transformed among the saccharomyces cerevisiae Mav203:pGADGAL4 (2TAG), produces～10 8Individual independently transformant.
With the selected plant of this library transformed saccharomyces cerevisiae [MaV203:pGADGAL4 (2TAG) (and referring to, for example, M.Vidal, etc., (1996), PNAS United States of America 93:10321-6; M.Vidal, etc., (1996), PNAS United States of America 93:10315-201 and Chin etc., (2003) Chem.Biol.10:511)] to provide 10 8Individual independently transformant, growth in the presence of the 1mM alpha-non-natural amino acid (Fig. 8, C group).In transcriptional activator GAL4, suppress two kinds of permission amber codons and cause the generation of total length GAL4 and the transcriptional activation of GAL4-reactive HIS3, URA3 and lacZ reporter (Fig. 8, A group).For example, allowing codon is T44 and the R110 that is used for Gal4.HIS3 and URA3 lack uracil (ura) or contain 20mM 3-aminotriazole(ATA) (referring to, for example, G.M.Kishore and D.M.Shah, (1988), Annual Review of Biochemistry57,627-63) (3-AT, the competitive inhibitor of His3 albumen) and lack histidine (expression in nutrient culture media his) allows just to select expression activity aaRS-tRNA CUARight clone.If carrying, mutation T yrRS has amino acid whose tRNA CUA, cell biological synthesizes histidine and uracil so, and survival.The survivaling cell that increases under the situation that does not have 3-AT and alpha-non-natural amino acid is removed total length GAL4 from selectivity is mixed the cell of alpha-non-natural amino acid.Mix the amino acid whose clone of endogenous for removing in response to amber codon, cellular incubation is lacked on the nutrient culture media of alpha-non-natural amino acid in containing 0.1%5-fluororotic acid (5-FOA).As the result who suppresses the GAL4 amber mutation with natural amino acid, those cells of expressing URA3 are converted into toxic products with 5-FOA, cell killing.Referring to, for example, J.D.Boeke, etc., (1984), Molecular﹠amp; General Genetics 197:345-6.Amplification survival clone is applied to just select again in the presence of alpha-non-natural amino acid.The LacZ reporter allows with the colorimetric method difference active and inactivation synzyme-tRNA to (Fig. 8, B organizes).
By using this method, five amino acids (Fig. 7, B group) with different spaces and electronic property are added in the genetic code of saccharomyces cerevisiae independently.These amino acid comprise right-acetyl group-L-phenylalanine (1), right-benzoyl-L-phenylalanine (2), right-azido-L-phenylalanine (3), oxygen-methyl-L-tyrosine (4) and right-iodo-L-phenylalanine (5) (at Fig. 7, in the B group with numeral).The ketone of right-acetyl group-L-phenylalanine unique reactive allow with a series of reagent that contain hydrazine or azanol carry out in vitro and in vivo albumen selective modification (referring to, for example, V.W.Cornish, Deng, (on August 28th, 1996), Journal of the AmericanChemical Society 118:8150-8151; And Zhang, Smith, Wang, Brock, Schultz is in the preparation).Can prove that the heavy atom of right-iodo-L-phenylalanine can be used for phasing x-ray structure data (with the irregular diffraction of multi-wavelength).The benzophenone of right-benzoyl-L-phenylalanine and right-azido-L-phenylalanine and phenylazide side chain allow albumen in vivo with external effective photo-crosslinking (referring to for example, Chin etc., (2002) J.Am.Chem.Soc., 124:9026; Chin and Schultz, (2002) Chem.Bio.Chem.11:1135; With Chin etc., (2002) PNAS, USA 99:11020).Available isotope-labeled methyl easily replaces the methyl of oxygen-methyl-L-tyrosine, is used as partial structurtes and dynamic (dynamical) probe in using nuclear magnetic resonance and vibrational spectroscopy.Three-wheel separates several bacterium colonies after selecting (Negative-Positive-Negative), and they depend on the adding of the alpha-non-natural amino acid of selection in-ura or the survival strictness on 20mM 3-AT-his nutrient culture media.Referring to, Fig. 8, D group.Identical clone only is blue on x-gal under the situation that the 1mM alpha-non-natural amino acid is arranged.These phenotypes that experimental results show that observation are by the aminoacyl-tRNA synthetase-tRNA that develops CUATo with their combination results (referring to, table 4) of cognate amino acid.
For example, for selecting the mutant synzyme, with cell (～10 9) liquid SD-leu ,-cultivated 4 hours in the trp+1mM amino acid.Centrifugal collecting cell is resuspended in 0.9%NaCl then, pave plate in SD-leu ,-trp ,-his+20mM 3-AT ,+1mM alpha-non-natural amino acid or SD-leu ,-trp ,-ura ,+the 1mM alpha-non-natural amino acid on.After 30 ℃ 48 to 60 hours, scrape on the slave plate liquid SD-leu ,-trp in, cultivated 15 hours at 30 ℃.Centrifugal collecting cell is resuspended in 0.9%NaCI, pave plate in SD-leu ,-trp+0.1%5-FOA on.30 ℃ after 48 hours, with cell scrape liquid SD-leu ,-the trp+1mM alpha-non-natural amino acid in, cultivated 15 hours.Centrifugal collecting cell is resuspended in 0.9%NaCl then, pave plate in SD-leu ,-trp ,-his+20mM 3-AT ,+1mM alpha-non-natural amino acid or SD-leu ,-trp ,-ura is on+1mM the alpha-non-natural amino acid.Be that screening selects the phenotype of cell, will from the bacterium colony (192) of each selection transfer to contain 0.5 milliliter of SD-leu ,-hole of 96 orifice plates of trp in, cultivated 24 hours at 30 ℃.Add glycerine (50%v/v to every hole; 0.5 milliliter), exist or do not have under the situation of 1mM alpha-non-natural amino acid, with cellular replication be plated on agar (SD-leu ,-trp; SD-leu ,-trp ,-his ,+20mM 3-AT; SD-leu ,-trp ,-ura) on.With the agarose cladding process SD-leu ,-carry out X-Gal on the trp flat board to measure.
In order to prove that further the phenotype of observing is because quadrature mutant TyrRS/tRNA loci specificity is mixed alpha-non-natural amino acid, produce and characterize the human superoxide dismutase 1 (hSOD) that contains each alpha-non-natural amino acid mutant (referring to, for example, H.E.Parge, Deng, (1992), PNAS United States of America 89:6109-13).
For example,, add the DNA of terminal six histidine marks of coding C-and be amber codon as template with PS356 (ATCC) by overlapping PCR with the Trp33 codon mutation in the human superoxide dismutase gene.HSOD (Trp33TAG) HIS is cloned between from the GAL1 promoter of pYES2.1 (Invitrogen, Carlsbad, CA USA) and CYC1 terminator.With pYES2.1hSOD (Trp33 TAG) HIS with pECTyrRS-tRNA CUAThe mutant synzyme and the tRNA gene corotation of deriving on the plasmid dissolve in the InvSc strain (Invitrogen).For protein expression, with cellular incubation in SD-trp, in-ura+ the gossypose, by adding galactose at OD 6600.5 abduction delivering.By Ni-NTA chromatography (Qiagen, Valencia, CA, USA) purifying HSOD mutant.
Depend on right-acetyl group PheRS-1-tRNA from 33 hSOD strictnesses that contain the genes produce six-histidine-mark of amber codon CUAWith 1mM right-acetyl group-L-phenylalanine (density measurement＜0.1% is under the situation without any an assembly) (referring to Fig. 9).Purifying contains right-acetyl group-L-phenylalanine (for example, by the Ni-NTA affinity chromatography) of total length hSOD, and productive rate is 50 nanograms/milliliter, and from containing Escherichia coli TyrRStRNA CUACell in the productive rate of purifying be more or less the same.In order to compare, under the same conditions, the purifying productive rate of wild type hSODHIS is 250 nanograms/milliliter.
HSOD (33TAG) HIS that Fig. 9 illustrates the genetic coding alpha-non-natural amino acid the protein expression in the saccharomyces cerevisiae (shown in Fig. 7 B group, in Fig. 9 with them at Fig. 7, the numbering in the B group is represented).There is (+) in the explanation of the top of Fig. 9 and have under (-) alpha-non-natural amino acid situation the SDS-polyacrylamide gel electrophoresis of the hSOD of purifying from yeast, alpha-non-natural amino acid is with numeral, with Fig. 7, in the B group with the alpha-non-natural amino acid phase of Coomassie blue stain-cause.Cell contain promising shown in mutant synzyme-tRNA of selecting of amino acid right.The Western trace that the middle part explanation of Fig. 9 is surveyed with anti-hSOD antibody.The Western trace that the bottom explanation of Fig. 9 is surveyed with the antibody of the terminal His6 mark of anti-C-.
Carry out liquid chromatography and the tandem mass spectrum analysis determines to mix amino acid whose identity by trypsinization thing with mutain.For example, make the colour developing of mass spectrum protein band with the dyeing of colloid coomassie.To downcut from polyacrylamide gel with wild type and the corresponding gel band of saltant SOD, and be cut into 1.5 millimeters cube, reduction and alkanisation carry out aforesaid substantially trypsin hydrolysis then.Referring to, for example, A.Shevchenko, etc., (1996), Analytical Chemistry 68,850-858.The tryptic peptide that contains alpha-non-natural amino acid by nanometer stream reversed-phase HPLC/μ ESI/MS and the analysis of LCQ ion trap mass spectrometer.On the Finnigan LCQ Deca ion trap mass spectrometer (Thermo Finnigan) that nanometer spraying HPLC (Agilent 1100 series) is housed, carry out liquid chromatography tandem mass spectrum (LC-MS/MS) analysis.Referring to, for example, Figure 10, A-H group.
Separate and the fragmentation precursor ion with ion trap mass spectrometer, it is corresponding with the peptide Val-Y*-Gly-Ser-Ile-Lys that contains alpha-non-natural amino acid (being designated as Y*) (SEQ ID NO:87) with single electric charge or double-charge ion.The fragment ions quality can be clearly appointment, confirmed that the locus specificity of right-acetyl group-L-phenylalanine mixes (referring to, Figure 10, A group).Do not observe tyrosine or other amino acid and replace right-acetyl group-L-phenylalanine, obtain the purity of mixing of minimum 99.8% from the signal to noise ratio (S/N ratio) of peptide spectrum.When right-benzoyl PheRS-1, right-azido PheRS-1, oxygen-meTyrRS-1 or right-iodo PheRS-1 be used for right-benzoyl-L-phenylalanine, right-azido-the L-phenylalanine, oxygen-methyl-L-lysine or right-iodo-L-phenylalanine mix hSOD (referring to; Fig. 9 and Figure 10; the A-H group) in the time of in, observes the fidelity and the efficient of similar protein expression.In the specimen preparation of experiment, right-azido-the L-phenylalanine is reduced into right-amino-L-phenylalanine, in mass spectrum, observed the latter.This reduction is not right by containing-and the chemically derived of purifying SOD of azido-L-phenylalanine take place in vivo.In control experiment, preparation is carried out mass spectroscopy (referring to Figure 10, F, G and H group) at 33 hSOD that contain tryptophane, tyrosine and leucic six-histidine-mark.The ion that contains amino acid 33 is high-visible in the mass spectrum of these samples.
The genetic code that 5 alpha-non-natural amino acids are added saccharomyces cerevisiae has independently proved the versatility of our methods, points out it to can be applicable to other alpha-non-natural amino acid, but comprises the amino acid of spin labeling, the metal combination or light isomery.This method can produce has albumen new or that improve character, and is easy to control protein function in yeast.And, in mammalian cell, Escherichia coli tyrosyl-tRNA synthetase and bacillus stearothermophilus tRNA CUAThe formation quadrature is right.Referring to, for example, Sakamoto etc., (2002) Nucleic Acids Res.30:4692.Therefore, people can use the aminoacyl-tRNA synthetase of developing in yeast that alpha-non-natural amino acid is added in the genetic code of higher eucaryote.
The sequence of aminoacyl-TRNA synzyme that table 4. is selected
Figure A20048002115501051
aThese clones also contain the Aspl65Gly sudden change
Embodiment 3: the amino acid that will have new reaction is added in the Eukaryotic genetic code
Proved a method based on [3+2] cycloaddition and protein biology conjugation, this method is a locus specificity, and is fast, reliable and reversible.Be starved of under physiological condition chemical reaction with height selection mode modified protein.Referring to, for example, Lemineux and Bertozzi, (1996) TIBTECH, 16:506-513.The major part reaction that is used for the albumen selective modification at present all comprises between nucleophilic and the electrophilic reaction spouse and forms covalent bond, for example reaction of α-Lu Daitong and histidine or cysteine side chain.In these cases, selectivity is by the quantity and the accessibility decision of nucleophilic residues in the albumen.Under the situation of synthetic or semi-synthetic albumen, can use other to have more optionally reaction, as the reaction of non-natural keto-acid-amino acid and hydrazides or aminooxy group compound.Referring to, for example, Cornish, etc., (1996) Am.Chem.Soc., 118:8150-8151; And Mahal, etc., (1997) Science, 276:1125-1128.Recently, in bacterium and yeast apparatus change the specific quadrature tRNA-of amino acid synzyme to may genetic coding alpha-non-natural amino acid (referring to, for example, Wang is etc., (2001) Science 292:498-500; Chin, etc., (2002) Am.Chem.Soc.124:9026-9027; And Chin, etc., (2002) Proc.Natl.Acad.Sci., 99:11020-11024), comprise the amino acid that contains ketone (referring to, for example, Wang, etc., (2003) Proc.Natl.Acad.Sci., 100:56-61; Zhang, etc., (2003) Biochemistry, 42:6735-6746; And Chin, etc., (2003) Science is in the printing).This method has made becomes possibility with the many reagent selectivity marks that comprise fluorophore, crosslinking chemical and cell toxicant molecule what protein of taking up an official post substantially.
A kind of high efficiency method that is used for the albumen selective modification has been described, it comprise in response to, for example, amber nonsense codon TAG, the alpha-non-natural amino acid heredity that will contain azide or acetylene is mixed in the protein.Can pass through Huisgen[3+2 with alkynyl (acetylene) or azide derivatives respectively then] cycloaddition reaction (referring to, for example, Padwa, A. " comprehensive organic synthesis " (Comprehensive Organic Synthesis), the 4th volume, (1991) Trost, B.M. compile, Pergamon, Oxford, 1069-1109 page or leaf; And Huisgen, R. publishes in " the bipolar cycloaddition chemistry of 1.3-" (1,3-Dipolar Cycloaddition Chemistry), (1984) Padwa, A. compile, Wiley, New York, 1-176 page or leaf) modify these amino acid side chains.Because this method comprises cycloaddition but not nucleophilic displacement of fluorine, so (operable other method is the ligand exchange that has on two arsenic compounds of four halfcystine motifs can to come modifying protein with high selectivity, referring to, for example, Griffin, Deng, (1998) Science 281:269-272).This reaction can be under room temperature, aqueous conditions joins in the reaction mixture by Cu (I) salt with catalytic amount with fabulous regioselectivity (1,4＞1,5) to be carried out.Referring to, for example, Tornoe, etc., (2002) Org.Chem.67:3057-3064; And Rostovtsev, etc., (2002) Angew.Chem.Int.Ed.Engl.41:2596-2599.In fact, Finn and colleagues' verified this azide-alkynes [3+2] cycloaddition can be carried out on complete cowpea mosaic virus surface.Referring to, for example, Wang, etc., (2003) J.Am.Chem.Soc., 125:3192-3193.Another nearest embodiment introduces albumen and [3+2] cycloaddition subsequently with the azido electrophilic, referring to, for example, Speers, etc., (2003) J.Am.Chem.Soc., 125:4686-4687.
Unique site for alkynyl (acetylene) or azide functional group selectivity introducing eukaryotic protein produces the quadrature TyrRS/tRNA that develops in yeast CUARight, its genetic coding acetylene and azido amino acid, respectively as Figure 11 1 and 2 shown in.Can be in follow-up also addition reaction, under physiological condition with the fluorophore albumen of mark gained effectively and optionally.
In the past, in yeast proof Escherichia coli tyrosyl tRNA-tRNA synzyme to being quadrature, that is, and tRNA or synzyme all not with endogenous yeast tRNA or synzyme cross reaction.Referring to, for example, Chin, etc., (2003) Chem.Biol., 10:511-519.This quadrature tRNA-synzyme is to being used for the codon in response to TAG, many alpha-non-natural amino acids optionally and are effectively mixed in the yeast (for example, Chin, etc., (2003) Science is in the printing).In order to change the amino acid specificity of Escherichia coli tyrosyl-tRNA synthetase, with the amino acid/11 or 2 of accepting Figure 11, by randomization Tyr 37, Asn 126, Asp 182, Phe 183And Leu 186Codon produce～10 7The library of mutant.Select this five residues according to crystal structure from the homology synzyme of bacillus stearothermophilus.For obtaining concrete amino acid as the synzyme of substrate, used-kind of selection scheme, wherein the Thr44 of the gene of transcriptional activator GAL4 and the codon of Arg110 are converted into amber nonsense codon (TAG).Referring to, for example, Chin, etc., (2003) Chem.Biol., 10:511-519.In MaV203:pGADGAL4 (2TAG) yeast strains, suppress these amber codons cause producing total length GAL4 (referring to, for example, Keegan, etc., (1986) Science, 231:699-704; And Ptashne, (1988) Nature, 335:683-689), it drives the expression of HIS3 and URA3 reporter conversely.Gene outcome supplementation group propylhomoserin in back and uracil auxotrophy, allow Figure 11 1 or 2 in the presence of the clone of selection carrying active synthase mutant.By lack Figure 11 1 or 2 but contain on the nutrient culture media of 5-fluororotic acid growth and remove and load the amino acid whose synzyme of endogenous, URA3 is converted into toxic products with the 5-fluororotic acid.Select by this library being carried out three-wheel (positive and negative, just), we have identified selectivity at 1 (pPR-EcRS1-5) of Figure 11 with at the synzyme of Figure 11 2 (pAZ-EcRS1-6), and are as shown in table 8.
All synzyme have all shown strong sequence similarity, comprise conservative Asn 126, this points out this residue to have the important function effect.Surprisingly, synzyme pPR-EcRS-2 and pAZ-EcRS-6 progressively form with respectively in conjunction with 1 and 2 of Figure 11, are converged to identical sequence (Tyr 37→ Thr 37, Asn 126→ Asn 126, Asp 182→ Ser 182And Phe 183→ Ala 183, Leu 186→ Leu 186).Phenolic hydroxyl group and Tyr in conjunction with tyrosine 37And Asp 182Between hydrogen bond owing to be mutated into Thr and Ser and destroyed respectively.Phe 183Be converted into Ala, may provide more spaces for holding alpha-non-natural amino acid.For confirming that this synzyme (with other synzyme) accepts the ability of amino acid as substrate, the selected plant that is loaded with the synzyme plasmid is incubated at lacks uracil (from the nutrient culture media that lacks histidine, having obtained identical result) but be supplemented with on 1 or 2 the nutrient culture media of Figure 11.Four in five alkynes synzyme of growth result announcement can be added to two kinds of alpha-non-natural amino acids on its tRNA.As if the azido synzyme has more selectivity, because have only 1 and 2 its tRNA of aminoacylation of the enough Figure 11 of pAZ-EcRS-6 (identical with pPR-EcRS-2) energy.Do not have not detect under 1 or 2 situations of Figure 11 the fact prompting of growth, synzyme is not accepted in 20 kinds of common amino acids any one as substrate.Referring to Figure 14.
Other uses the experiment of pPR-EcRS-2 (pAZ-EcRS-6) for all, allows people to contain the nutrient culture media of expressing strain and control which alpha-non-natural amino acid is mixed by 1 or 2 of Figure 11 is added simply.For protein production, be TAG with the codon mutation of permission residue Trp33 that has merged the human superoxide dismutase-1 (SOD) of the terminal 6xHis mark of C-.For example, with human superoxide dismutase (Trp 33TAG) HIS clones between the GAL1 promoter of pYES2.1 (Invitrogen, Carlsbad, CA USA) and CYC1 terminator.With pYES2.1SOD (Trp 33TAG) HIS is with pECTyrRS-tRNA CUAThe mutant synzyme and the tRNA gene corotation of deriving on the plasmid dissolve in the InvSc strain (Invitrogen).For protein expression, with cell grow in SD-tr ,-the ura+ gossypose in, by adding galactose at OD 6600.5 abduction delivering.Existing or not having expressing protein under the 1mM 1 of Figure 11 or 2 the situation, by Ni-NTA chromatography (Qiagen, Valencia, CA, USA) purifying.
SDS-PAGE and Western engram analysis disclose the alpha-non-natural amino acid dependence protein and express, and compare with the protein expression under 1 or 2 situations that do not have Figure 11, and its fidelity＞99% is determined in density measurement.Referring to Figure 12.Mix amino acid whose identity for further confirming, the trypsinization thing is carried out liquid chromatography and tandem mass spectrum analysis.
For example, with nickel affinity column purifying wild type and saltant hSOD, make the protein band colour developing with the dyeing of colloid coomassie.To downcut from polyacrylamide gel with wild type and the corresponding gel band of saltant SOD, and be cut into 1.5 millimeters cube, reduction and alkanisation carry out aforesaid substantially trypsin hydrolysis then.Referring to, for example, Shevchenko, A etc., (1996) Anal.Chem.68:850-858.The tryptic peptide that contains alpha-non-natural amino acid by nanometer stream reversed-phase HPLC/μ ESI/MS and the analysis of LCQ ion trap mass spectrometer.Referring to, Figure 15, A and B group.On the Finnigan LCQ Deca ion trap mass spectrometer (Thermo Finnigan) that nanometer spraying HPLC (Agilent 1100 series) is housed, carry out liquid chromatography tandem mass spectrum (LC-MS/MS) analysis.
Separate and the fragmentation precursor ion with ion trap mass spectrometer, it is with corresponding with single electric charge or doubly charged peptide VY*GSIK (the SEQ ID NO:87) precursor ion that contains alpha-non-natural amino acid (being designated as Y*).The fragment ions quality can be clearly appointment, confirms that the locus specificity of each alpha-non-natural amino acid mixes.LCMS/MS does not show in this position and mixes any natural amino acid.The signal to noise ratio (S/N ratio) of all mutant peptides＞1000, the fidelity that this prompting is mixed is better than 99.8%.Referring to, Figure 15, A and B group.
For proof can pass through nitrine-alkynes [3+2] cycloaddition reaction with little organic molecule conjugation to protein, the dyestuff 3-6 shown in A organizes among synthetic Figure 13, they contain ethinyl or azido and have red sulphonyl or fluorescein fluorophore (referring to the embodiment 5 of this paper).Cycloaddition itself uses 0.01mM albumen in the phosphate buffer (PB) of pH8, at 2mM 3-6,1mM CuSO shown in Figure 13 A group 4Under the existence of～1 milligram of copper cash, 37 ℃ of reactions 4 hours (referring to Figure 13, the B group) are carried out.
For example, in the PB damping fluid (pH=8) of 45 microlitre albumen, add 1 microlitre CuSO 4(at H 250mM in 0), 2 microlitre dyestuffs (50mM in EtOH), 2 microlitres three (1-benzyl-1H-[1,2,3] triazole-4-ylmethyl) amine (50mM in DMSO) and copper cash.Room temperature or 37 ℃ added 450 microlitre H after spending the night in following 4 hours or 4 ℃ 2O is centrifugal by dialysis membrane (10kDa blocks) with potpourri.By behind the centrifuge washing supernatant, liquor capacity is 50 milliliters with the 2x500 microlitre.Sample by 20 milliliters of SDS-PAGE analyses.Can pass through at H 2Soaked overnight is removed remaining once in a while dyestuff among the O/MeOH/AcOH (5: 5: 1) from gel.Usually cause labeling effciency lower as reductive agent three (carboxyethyl) phosphine.With observation (for example, Wang, Q. etc., (2003) J.Am.Chem.Soc.125:3192-3193) difference early, exist or do not have a not result of substantial effect reaction of three (triazolyl) amine ligand.
After the dialysis, with SDS-PAGE evaluation of markers albumen, at the red sulphonyl dye (λ of 3-4 shown in Figure 13 A group Ex=337 nanometers, λ Em=506 nanometers) optical density meter or at 5-6 fluorescein(e) dye (λ shown in Figure 13 A group under the situation Ex=483 nanometers, λ Em=516 nanometers) use the imaging in gel of photosensitive imaging instrument under the situation.Referring to, for example, Blake, (2001) Curr.Opin.Pharmacol., 1:533-539; Wouters, etc., (2001) Trendsin Cell Biology 11:203-211; And Zacharias, etc., (2000) Curr.Opin.Neurobiol., 10:416-421.Analyze the trypsinization thing by LC MS/MS and characterize labelled protein, shown that the locus specificity of fluorophore adheres to, conversion ratio average out to 75% is (for example, by relatively using Figure 13, the A of the SOD of 5 or 6 marks shown in the A group 280/ A 495Value is determined).Figure 13, shown in the A group 3 and alkynes albumen or Figure 13, shown in the A group 4 and azido albumen between do not have the fact of observable reaction to prove conclusively the selectivity of this biological conjugation.
The synzyme that table 8 is evolved and formed
Be 1 pPR-EcRS that selects and be 2 pAZEcRS (as shown in figure 11) that select
Figure A20048002115501091
Embodiment 4: alkynyl amino acid synthetic
In one aspect of the invention, the invention provides alkynyl amino acids.Formula IV has illustrated an example of structure of alkynyl amino acid:
Figure A20048002115501092
Alkynyl amino acid generally is the arbitrary structures with formula IV, wherein R 1Be the substituting group that is used for one of 20 kinds of natural amino acids, R 2It is the alkynyl substituted base.For example, the structure of the right-propargyloxy phenylalanine of 1 explanation among Figure 11.Can synthesize right-propargyloxy phenylalanine, for example, as described below.In this embodiment, the synthetic of right-propargyloxy phenylalanine can be finished in originating in three steps of commercially available N-Boc-tyrosine.
For example, with uncle N--butoxy carbonyl-tyrosine (2 grams, 7 mMs, 1 equivalent) and K 2CO 3(3 grams, 21 mMs, 3 equivalents) are suspended in dry DMF (15 milliliters).Propargyl bromide (2.1 milliliters, 21 mMs, 3 equivalents, 80% toluene solution) is slowly added, and stirred reaction mixture is 18 hours under the room temperature.Add entry (75 milliliters and Et 2O (50 milliliters), Et is used in layering 2O (2x50 milliliter) extracts water.Dry (MgSO 4) organic layer that mixes, solvent is removed in decompression.Obtain yellow oil product (2.3 grams, 91%), need not to be further purified and just be used for next step.Product with the 8 explanation Boc-protections of following chemical constitution:
Figure A20048002115501101
Uncle 2--butoxy carbonyl amino-3-[4-(Propargyl oxygen base) phenyl]-the propionic acid propargyl ester
Under 0 ℃, carefully acetyl chloride (7 milliliters) is added in the methyl alcohol (60 milliliters), to produce the methanol solution of the anhydrous HCl of 5M.Add the product (2 grams, 5.6 mMs) of previous step, reaction stirred 4 hours allows to be heated to environment temperature this moment.After volatile matter is removed in decompression, obtain faint yellow solid (1.6 grams, 98%) (referring to chemical constitution 9), it is directly used in next step.
2-amino-3-[4-(Propargyl oxygen base) phenyl]-the propionic acid propargyl ester
To be dissolved in the aqueous mixture of 2N NaOH (14 milliliters) and MeOH (10 milliliters) from the propargyl ester (1.6 grams, 5.5 mMs) of previous step.Stir after 1.5 hours under the room temperature, pH is adjusted to 7 by adding dense HCI.Add entry (20 milliliters), place 4 ℃ to spend the night in potpourri.Filtering-depositing is with ice-cold H 2The O washing, vacuum drying produces 1 (2-amino-3-phenylpropionic acid (1) (being also referred to as right-propargyloxy phenylalanine), white solid among 1.23 gram (90%) Figure 11. 1HNMR (400MHz, D 2O) (as D 2Sylvite among the O) δ 7.20 (d, J=8.8Hz, 2H), 6.99 (d, J=8.8Hz, 2H) 4.75 (s, 2H), 3.50 (dd, J=5.6,7.2Hz, 1H), 2.95 (dd, J=5.6,13.6Hz, 1H), 2.82 (dd, J=7.2,13.6Hz, 1H); 13C NMR (100MHz, D 2O) δ 181.3,164.9, and 155.6,131.4,130.7,115.3,57.3,56.1,39.3; HRMS (CI) m/z220.0969[C 12H 13NO 3(M+1) need 220.0968].
Embodiment 5: by [3+2] cycloaddition molecule is joined in the albumen with alpha-non-natural amino acid
In one aspect, the invention provides and to contain the protein and the method for adding the coupling of replacement molecule and compositions related of alpha-non-natural amino acid.For example, can will add substituting group by [3+2] cycloaddition and add alpha-non-natural amino acid.Referring to, for example, Figure 16.For example, can be with [3+2] cycloaddition (for example, comprising second reactive group) of desired molecule (for example to albumen with alpha-non-natural amino acid as alkynes triple bond or azido according to the condition of following public [3+2] cycloaddition reaction, have first reactive group, as azido or triple bond) in.PB damping fluid (pH=8) the adding CuSO that for example, will comprise the albumen of alpha-non-natural amino acid 4, in desired molecule and the copper cash.Potpourri is hatched back (for example, room temperature or 37 ℃ about 4 hours down, or 4 ℃ spent the night), adds H 2O is by the dialysis membrane filtering mixt.Can pass through, the adding sample is analyzed in the example gel analysis.
The example of described molecule includes but not limited to for example, have the molecule of triple bond or azido, as have Figure 13, formula 3,4, the 5 and 6 isostructural molecules of A group.And, triple bond or azido can be mixed other molecule (s) of interest, for example in the structure of polymkeric substance (as poly-(ethylene glycol) and derivant), crosslinking chemical, additional dye, photocrosslinking agent, cytotoxic compound, affinity labeling, biotin, sugar, resin, pearl, second kind of albumen or polypeptide, metal-chelator, co-factor, fatty acid, carbohydrates, polynucleotide (for example DNA, RNA etc.) etc., also can be used for [3+2] cycloaddition then.
In one aspect of the invention, can be as described below synthetic have Figure 13, the formula 3,4,5 of A group or a molecule of 6.For example, by under 0 ℃, propargyl amine (250 microlitres, 3.71 mMs, 3 equivalents) being added the CH of dansyl Cl (500 milligrams, 1.85 mMs, 1 equivalent) and triethylamine (258 microlitres, 1.85 mMs, 1 equivalent) 2Cl 2(10 milliliters) solution, synthetic Figure 13, the A group 3 in and shown alkynes dyestuff in the following chemical constitution 3.Stir after 1 hour, reaction mixture is heated to room temperature, stirred again 1 hour.Vacuum is removed volatile matter, by silica gel column chromatography (Et 2O/ hexane=1: 1) purifying crude product, Figure 13 of generation yellow solid, 3 (418 milligrams, 78%) of A group.Analyze report in data and the document identical.Referring to, for example, Bolletta, F etc., (1996) Organometallics 15:2415-17.Shown the example of structure of spendable alkynes dyestuff among the present invention in the chemical constitution 3:
Figure A20048002115501111
By under 0 ℃ with 3-azido propylamine (for example, as Carboni, B etc., (1993) described in the J.Org.Chem.58:3736-3741) (371 milligrams, 3.71 mMs, 3 equivalents) add (500 milligrams of dansyl Cls, 1.85 mM, 1 equivalent) and the CH of triethylamine (258 microlitres, 1.85 mMs, 1 equivalent) 2Cl 2(10 milliliters) solution synthesizes Figure 13, A group 4 in and shown azido dyestuff in the following chemical constitution 4.Stir after 1 hour, reaction mixture is heated to room temperature, stirred again 1 hour.Vacuum is removed volatile matter, by silica gel column chromatography (Et 2O/ hexane=1: 1) purifying crude product, Figure 13 of generation yellow oily, 4 (548 milligrams, 89%) of A group. 1HNMR (400MHz, CDCl 3) δ 8.55 (d, J=8.4Hz, 1H), 8.29 (d, J=8.8Hz, 1H), 8.23 (dd, J=1.2,7.2Hz, 1H), 7.56-7.49 (comp, 2H), 7.18 (d, J=7.6Hz, 1H), 5.24 (brs, 1H), 3.21 (t, J=6.4Hz, 2H), 2.95 (dt, J=6.4Hz, 2H), 2.89 (s, 6H), 1.62 (quin, J=6.4Hz, 2H); 13C NMR (100MHz, CDCl 3) δ 134.3,130.4,129.7,129.4,128.4,123.3,118.8,115.3,48.6,45.4,40.6,28.7 ( 13In the C NMR spectrum be not all quaternary carbon atom signal all as seen); HRMS (CI) m/z 334.1336[C 15H 20N 5O 2S (M+1) needs 334.1332].The example of structure that has shown the azido dyestuff in the chemical constitution 4:
Figure A20048002115501121
By at room temperature with (83 milligrams of EDCI (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride), 0.43 mM, 1 equivalent) adds (150 milligrams of fluorescein amine, 0.43 mM, 1 equivalent) and pyridine (2 milliliters) solution of 10-undecynoic acid (79 milligrams, 0.43 mM, 1 equivalent), synthetic Figure 13, the A group 5 in and shown alkynes dyestuff in the following chemical constitution 5.The suspension stirring is spent the night, with reaction mixture impouring H 2Among the O (15 milliliters).By adding dense HCl with this solution acidifying (pH＜2).Stir after 1 hour, filter out precipitation, use H 2O (5 milliliters) washing is dissolved in a spot of EtOAc.The adding of hexane causes Figure 13, and 5 of A group is separated out with orange crystal, collects and under vacuum dry (138 milligrams, 63%).Analyze report in data and the document identical.Referring to, for example, Crisp, G.T. and Gore, J. (1997) Tetrahedron 53:1505-1522.The example of structure that has shown the alkynes dyestuff in the chemical constitution 5:
Figure A20048002115501122
By at room temperature with (83 milligrams of EDCI (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride); 0.43 mM; 1 equivalent) adds (150 milligrams of fluorescein amine; 0.43 mM; 1 equivalent) and 4-(3-azido propyl group carbamyl)-butyric acid (for example; reaction by 3-azido propylamine and glutaric anhydride is synthetic) (92 milligrams; 0.43 mM; 1 equivalent) pyridine (2 milliliters) solution synthesizes Figure 13, A group 6 in and shown azido dyestuff in the following chemical constitution 6.The suspension stirring is spent the night, with reaction mixture impouring H 2Among the O (15 milliliters).By adding dense HCl with this solution acidifying (pH＜2).Stir after 1 hour, filter out precipitation,, be dissolved in a spot of EtOAc with 1N HCl (3x3 milliliter) washing.The adding of hexane causes Figure 13, and 6 of A group is separated out with orange crystal, collects and under vacuum dry (200 milligrams, 86%). 1H NMR (400MHz, CD 3OD) δ 8.65 (s, 1H), 8.15 (d, J=8.4Hz, 1H), 7.61-7.51 (comp, 2H), 7.40 (d, J=8.4Hz, 1H), 7.35 (brs, 2H), 7.22-7.14 (comp, 2H), 6.85-6.56 (comp, 3H), and 3.40-3.24 (comp, 4H), 2.54 (t, J=7.2Hz, 2H), 2.39-2.30 (comp, 2H), 2.10-1.99 (comp, 2H), 1.82-1.72 (comp, 2H); 13CNMR (100MHz, CD 3OD) δ 175.7,174.4,172.4,167.9,160.8,143.0,134.3,132.9,131.8,129.6,124.4,123.3,121.1,118.5,103.5,50.2,38.0,37.2,36.2,29.8,22.9 ( 13In the C NMR spectrum be not all quaternary carbon atom signal all as seen); HRMS (CI) m/z544.1835[C 28H 25N 5O 7(M+1) need 544.1827].The example of structure that has shown the azido dyestuff in the chemical constitution 6:
Figure A20048002115501131
In one embodiment, also the PEG molecule can be joined and have alpha-non-natural amino acid, for example in the amino acid whose protein of azido amino acid or propargyl.For example, can propargyl acid amides PEG (as Figure 17, A group shown in) be joined by [3+2] cycloaddition and have in the amino acid whose protein of azido.Referring to for example, Figure 17, A group.Figure 17, the B group profile have a gel analysis of the substituent protein of PEG of adding.
In one aspect of the invention, can synthetic propargyl acid amides PEG as described below (as Figure 17, shown in the A group).For example, with the CH of propargyl amine (30 microlitre) 2Cl 2(1 milliliter) solution adds in the 20kDa PEG-hydroxysuccinimide eater (120 milligrams, available from Nektar).Stirring reaction is 4 hours under the room temperature.Add Et then 2O (10 milliliters) filters out precipitation, by adding Et 2O (10 milliliters) is secondary recrystallization from MeOH (1 milliliter).Product is dry under vacuum, produce white solid (105 milligrams, productive rate 88%).Referring to, for example, Figure 17, C group.
Embodiment 6: exemplary O-RS and 0-tRNA
Exemplary O-tRNA comprises SEQ ID NO.:65 (referring to, table 5).The O-RS example comprises SEQ ID NOs.:36-63,86 (referring to, table 5).The example of the polynucleotide of coding O-RS or its part (as avtive spot) comprises SEQ ID NOs.:3-35.The amino acid change of exemplary O-RS has been described in the table 6 in addition.
Table 6: the EcTyrRS variant of evolving and forming
Figure A20048002115501141
aThese clones also contain the Asp165Gly sudden change
Be appreciated that embodiment described herein and embodiment are only as exemplary purposes, those skilled in the art can carry out various modifications or change to them, and still are included within the scope of the application's spirit and scope and claims.
Though, to those skilled in the art, can make various changes in form and details by reading the disclosure, and not deviate from scope of the present invention for illustrating and understanding aforementioned invention is being described in detail in detail aspect some details.For example, all technology described herein and device may be used to various combination.As if all publications of quoting among the application, patent, patented claim and/or other file are all for all purposes are incorporated herein by reference with same degree integral body, and each independent publication, patent, patented claim and/or other file are introduced separately into as a reference for all purposes.
Table 5
SEQ ID NO.： Mark Sequence
SEQ ID NO.：1 Escherichia coli wild type TyrRS (synzyme) polynucleotide ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTAG CCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGC CCGATCGCGCTCTATTGCGGCTTCGATCCTACCGCTGACAGCTTGCATTT GGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGC CACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGAC CCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTT CAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATT TCGACTGTGGAGAAAACTCTGCTATCGCGGCGAACAACTATGACTGGTT CGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCT CCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACC GTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGTTGCA GGGTTATGACTTCGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAA ATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGAC CCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCA CTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCT GGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGAT CAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTA TGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCG GTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCT GGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGC CTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGC TGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACC TGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGC ACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAG TCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTT TACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAAT AA
SEQ ID NO.：2 Escherichia coli wild type TyrRS (synzyme) amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALYCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYDFACLNKQYGVVLQIGGSDQWG NITSGIDITRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：3 POMe-1 synzyme polynucleotide ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTAG CCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCC CGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTG GGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA CAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCG AGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAG
GAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCG ACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGC AATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTT AACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAA GATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTA TAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGT GGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCG TCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAG CAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGA TCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACT GCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCAT TGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGC ACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCAC GGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCA GCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCA GGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCA GGCACTGGTCGATTCTGAACTGCAACCTTCCcGTGGTCAGGCACGTAAA ACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCC TGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACT GCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAATAA
SEQ ID NO.：4 POMe-2 synzyme polynucleotide ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTAg CCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCC CGATCGCACTCACTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTG GGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA CAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCG AGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAG GAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCG ACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGC AATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTT AACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAA GATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTA TACGTATGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGT GGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCG TCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAG CAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGA TCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACT GCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCAT TGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGC ACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCAC GGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCA GCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCA GGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCA GGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAA ACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCC TGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACT GCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAATAA
SEQ ID NO.：5 POMe-3 synzyme polynucleotide ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTAG CCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCC CGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTG GGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA CAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCG AGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAG GAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCG ACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGC AATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTT AACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAA GATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTA TAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGT GGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCG TCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAG CAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGA TCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACT
GCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCAT TGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGC ACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCAC GGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCA GCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCA GGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCA GGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAA ACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCC TGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACT GCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAATAA
SEQ ID NO.：6 POMe-4 synzyme polynucleotide ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAgCGGGGGCTGGTAG CCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCC CGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTG GGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA CAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCG AGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAG GAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCG ACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGC AATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTT AACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAA GATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTA TAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGT GGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCG TCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAG CAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGA TCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACT GCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCAT TGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGC ACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCAC GGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCA GCGGTTCTTTGAGTGCGCTGAGTGAAgCGGACTTCGAACAGCTGGCGCA GGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCA GGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAA ACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCC TGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACT GCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAATAA
SEQ ID NO.：7 POMe-5 synzyme polynucleotide ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTAg CCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCC CGATCGCACTCACGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTG GGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA CAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCG AGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAG GAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCG ACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGC AATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTT AACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAA GATCAGGGGATTTCGTTCACTGAGTTTTCCTACAGCCTGCTGCAGGGTTA TACGATGGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGT GGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCG TCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAG CAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGA TCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACT GCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCAT TGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGC ACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCAC GGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCA GCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCA GGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCA GGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAA ACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCC TGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACT
GCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAATAA
SEQ ID NO.：8 POMe-6 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACGGTGTG
SEQ ID NO.：9 POMe-7 (avtive spot) synzyme polynucleotide CGGGGGCTGGTACCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACGGTGTG
SEQ ID NO.：10 POMe-8 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACGGTGTG
SEQ ID NO.：11 POMe-9 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATTCGTATGCCTGTGCGAACAAACAGTACGGTGTG
SEQ ID NO.：12 POMe-10 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACGGTGTG
SEQ ID NO.：13 POMe-11 (avtive spot) synzyme polynucleotide CGGGGGCTGGTACCcCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCCTTTGTGGCTTCGATCCTACCGCTGA CAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCC AGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTC TGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGA
AGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCC GTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATT ATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATTCTATTGCCTGTTCGAACAAACAGTACGGTGTG
SEQ ID NO.：14 POMe-12 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTAGTATTGCCTGTTTGAACAAACAGTACGGTGTG
SEQ ID NO.：15 POMe-13 (avtive spot) synzyme polynucleotide CGGGGGCTGGTACCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATAGTATTGCCTGTTTGAACAAACAGTACGGTGTG
SEQ ID NO.：16 POMe-14 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCTGGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAGGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATTGT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATATGCGTGCCTGTGAGAACAAACAGTACGGTGTG
SEQ ID NO.：17 Right-acetyl group Phe-1 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCATTTGTGGCTTCGATCCTACCGCTGA CAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCC AGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTC TGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGA AGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCC GTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATT ATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGGTCAGGGGATTTCGTTCACTGAGTTTTCCTACAA CCTGCTGCAGGGTTATGGTATGGCCTGTGCTAACAAACAGTACGGTGTGG TGCTGCAAATTGGTGGTTCTGACCAATGGGGTAACATCACTTCTGGTATC GACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：18 To benzophenone-1 (avtive spot) synzyme polynucleotide CAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCG ATCGCACTCGGTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGG GCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACA AGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGA GCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGG AGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGA CTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCA ATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTA ACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAG ATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTAT
GGTTTTGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTG GTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGT CTGCATCAGAATCAGGTG
SEQ ID NO.：19 To benzophenone-2 (avtive spot) synzyme polynucleotide GCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGGGTGTGGCT TCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTAT GCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAG GCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGC GTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCC GTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCT ATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTC CTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGA AGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACT GAGTTTTCCTACAACCTGCTGCAGGGTTATGGTTATGCCTGTATGAACAA ACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAAC ATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：20 To azido Phe-1 (avtive spot) synzyme polynucleotide GGGCTGGTAGCCCAGGTGACGGACGNAGAAGCGTTAGCAGAGCGACTG GCGCAAGGCCCGATCGCACTCCTTTGTGGCTTCGATCCTACCGCTGACA GCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAG CAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTG ATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAA GAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGT TCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTAT GACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAA ACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGT CTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCT GCTGC AGGGTTATTCTATGGCCTGTGCGAACAAACAGTACGGTGTGGTG CTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGA CCTGACCCGTCGTCTGCATCANAATCANGTG
SEQ ID NO.：21 To azido Phe-2 (avtive spot) synzyme polynucleotide TTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCTTCG ATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCC TGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCG GCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAA GCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAA GCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCG CGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTG CGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAG CGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGA GTTTTCCTACAACCTGCTGCAGGGTTATTCTGCGGCCTGTGCGAACAAAC AGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACAT CACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：22 To azido Phe-3 (avtive spot) synzyme polynucleotide GACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTC CTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGT TCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTT GCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAA GCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTG GACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAG AAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAAT GTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGAT GATCAACAAANAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGG GATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGGCTG CCTGTGCGAACAAACAGTACGGNGNGGNGCTGCAAATTGGNGGTTCTG ACCAGGGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCAT CAAAATCAGGTG
SEQ ID NO.：23 To azido Phe-4 (avtive spot) synzyme polynucleotide GCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCT TCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTGT GCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAG GCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGC GTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCC GTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCT ATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTC CTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGA
AGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACT GAGTTTTCCTACAACCTGCTGCAGGGTTATAGTGCGGCCTGTGTTAACAA ACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAAC ATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCANGTG
SEQ ID NO.：24 To azido Phe-5 (avtive spot) synzyme polynucleotide GACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTC ATTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTT CCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTG CGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGC TGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGA CAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAA AACTCTGCTATCGCGGCCAATGATTATGACTGGTTCGGCAATATGAATGT GCTGACCTTCCTGCGCGATTTGGCAAACACTTCTCCGTTAACCAGATGA TCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGA TTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAATTTTGCCT GTGTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCA GTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGA ATCAGGTG
SEQ ID NO.：25 To azido Phe-6 (avtive spot) synzyme polynucleotide CGACTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCG CTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGC TTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACG GGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACA CCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTG CCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAAT AATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATTT GGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGC AGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTAC AATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGT GGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTA TCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：26 PPR-EcRS-1 (propargyloxy phenylalanine synzyme) (avtive spot) synzyme polynucleotide CGGGGGCTGGTANCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCGGGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATTCTATGGCCTGTTTGAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG ACCTGANCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：27 PPR-EcRS-2 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAT CTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG AACCTGANCCGTCGTCTGCATCAAAATCAAGTG
SEQ ID NO.：28 PPR-EcRS-3 (avtive spot) synzyme polynucleotide CGGGGGCTGGTACCCCAAGTGACGGACGAGGAAACGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCTCTTGTGGCTTCGATCCTACCGCTGA CAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCC AGCAGGCAGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTC TGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGA AGAAA CTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCC GTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATT
ATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGATGGCCTGTGTGAACAAACAGTACGGTGTGG TGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATC GACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：29 PPR-EcRS-4 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCGCGTGCGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAGGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATGAATGTGCTGACCTTCCTGCGCGATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATTCTTATGCCTGTCTTAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG ACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：30 PPR-EcRS-5 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCGCGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGATGGCCTGTTGTAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG ACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：31 PPR-EcRS-6 (avtive spot) synzyme polynucleotide CGGGGGCTGGTACCCCAAGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCGCTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGTTTGCCTGTATGAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG ACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：32 PPR-EcRS-7 (avtive spot) synzyme polynucleotide GTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATC GCACTCACGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCA TCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGC CGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTT CAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTG GGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGT GGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATAT GAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACC AGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCA GGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGG CTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCT GACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCA TCAGAATCAGGTG
SEQ ID NO.：33 PPR-EcRS-8 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCTTCGATCCTACCGCTGA CAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCC AGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTC
TGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGA AGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCC GTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATT ATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATTCGATGGCCTGTACGAACAAACAGTACGGTGTGG TGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATC GACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：34 PPR-EcRS-9 (avtive spot) synzyme polynucleotide CGGGGGCTGGTANCCCAAGTGACGGACGGGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCAGTTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATCTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATAGTTTTGCCTGTCTGAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG ACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：35 PPR-EcRS-10 (avtive spot) synzyme polynucleotide CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCG AAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCC CGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAAT TATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGC AAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGC GTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAAC CTGCTGCAGGGTTATACGTTTGCCTGTACTAACAAACAGTACGGTGTGGT GCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCG ACCTGACCCGTCGTCTGCATCAGAATCAGGTG
SEQ ID NO.：36 Right-iodo PheRS-1 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSYACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：37 Right-iodo PheRS-2 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWG NITSGIILTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDUFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：38 Right-iodo PheRS-3 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEETNALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITTNGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：39 OMeTyrRS-1 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：40 OMeTyrRS-2 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYTMACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：41 OMeTyrRS-3 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIAITCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYTYACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：42 OMeTyrRS-4 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACSNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：43 OMeTyrRS-5 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：44 OMeTyrRS-6 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYRMACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：45 Right-acetyl group PheRS-1 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHL VPLLCLKRFQQAGHKKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYGMACANKQYGVVLQIGGSDQW GNITSGIDLTRRLHQNQVFGITVPLITKADGTKFGKTEGGAVWLDPKKTSP YKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLA EQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEME KGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF
GRFTLLRRGKKNYCLICWK
SEQ ID NO.：46 Right-benzoyl PheRS-1 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYGFACANKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWITADADVYRFLKFFTFMSIEEIALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：47 Right-benzoyl PheRS-2 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYGYACMNKQYGVVLQIGGSDQW GNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSP YKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLA EQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEME KGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWK
SEQ ID NO.：48 Right-azido PheRS-1 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWG NITSGIDTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：49 Right-azido PheRS-2 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSAACANKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：50 Right-azido PheRS-3 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSAACANKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：51 Right-azido PheRS-4 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSAACVNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：52 Right-azido PheRS-5 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANDYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYNFACVNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE
QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：53 Right-azido PheRS-6 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSALAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSAACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGITVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：54 PPR-EcRS-1 synzyme amino acid (aa) is right-propargyloxy phenylalanine synzyme MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWG NITSGIDLTRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.55 PPR-Ec RS-2 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSAACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：56 PPR-EcRS-3 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALSCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYTMACVNKQYGVVLQIGGSDQW GNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSP YKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLA EQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEME KGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWK
SEQ ID NO.：57 PPR-EcRS-4 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALACGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSYACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：58 PPR-EcRS-5 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALACGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYTMACCNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSG SLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：59 PPR-EcRS-6 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYTFACMNKQYGVVLQIGGSDQWG
NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：60 PPR-EcRS-7 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSVACLNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：61 PPR-EcRS-8 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSMACTNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：62 PPR-EcRS-9 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALSCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVA LVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYSFACLNKQYGVVLQIGGSDQWG NITSGIDITRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：63 PPR-EcRS-10 synzyme amino acid (aa) MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREDQGISFTEFSYNLLQGYTFACTNKQYGVVLQIGGSDQWG NITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPY KFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAE QVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEK GADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGR FTLLRRGKKNYCLICWK
SEQ ID NO.：64 The tRNA/Tyr polynucleotide AGCTTCCCGATAAGGGAGCAGGCCAGTAAAAAGCATTACCCCGTGGTG GGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCG ACCTCGAAGGTTCGAATCCTTCCCCCACCACCA
SEQ ID NO.：65 tRNA/Tyr AGCUUCCCGAUAAGGGAGCAGGCCAGUAAAAAGCAUUACCCCGUGGU GGGGUUCCCGAGCGGCCAAAGGGAGCAGACUCUAAAUCUGCCGUCAU CGACCUCGAAGGUUCGAAUCCUUCCCCCACCACCA
SEQ ID NO.：66 Amber mutant L3TAG 5’-ATGAAGTAGCTGTCTTCTATCGAACAAGCATGCG-3’
SEQ ID NO.：67 Amber mutant I13TAG 5’-CGAACAAGCATGCGATTAGTGCCGACTTAAAAAG-3’
SEQ ID NO.：68 Amber mutant T44TAG 5’-CGCTACTCTCCCAAATAGAAAAGGTCTCCGCTG-3’
SEQ ID NO.：69 Amber mutant F68TAG 5’-CTGGAACAGCTATAGCTACTGATTTTTCCTCG-3’
SEQ ID NO.：70 Amber mutant R110TAG 5’-GCCGTCACAGATTAGTTGGCTTCAGTGGAGACTG-3’
SEQ ID NO.：71 Amber mutant V114TAG 5’-GATTGGCTTCATAGGAGACTGATATGCTCTAAC-3’
SEQ ID NO.：72 Amber mutant T121TAG 5’-GCCTCTATAGTTGAGACAGCATAGAATAATGCG-3’
SEQ ID NO.：73 Amber mutant I127TAG 5’-GAGACAGCATAGATAGAGTGCGACATCATCATCGG-3’
SEQ ID NO.：74 Amber mutant S131TAG 5’-GAATAAGTGCGACATAGTCATCGGAAGAGAGTAGTAG-3’
SEQ ID NO.：75 Amber mutant T145TAG 5’-GGTCAAAGACAGTTGTAGGTATCGATTGACTCGGC-3’
SEQ ID NO.：76 Allow site mutation body T44F 5’-CGCTACTCTCCCCAAATTTAAAAGGTCTCCGCTG-3’
SEQ ID NO.：77 Allow site mutation body T44Y 5’-CGCTACTCTCCCCAAATATAAAAGGTCTCCGCTG-3’
SEQ ID NO.：78 Allow site mutation body T44W 5’-CGCTACTCTCCCCAAATGGAAAAGGTCTCCGCTG-3’
SEQ ID NO.：79 Allow site mutation body T44D 5’-CGCTACTCTCCCCAAAGATAAAAGGTCTCCGCTG-3’
SEQ ID NO.：80 Allow site mutation body T44K 5’-CGCTACTCTCCCCAAAAAAAAAAGGTCTCCGCTG-3’
SEQ ID NO.：81 Allow site mutation body R110F 5’-GCCGTCACAGATTTTTTGGCTTCAGTGGAGACTG-3’
SEQ ID NO.：82 Allow site mutation body R110Y 5’-GCCGTCACAGATTATTTGGCTTCAGTGGAGACTG-3’
SEQ ID NO.：83 Allow site mutation body R110W 5’-GCCGTCACAGATTGGTTGGCTTCAGTGGAGACTG-3’
SEQ ID NO.：84 Allow site mutation body R110D 5’-GCCGTCACAGATGATTTGGCTTCAGTGGAGACTG-3’
SEQ ID NO.：85 Allow site mutation body R110K 5’-GCCGTCACAGATAAATTGGCTTCAGTGGAGACTG-3’
SEQ ID NO.：86 Right-acetyl group PheRS-1 synzyme amino acid (aa) a MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHL VPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVD KIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMIN KEAVKQRLNREGQGISFTEFSYNLLQGYGMACANKQYGVVLQIGGSDQW GNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSP YKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLA EQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEME KGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWK
aThese clones also contain the Asp165Gly sudden change
Sequence table
＜110〉Scripps Research Inst (The Scipps Research Institute)
A. Dai Tesi (Deiters, Alexander)
A.T. Crow general (Cropp, T Ashton)
J.W. (Chin, Jason W) admires
C.J. Anderson (Anderson, J Chri stopher)
P.G. Shu Erci (Schultz, Peter G)
＜120〉the unnatural reactive amino acid genetic code increases
<130>54-000250US/PC
<160>104
<170>PatentIn version 3.3
<210>1
<211>1275
<212>DNA
＜213〉Escherichia coli (Escherichia ccli)
<400>1
atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcgctcta ttgcggcttc 120
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 180
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 240
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 300
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360
gctatcgcgg cgaacaacta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 480
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gttgcagggt 540
tatgacttcg cctgtctgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660
tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720
ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg 780
atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt 840
gaagagatca acgccctgga agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900
tatgtactgg cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca 960
aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020
gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg 1080
caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa aactatcgcc 1140
tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200
gaagatcgtc tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260
atttgctgga aataa 1275
<210>2
<211>424
<212>PRT
＜213〉Escherichia coli (Escherichia coli)
<400>2
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Tyr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Asp Phe Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu Hi s Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>3
<211>1275
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>3
atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcgt gtgtggcttc 120
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 180
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 240
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 300
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360
gctatcgcgg ccaataatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 480
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gctgcagggt 540
tatagtatgg cctgtttgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660
tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720
ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg 780
atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt 840
gaagagatca acgccctgga agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900
tatgtactgg cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca 960
aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggactt 1020
gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg 1080
caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa aactatcgcc 1140
tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200
gaagatcgtc tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260
atttgctgga aataa 1275
<210>4
<211>1275
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>4
atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcac ttgtggcttc 120
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 180
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 240
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 300
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360
gctatcgcgg ccaataatta tgactggttc agcaatatga atgtgctgac cttcctgcgc 420
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 480
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gctgcagggt 540
tatacgtatg cctgtctgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660
tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720
ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg 780
atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt 840
gaagagatca acgccctgga agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900
tatgtactgg cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca 960
aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020
gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg 1080
caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa aactatcgcc 1140
tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200
gaagatcgtc tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260
atttgctgga aataa 1275
<210>5
<211>1275
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>5
atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcgt gtgtggcttc 120
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 180
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 240
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 300
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360
gctatcgcgg ccaataatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 480
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gctgcagggt 540
tatagtatgg cctgtttgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660
tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720
ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg 780
atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt 840
gaagagatca acgccctgga agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900
tatgtactgg cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca 960
aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020
gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg 1080
caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa aactatcgcc 1140
tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200
gaagatcgtc tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260
atttgctgga aataa 1275
<210>6
<211>1275
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>6
atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcgt gtgtggcttc 120
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 180
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 240
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 300
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360
gctatcgcgg ccaataatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 480
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gctgcagggt 540
tatagtatgg cctgtttgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660
tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720
ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg 780
atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt 840
gaagagatca acgccctgga agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900
tatgtactgg cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca 960
aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020
gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg 1080
caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa aactatcgcc 1140
tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200
gaagatcgtc tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260
atttgctgga aataa 1275
<210>7
<211>1275
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>7
atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcac gtgtggcttc 120
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 180
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 240
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 300
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360
gctatcgcgg ccaataatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 480
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacagcct gctgcagggt 540
tatacgatgg cctgtctgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660
tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720
ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg 780
atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt tatgagcatt 840
gaagagatca acgccctgga agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900
tatgtactgg cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca 960
aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020
gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg 1080
caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa aactatcgcc 1140
tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200
gaagatcgtc tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260
atttgctgga aataa 1275
<210>8
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>8
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcagcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg tatgcctgtc tgaacaaaca gtacggtgtg 540
<210>9
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>9
cgggggctgg taccccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcagcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg tatgcctgtc tgaacaaaca gtacggtgtg 540
<210>10
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>10
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcagcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg tatgcctgtc tgaacaaaca gtacggtgtg 540
<210>11
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>11
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttattcg tatgcctgtg cgaacaaaca gtacggtgtg 540
<210>12
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>12
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcagcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg tatgcctgtc tgaacaaaca gtacggtgtg 540
<210>13
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>13
cgggggctgg taccccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcctttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttattct attgcctgtt cgaacaaaca gtacggtgtg 540
<210>14
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>14
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcgtgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatagt attgcctgtt tgaacaaaca gtacggtgtg 540
<210>15
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>15
cgggggctgg taccccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcgtgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatagt attgcctgtt tgaacaaaca gtacggtgtg 540
<210>16
<211>540
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>16
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tctggtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaagg ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaatt gttatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatatg cgtgcctgtg agaacaaaca gtacggtgtg 540
<210>17
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>17
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcatttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaaggtc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatggt atggcctgtg ctaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccaatgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>18
<211>609
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>18
caggtgacgg acgaggaagc gttagcagag cgactggcgc aaggcccgat cgcactcggt 60
tgtggcttcg atcctaccgc tgacagcttg catttggggc atcttgttcc attgttatgc 120
ctggaacgct tccagcaggc gggccacaag ccggttgcgc tggtaggcgg cgcgacgggt 180
ctgattggcg acccgagctt caaagctgcc gagcgtaagc tgaacaccga agaaactgtt 240
caggagtggg tggacaaaat ccgtaagcag gttgccccgt tcctcgattt cgactgtgga 300
gaaaactctg ctatcgcggc caataattat gactggttcg gcaatatgaa tgtgctgacc 360
ttcctgcgcg atattggcaa acacttctcc gttaaccaga tgatcaacaa agaagcggtt 420
aagcagcgtc tcaaccgtga agatcagggg atttcgttca ctgagttttc ctacaacctg 480
ctgcagggtt atggttttgc ctgtttgaac aaacagtacg gtgtggtgct gcaaattggt 540
ggttctgacc agtggggtaa catcacttct ggtatcgacc tgacccgtcg tctgcatcag 600
aatcaggtg 609
<210>19
<211>591
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>19
gcgttagcag agcgactggc gcaaggcccg atcgcactcg ggtgtggctt cgatcctacc 60
gctgacagct tgcatttggg gcatcttgtt ccattgttat gcctgaaacg cttccagcag 120
gcgggccaca agccggttgc gctggtaggc ggcgcgacgg gtctgattgg cgacccgagc 180
ttcaaagctg ccgagcgtaa gctgaacacc gaagaaactg ttcaggagtg ggtggacaaa 240
atccgtaagc aggttgcccc gttcctcgat ttcgactgtg gagaaaactc tgctatcgcg 300
gccaataatt atgactggtt cggcaatatg aatgtgctga ccttcctgcg cgatattggc 360
aaacacttct ccgttaacca gatgatcaac aaagaagcgg ttaagcagcg tctcaaccgt 420
gaagatcagg ggatttcgtt cactgagttt tcctacaacc tgctgcaggg ttatggttat 480
gcctgtatga acaaacagta cggtgtggtg ctgcaaattg gtggttctga ccagtggggt 540
aacatcactt ctggtatcga cctgacccgt cgtctgcatc agaatcaggt g 591
<210>20
<211>621
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<220>
<221>misc_feature
<222>(26)..(26)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(612)..(612)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(618)..(618)
＜223〉n is a, c, g or t
<400>20
gggctggtag cccaggtgac ggacgnagaa gcgttagcag agcgactggc gcaaggcccg 60
atcgcactcc tttgtggctt cgatcctacc gctgacagct tgcatttggg gcatcttgtt 120
ccattgttat gcctgaaacg cttccagcag gcgggccaca agccggttgc gctggtaggc 180
ggcgcgacgg gtctgattgg cgacccgagc ttcaaagctg ccgagcgtaa gctgaacacc 240
gaagaaactg ttcaggagtg ggtggacaaa atccgtaagc aggttgcccc gttcctcgat 300
ttcgactgtg gagaaaactc tgctatcgcg gccaataatt atgactggtt cggcaatatg 360
aatgtgctga ccttcctgcg cgatattggc aaacacttct ccgttaacca gatgatcaac 420
aaagaagcgg ttaagcagcg tctcaaccgt gaagatcagg ggatttcgtt cactgagttt 480
tcctacaacc tgctgcaggg ttattctatg gcctgtgcga acaaacagta cggtgtggtg 540
ctgcaaattg gtggttctga ccagtggggt aacatcactt ctggtatcga cctgacccgt 600
cgtctgcatc anaatcangt g 621
<210>21
<211>588
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>21
ttagcagagc gactggcgca aggcccgatc gcactcgttt gtggcttcga tcctaccgct 60
gacagcttgc atttggggca tcttgttcca ttgttatgcc tgaaacgctt ccagcaggcg 120
ggccacaagc cggttgcgct ggtaggcggc gcgacgggtc tgattggcga cccgagcttc 180
aaagctgccg agcgtaagct gaacaccgaa gaaactgttc aggagtgggt ggacaaaatc 240
cgtaagcagg ttgccccgtt cctcgatttc gactgtggag aaaactctgc tatcgcggcc 300
aataattatg actggttcgg caatatgaat gtgctgacct tcctgcgcga tattggcaaa 360
cacttctccg ttaaccagat gatcaacaaa gaagcggtta agcagcgtct caaccgtgaa 420
gatcagggga tttcgttcac tgagttttcc tacaacctgc tgcagggtta ttctgcggcc 480
tgtgcgaaca aacagtacgg tgtggtgctg caaattggtg gttctgacca gtggggtaac 540
atcacttctg gtatcgacct gacccgtcgt ctgcatcaga atcaggtg 588
<210>22
<211>600
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<220>
<221>misc_feature
<222>(403)..(403)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(513)..(513)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(515)..(515)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(518)..(518)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(531)..(531)
＜223〉n is a, c, g or t
<400>22
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcct gtgtggcttc 60
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 120
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 180
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 240
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 300
gctatcgcgg ccaataatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 360
gatattggca aacacttctc cgttaaccag atgatcaaca aanaagcggt taagcagcgt 420
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gctgcagggt 480
tattcggctg cctgtgcgaa caaacagtac ggngnggngc tgcaaattgg nggttctgac 540
caggggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca aaatcaggtg 600
<210>23
<211>591
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<220>
<221>misc_feature
<222>(588)..(588)
＜223〉n is a, c, g or t
<400>23
gcgttagcag agcgactggc gcaaggcccg atcgcactcg tttgtggctt cgatcctacc 60
gctgacagct tgcatttggg gcatcttgtt ccattgttgt gcctgaaacg cttccagcag 120
gcgggccaca agccggttgc gctggtaggc ggcgcgacgg gtctgattgg cgacccgagc 180
ttcaaagctg ccgagcgtaa gctgaacacc gaagaaactg ttcaggagtg ggtggacaaa 240
atccgtaagc aggttgcccc gttcctcgat ttcgactgtg gagaaaactc tgctatcgcg 300
gccaataatt atgactggtt cggcaatatg aatgtgctga ccttcctgcg cgatattggc 360
aaacacttct ccgttaacca gatgatcaac aaagaagcgg ttaagcagcg tctcaaccgt 420
gaagatcagg ggatttcgtt cactgagttt tcctacaacc tgctgcaggg ttatagtgcg 480
gcctgtgtta acaaacagta cggtgtggtg ctgcaaattg gtggttctga ccagtggggt 540
aacatcactt ctggtatcga cctgacccgt cgtctgcatc agaatcangt g 591
<210>24
<211>600
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>24
gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcat ttgtggcttc 60
gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc 120
ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg tctgattggc 180
gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg aagaaactgt tcaggagtgg 240
gtggacaaaa tccgtaagca ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 300
gctatcgcgg ccaatgatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc 360
gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt 420
ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct gctgcagggt 480
tataattttg cctgtgtgaa caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 540
cagtggggta acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 600
<210>25
<211>579
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>25
cgactggcgc aaggcccgat cgcactcacg tgtggcttcg atcctaccgc tgacagcttg 60
catttggggc atcttgttcc attgttatgc ctgaaacgct tccagcaggc gggccacaag 120
ccggttgcgc tggtaggcgg cgcgacgggt ctgattggcg acccgagctt caaagctgcc 180
gagcgtaagc tgaacaccga agaaactgtt caggagtggg tggacaaaat ccgtaagcag 240
gttgccccgt tcctcgattt cgactgtgga gaaaactctg ctatcgcggc caataattat 300
gactggttcg gcaatatgaa tgtgctgacc ttcctgcgcg atattggcaa acacttctcc 360
gttaaccaga tgatcaacaa agaagcggtt aagcagcgtc tcaaccgtga agatcagggg 420
atttcgttca ctgagttttc ctacaatctg ctgcagggtt attcggctgc ctgtcttaac 480
aaacagtacg gtgtggtgct gcaaattggt ggttctgacc agtggggtaa catcacttct 540
ggtatcgacc tgacccgtcg tctgcatcag aatcaggtg 579
<210>26
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉n is a, c, g or t
<220>
<221>misc_feature
<222>(599)..(599)
＜223〉n is a, c, g or t
<400>26
cgggggctgg tancccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcgggtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttattct atggcctgtt tgaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctganc 600
cgtcgtctgc atcagaatca ggtg 624
<210>27
<211>625
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<220>
<221>misc_feature
<222>(600)..(600)
＜223〉n is a, c, g or t
<400>27
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca atctgctgca gggttattcg gctgcctgtc ttaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgaacctgan 600
ccgtcgtctg catcaaaatc aagtg 625
<210>28
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>28
cgggggctgg taccccaagt gacggacgag gaaacgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tctcttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcaggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg atggcctgtg tgaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>29
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>29
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcgcgtgcgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaagg ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttattct tatgcctgtc ttaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>30
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>30
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcgcgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg atggcctgtt gtaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>31
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>31
cgggggctgg taccccaagt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcgctgag 480
ttttcctaca acctgctgca gggttatacg tttgcctgta tgaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>32
<211>606
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>32
gtgacggacg aggaagcgtt agcagagcga ctggcgcaag gcccgatcgc actcacgtgt 60
ggcttcgatc ctaccgctga cagcttgcat ttggggcatc ttgttccatt gttatgcctg 120
aaacgcttcc agcaggcggg ccacaagccg gttgcgctgg taggcggcgc gacgggtctg 180
attggcgacc cgagcttcaa agctgccgag cgtaagctga acaccgaaga aactgttcag 240
gagtgggtgg acaaaatccg taagcaggtt gccccgttcc tcgatttcga ctgtggagaa 300
aactctgcta tcgcggccaa taattatgac tggttcggca atatgaatgt gctgaccttc 360
ctgcgcgata ttggcaaaca cttctccgtt aaccagatga tcaacaaaga agcggttaag 420
cagcgtctca accgtgaaga tcaggggatt tcgttcactg agttttccta caatctgctg 480
cagggttatt cggctgcctg tcttaacaaa cagtacggtg tggtgctgca aattggtggt 540
tctgaccagt ggggtaacat cacttctggt atcgacctga cccgtcgtct gcatcagaat 600
caggtg 606
<210>33
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>33
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcgtttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttattcg atggcctgta cgaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>34
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉n is a, c, g or t
<400>34
cgggggctgg tancccaagt gacggacggg gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcagttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatctcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatagt tttgcctgtc tgaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>35
<211>624
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>35
cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60
ccgatcgcac tcacgtgtgg cttcgactct accgctgaca gcttgcattt ggggcatctt 120
gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta 180
ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg taagctgaac 240
accgaagaaa ctgttcagga gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300
gatttcgact gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat 360
atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420
aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag 480
ttttcctaca acctgctgca gggttatacg tttgcctgta ctaacaaaca gtacggtgtg 540
gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600
cgtcgtctgc atcagaatca ggtg 624
<210>36
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>36
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Tyr Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>37
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>37
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro LeuIle Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn 6ly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>38
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>38
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys ArgIle Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>39
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>39
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln GluArg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp ValAsp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn MetAsn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>40
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>40
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Thr Met Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu Hi s Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>41
<21l>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>41
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
ProIle Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Thr Tyr Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>42
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>42
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Ser Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>43
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>43
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
2l0 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>44
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>44
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Arg Met Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>45
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>45
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Gly Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val Hi s Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>46
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>46
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Gly Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Gly Phe Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu CysLeu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>47
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>47
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Gly Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Gly Tyr Ala Cys Met Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val Hi s Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala I1e
370 375 380
Thr Ile Asn Cly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>48
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>48
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>49
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>49
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Ala Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>50
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>50
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Ala Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>51
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>51
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Ala Ala Cys Val Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>52
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>52
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asp Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Asn Phe Ala Cys Val Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>53
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>53
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Ala Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>54
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>54
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Gly Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val Hi s Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>55
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>55
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu V8l Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Ala Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>56
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>56
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ser Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser AlaIle Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Thr Met Ala Cys Val Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val Hi s Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>57
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>57
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ala Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Tyr Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu Hi s Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg PhcThr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>58
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>58
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ala Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Thr Met Ala Cys Cys Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>59
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>59
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp LysIle Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Thr Phe Ala Cys Met Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala AspGly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>60
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>60
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Val Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>61
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>61
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu ValGly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Met Ala Cys Thr Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>62
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>62
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ser Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Ser Phe Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>63
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>63
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Thr Phe Ala Cys Thr Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>64
<211>129
<212>DNA
＜213〉Escherichia coli (Escherichia coli)
<400>64
agcttcccga taagggagca ggccagtaaa aagcattacc ccgtggtggg gttcccgagc 60
ggccaaaggg agcagactct aaatctgccg tcatcgacct cgaaggttcg aatccttccc 120
ccaccacca 129
<210>65
<211>129
<212>RNA
＜213〉Escherichia coli (Escherichia coli)
<400>65
agcuucccga uaagggagca ggccaguaaa aagcauuacc ccgugguggg guucccgagc 60
ggccaaaggg agcagacucu aaaucugccg ucaucgaccu cgaagguucg aauccuuccc 120
ccaccacca 129
<210>66
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>66
atgaagtagc tgtcttctat cgaacaagca tgcg 34
<210>67
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>67
cgaacaagca tgcgattagt gccgacttaa aaag 34
<210>68
<211>33
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>68
cgctactctc ccaaatagaa aaggtctccg ctg 33
<210>69
<211>32
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>69
ctggaacagc tatagctact gatttttcct cg 32
<210>70
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>70
gccgtcacag attagttggc ttcagtggag actg 34
<210>71
<211>33
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>71
gattggcttc ataggagact gatatgctct aac 33
<210>72
<211>33
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>72
gcctctatag ttgagacagc atagaataat gcg 33
<210>73
<211>35
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>73
gagacagcat agatagagtg cgacatcatc atcgg 35
<210>74
<211>37
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>74
gaataagtgc gacatagtca tcggaagaga gtagtag 37
<210>75
<211>35
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>75
ggtcaaagac agttgtaggt atcgattgac tcggc 35
<210>76
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>76
cgctactctc cccaaattta aaaggtctcc gctg 34
<210>77
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>77
cgctactctc cccaaatata aaaggtctcc gctg 34
<210>78
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>78
cgctactctc cccaaatgga aaaggtctcc gctg 34
<210>79
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>79
cgctactctc cccaaagata aaaggtctcc gctg 34
<210>80
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>80
cgctactctc cccaaaaaaa aaaggtctcc gctg 34
<210>81
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>81
gccgtcacag attttttggc ttcagtggag actg 34
<210>82
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>82
gccgtcacag attatttggc ttcagtggag actg 34
<210>83
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>83
gccgtcacag attggttggc ttcagtggag actg 34
<210>84
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>84
gccgtcacag atgatttggc ttcagtggag actg 34
<210>85
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>85
gccgtcacag ataaattggc ttcagtggag actg 34
<210>86
<211>424
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic enzyme
<400>86
Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val
1 5 10 15
Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30
Pro Ile Ala Leu Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His
35 40 45
Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala
50 55 60
Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly
65 70 75 80
Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95
Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu
100 105 110
Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp
115 120 125
Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys
130 135 140
His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg
145 150 155 160
Leu Asn Arg Glu Gly Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn
165 170 175
Leu Leu Gln Gly Tyr Gly Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190
Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly
195 200 205
Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr
210 215 220
Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu
225 230 235 240
Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe
245 250 255
Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270
Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu
275 280 285
Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala
290 295 300
Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala
305 310 315 320
Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335
Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu
340 345 350
Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu
355 360 365
Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile
370 375 380
Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu
385 390 395 400
Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys
405 410 415
Asn Tyr Cys Leu Ile Cys Trp Lys
420
<210>87
<211>6
<212>PRT
＜213〉artificial
<220>
＜223〉comprise the tryptic peptide of alpha-non-natural amino acid
<220>
<221>MISC_FEATURE
<222>(2)..(2)
＜223〉X is alpha-non-natural amino acid (right-acetyl group-the L-phenylalanine, right-benzoyl-the L-phenylalanine, right-azido-the L-phenylalanine, 0-methyl-L-tyrosine or right-iodo-L-phenylalanine) or tryptophane, tyrosine or leucine
<400>87
Val Xaa Gly Ser Ile Lys
1 5
<210>88
<211>11
<212>DNA
＜213〉artificial
<220>
＜223〉B frame
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉n is a, c, g or t
<400>88
ggttcgantc c 11
<210>89
<211>82
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>89
ggggggaccg gtggggggac cggtaagctt cccgataagg gagcaggcca gtaaaaagca 60
ttaccccgtg gtgggttccc ga 82
<210>90
<211>90
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>90
ggcggcgcta gcaagcttcc cgataaggga gcaggccagt aaaaagggaa gttcagggac 60
ttttgaaaaa aatggtggtg ggggaaggat 90
<210>91
<211>68
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<220>
<221>misc_feature
<222>(1)..(1)
<223>n＝I
<220>
<221>misc_feature
<222>(14)..(14)
<223>n＝I
<400>91
nggggggacc ggtngggggg accggtcggg atcgaagaaa tgatggtaaa tgaaatagga 60
aatcaagg 68
<210>92
<211>62
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>92
gggggggaat tcagttgatt gtatgcttgg tatagcttga aatattgtgc agaaaaagaa 60
ac 62
<210>93
<211>86
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>93
tcataacgag aattccggga tcgaagaaat gatggtaaat gaaataggaa atctcataac 60
gagaattcat ggcaagcagt aacttg 86
<210>94
<211>72
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>94
ttactacgtg cggccgcatg gcaagcagta acttgttact acgtgcggcc gcttatttcc 60
agcaaatcag ac 72
<210>95
<211>28
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>95
ccgatcgcgc tcgcttgcgg cttcgatc 28
<210>96
<211>27
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>96
atcgcggcga acgcctatga ctggttc 27
<210>97
<211>40
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>97
gttgcagggt tatgccgccg cctgtgcgaa caaacagtac 40
<210>98
<211>26
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>98
gccgctttgc tatcaagtat aaatag 26
<210>99
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>99
caagccgaca accttgattg g 21
<210>100
<211>60
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>100
ggggacaagt ttgtacaaaa aagcaggcta cgccaatttt aatcaaagtg ggaatattgc 60
<210>101
<211>60
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>101
ggggacaagt ttgtacaaaa aagcaggcta ggccaatttt aatcaaagtg ggaatattgc 60
<210>102
<211>58
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>102
ggggaccact ttgtacaaga aagctgggtt actctttttt tgggtttggt ggggtatc 58
<210>103
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>103
aagctatacc aagcatacaa tc 22
<210>104
<211>49
<212>DNA
＜213〉artificial
<220>
＜223〉Oligonucleolide primers
<400>104
acaaggcctt gctagcttac tctttttttg ggtttggtgg ggtatcttc 49

Claims (61)

1. composition that contains albumen, wherein this albumen comprises at least a alpha-non-natural amino acid and at least one posttranslational modification, and wherein said at least one posttranslational modification is that the molecule that will contain second reactive group is attached on the described at least a alpha-non-natural amino acid that contains first reactive group by [3+2] cycloaddition.
2. composition as claimed in claim 1, it is characterized in that described molecule is dyestuff, polymkeric substance, polyethyleneglycol derivative, photocrosslinking agent, cytotoxic compound, affinity labeling, biotin derivative, resin, second kind of protein or polypeptide, metal-chelator, co-factor, fatty acid, carbohydrates or polynucleotide.
3. composition as claimed in claim 1 is characterized in that, described first reactive group is alkynyl or azido part, and described second reactive group is azido or alkynyl part.
4. composition as claimed in claim 3 is characterized in that, first reactive group is the alkynyl part, and second reactive group is the azido part.
5. composition as claimed in claim 4 is characterized in that, described alpha-non-natural amino acid comprises right-propargyloxy phenylalanine.
6. composition as claimed in claim 3 is characterized in that, described first reactive group is the azido part, and described second reactive group is the alkynyl part.
7. composition as claimed in claim 6 is characterized in that, described alpha-non-natural amino acid comprises right-azido-L-phenylalanine.
8. composition as claimed in claim 6 is characterized in that, described at least one posttranslational modification is carried out in the body in eukaryotic.
9. composition that comprises alpha-non-natural amino acid with following chemical constitution:
Figure A2004800211550002C1
10. composition as claimed in claim 9 also comprises quadrature tRNA.
11. composition as claimed in claim 10 is characterized in that, described alpha-non-natural amino acid is covalently bound to quadrature tRNA.
12. composition as claimed in claim 10 is characterized in that, described alpha-non-natural amino acid is covalently bound to quadrature tRNA by amino-acyl bond.
13. composition as claimed in claim 10 is characterized in that, described alpha-non-natural amino acid is covalently bound to the 3 ' OH or the 2 ' OH of the terminal ribose of quadrature tRNA.
14. protein that contains the described alpha-non-natural amino acid of claim 9.
15. cell that contains the described alpha-non-natural amino acid of claim 9.
16. composition that comprises azido dyestuff with following structure:
17. composition that comprises azido dyestuff with following structure:
Figure A2004800211550003C2
18. protein that contains claim 16 or 17 described azido dyestuffs.
19. protein as claimed in claim 18 also comprises at least a alpha-non-natural amino acid, wherein the azido dyestuff is attached on this alpha-non-natural amino acid by [3+2] cycloaddition.
20. protein as claimed in claim 19 is characterized in that, described alpha-non-natural amino acid comprises alkynyl amino acid.
21. composition that comprises alkynyl polyglycol with following structure:
Figure A2004800211550003C3
Wherein n is the integer between 100 and 2,000.
22. composition as claimed in claim 21 is characterized in that, the molecular weight of described alkynyl polyglycol is about 5,000 to about 100,000Da.
23. protein that contains the described alkynyl polyglycol of claim 21.
24. protein as claimed in claim 23 also comprises at least a alpha-non-natural amino acid, wherein said alkynyl polyglycol is attached on this alpha-non-natural amino acid by [3+2] cycloaddition.
25. protein as claimed in claim 24 is characterized in that, described alpha-non-natural amino acid comprises azido amino acid.
26. the method for synthetic right-(propargyloxy) phenylalanine compound, this method comprises:
With uncle N--butoxy carbonyl-tyrosine and K 2CO 3Be suspended in the dry DMF;
Propargyl bromide is added in the reaction mixture of (a), alkanisation hydroxyl and carboxylic group produce the protection intermediate compound with following structure:
Figure A2004800211550004C1
With
To protect intermediate compound in MeOH, to mix, and make amine moiety go protection with anhydrous HCl, thus synthetic right-(propargyloxy) phenylalanine compound.
27. method as claimed in claim 26 also comprises:
Right-(propargyloxy) phenylalanine HCl is dissolved in NaOH and the MeOH solution stirring at room;
PH is adjusted to 7; With
Precipitate right-(propargyloxy) phenylalanine compound.
28. the method for a synthetic azido dyestuff, this method comprises:
The dye composition that contains sulfonyl halogenide part is provided;
In the presence of 3-azido propylamine and triethylamine, dye composition is heated to room temperature; With
The amine moiety of 3-azido propylamine is coupled to the halogen position of dye composition, thereby synthesizes the azido dyestuff.
29. method as claimed in claim 28, wherein said dye composition contains dansyl Cl, and wherein said azido dyestuff contains the described composition of claim 16.
30. method as claimed in claim 28 also comprises:
The described azido dyestuff of purifying from reaction mixture.
31. the method for a synthetic azido dyestuff, this method comprises:
Provide and contain the amine dye composition;
To contain the amine dye composition and in suitable solvent, mix with carbodiimide and 4-(3-azido propyl group carbamyl)-butyric acid, with the amine moiety coupling of the carbonyl and the dye composition of acid, thus synthetic azido dyestuff.
32. method as claimed in claim 31 is characterized in that, described carbodiimide comprises 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCI).
33. method as claimed in claim 31 is characterized in that, the described amine dyestuff that contains comprises fluorescein amine, and described suitable solvent comprises pyridine.
34. method as claimed in claim 31 is characterized in that, the described amine dyestuff that contains comprises fluorescein amine, and described azido dyestuff comprises the described composition of claim 17.
35. method as claimed in claim 31 also comprises:
Precipitation azido dyestuff;
Use the HCl washing precipitation;
With washed resolution of precipitate in EtOAc; With
Precipitation azido dyestuff in hexane.
36. the method for a synthetic propargyl acid amides polyglycol, this method comprises: at room temperature propargyl amine and polyglycol (PEG)-hydroxysuccinimide eater are reacted in organic solvent, produce the described propargyl acid amides of claim 21 polyglycol.
37. method as claimed in claim 36 is characterized in that, described organic solvent comprises CH 2Cl 2
38. method as claimed in claim 36 also comprises: with ethyl acetate precipitation propargyl acid amides polyglycol.
39. method as claimed in claim 38 also comprises: crystallization propargyl acid amides polyglycol again in methyl alcohol; With desciccate under the vacuum.
40. an eukaryotic that contains quadrature aminoacyl-tRNA synthetase (O-RS), wherein, O-RS in eukaryotic preferably aminoacylation have the quadrature tRNA (O-tRNA) of at least one alpha-non-natural amino acid, wherein:
(a.) described O-RS or its part are by arbitrary listed polynucleotide sequence, their complementary polynucleotide sequence or their conservative variant coding among the SEQ ID NO.:20-25;
(b.) described O-RS comprises arbitrary listed amino acid sequence among the SEQ ID NO.:48-63, or its conservative variant;
(c.) described O-RS comprises the amino acid sequence identical with the tyrosyl aminoacyl-tRNA synthetase (TyrRS) at least 90% of natural generation, and comprises two or more amino acid that are selected from down group: with glycocoll, serine or the alanine on the Tyr37 opposite position of Escherichia coli TyrRS; With the aspartic acid on the Asn126 opposite position of Escherichia coli TyrRS; With the asparagine on the Asp182 opposite position of Escherichia coli TyrRS; With alanine or the valine on the Phe183 opposite position of Escherichia coli TyrRS; With methionine, valine, halfcystine or the threonine on the Leu186 opposite position of Escherichia coli TyrRS;
(d.) efficient of O-RS aminoacylation with O-tRNA of at least one alpha-non-natural amino acid be equivalent at least to have listed amino acid sequence among the SEQ ID NO.:45 O-RS 50%.
41. cell as claimed in claim 40; it is characterized in that; described cell also comprises quadrature tRNA (O-tRNA); this O-tRNA identification selection codon wherein; and preferably has at least one alpha-non-natural amino acid by the O-RS aminoacylation; wherein this O-tRNA produces by cell processing and the corresponding nucleic acid of SEQ ID NO.:65 in cell, and this O-RS comprises the peptide sequence that is selected from SEQ ID NO.:48-63 and their conservative variant.
42. a peptide species, it is selected from:
(a) comprise the polypeptide of arbitrary listed amino acid sequence among the SEQ ID NO.:48-63;
(b) comprise polypeptide by the amino acid sequence of arbitrary listed polynucleotide sequence coding among the SEQ ID NO.:20-35;
(c) polypeptide (a) or specific antibody (b) had the polypeptide of specific immune activity;
(d) comprise the amino acid sequence identical and comprise two or more amino acid whose polypeptide that are selected from down group: with glycocoll, serine or the alanine on the Tyr37 opposite position of Escherichia coli TyrRS with the tyrosyl aminoacyl-tRNA synthetase (TyrRS) at least 90% of natural generation; With the aspartic acid on the Asn126 opposite position of Escherichia coli TyrRS; With the asparagine on the Asp182 opposite position of Escherichia coli TyrRS; With alanine or the valine on the Phe183 opposite position of Escherichia coli TyrRS; With methionine, valine, halfcystine or the threonine on the Leu186 opposite position of Escherichia coli TyrRS;
(e) contain at least 20 continuous amino acids of SEQ ID NO.:36-48 or 86 and be selected from down the polypeptide of two or more aminoacid replacement of organizing: with glycocoll, serine or the alanine on the Tyr37 opposite position of Escherichia coli TyrRS; With the aspartic acid on the Asn126 opposite position of Escherichia coli TyrRS; With the asparagine on the Asp182 opposite position of Escherichia coli TyrRS; With alanine or the valine on the Phe183 opposite position of Escherichia coli TyrRS; With methionine, valine, halfcystine or the threonine on the Leu186 opposite position of Escherichia coli TyrRS; With
(f) contain the amino acid sequence of (a) and (b), (c), (d) or conservative variant (e).
43. composition that comprises described polypeptide of claim 42 and excipient.
44. one kind has the antibody or the antiserum of specific immune activity with the described polypeptide of claim 42.
45. composition that comprises described polypeptide of claim 42 and excipient.
46. one kind has the antibody or the antiserum of specific immune activity with the described polypeptide of claim 42.
47. polynucleotide that are selected from down group:
(a) comprise the polynucleotide of arbitrary listed nucleotide sequence among the SEQ ID NO.:20-35;
(b) polynucleotide of the polynucleotide sequence of or coding (a) complementary with (a) polynucleotide sequence;
(c) coding contains the polynucleotide of the polypeptide of arbitrary listed amino acid sequence among the SEQ ID NO.:48-63 or its conservative variant;
(d) polynucleotide of the described polypeptide of coding claim 42;
(e) nucleic acid of hybridizing under highly rigorous condition with total length nucleic acid and (a) and (b), (c) or polynucleotide (d) basically;
(f) polynucleotide of coded polypeptide, described polypeptide comprise the amino acid sequence identical with the sequence at least 90% of the tyrosyl aminoacyl-tRNA synthetase (TyrRS) of natural generation and contain two or more sudden changes that are selected from down group: with glycocoll, serine or the alanine on the Tyr37 opposite position of Escherichia coli TyrRS; With the aspartic acid on the Asn126 opposite position of Escherichia coli TyrRS; With the asparagine on the Asp182 opposite position of Escherichia coli TyrRS; With alanine or the valine on the Phe183 opposite position of Escherichia coli TyrRS; With methionine, valine, halfcystine or the threonine on the Leu186 opposite position of Escherichia coli TyrRS;
(g) polynucleotide identical with (a) and (b), (c), (d), (e) or polynucleotide (f) at least 98%; With,
(h) comprise the polynucleotide of (a) and (b), (c), (d), (e), (f) or conservative variant (g).
48. carrier that comprises the described polynucleotide of claim 47.
49. carrier as claimed in claim 48 is characterized in that, described carrier comprises plasmid, clay, bacteriophage or virus.
50. carrier as claimed in claim 48 is characterized in that, described carrier is an expression vector.
51. cell that comprises the described carrier of claim 48.
52. produce at least a method of protein that contains at least one alpha-non-natural amino acid for one kind in eukaryotic, this method comprises:
Cultivate the eukaryotic that contains nucleic acid in proper culture medium, this nucleic acid comprises at least one and selects codon and encoding said proteins; Wherein said nutrient culture media contains alpha-non-natural amino acid, and described eukaryotic comprises:
The also quadrature tRNA (O-tRNA) of identification selection codon works in cell; With
Preferably aminoacylation has the quadrature aminoacyl tRNA synthetase (O-RS) of the O-tRNA of alpha-non-natural amino acid, and wherein this O-RS comprises the corresponding amino acid sequence with SEQ ID NO.:48-53.
53. produce at least a method of protein that contains at least one alpha-non-natural amino acid for one kind in eukaryotic, this method comprises:
Cultivate the eukaryotic that contains nucleic acid in proper culture medium, this nucleic acid comprises at least one and selects codon and encoding said proteins; Wherein said nutrient culture media contains alpha-non-natural amino acid, described eukaryotic be included in work in the cell and the quadrature tRNA (O-tRNA) of identification selection codon and preferably aminoacylation have the quadrature aminoacyl tRNA synthetase (O-RS) of the O-tRNA of alpha-non-natural amino acid;
In this eukaryotic alpha-non-natural amino acid is mixed described protein, wherein this alpha-non-natural amino acid comprises first reactive group; With
This protein is contacted with the molecule that comprises second reactive group; Wherein first reactive group and second reactive group reaction makes this molecule be attached on the described alpha-non-natural amino acid by [3+2] cycloaddition.
54. method as claimed in claim 53, it is characterized in that described molecule is dyestuff, polymkeric substance, polyethyleneglycol derivative, photocrosslinking agent, cytotoxic compound, affinity labeling, biotin derivative, resin, second kind of protein or polypeptide, metal-chelator, co-factor, fatty acid, carbohydrates or polynucleotide.
55. method as claimed in claim 53 is characterized in that, described first reactive group is alkynyl or azido part, and described second reactive group is azido or alkynyl part.
56. method as claimed in claim 55 is characterized in that, described first reactive group is the alkynyl part, and described second reactive group is the azido part.
57. method as claimed in claim 56 is characterized in that, described alpha-non-natural amino acid comprises right-propargyloxy phenylalanine.
58. method as claimed in claim 55 is characterized in that, described first reactive group is the azido part, and described second reactive group is the alkynyl part.
59. method as claimed in claim 58 is characterized in that, described alpha-non-natural amino acid comprises right-azido-L-phenylalanine.
60. protein of producing by the described method of claim 53.
61. protein as claimed in claim 60; it is characterized in that; described protein is modified by posttranslational modification at least a body, and wherein said posttranslational modification is selected from: N-glycosylation, O-glycosylation, acetylation, acidylate, lipid-modification, palmitoylation, palmitate addition, phosphorylation and glycolipid-be connected modification.
CNA2004800211558A 2003-06-18 2004-04-16 Unnatural reactive amino acid genetic code additions Pending CN101160525A (en)

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Cited By (5)

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CN102307905A (en) * 2008-12-10 2012-01-04 斯克利普斯研究院 Production of carrier-peptide conjugates using chemically reactive unnatural amino acids
CN104203971A (en) * 2012-01-20 2014-12-10 医药研究委员会 Polypeptides and methods
CN104328086A (en) * 2006-09-08 2015-02-04 Ambrx公司 Site Specific Incorporation of Non-Natural Amino Acids by Vertebrate Cells
CN108752452A (en) * 2018-06-12 2018-11-06 中国医学科学院北京协和医院 The application of SARS and its mutant
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328086A (en) * 2006-09-08 2015-02-04 Ambrx公司 Site Specific Incorporation of Non-Natural Amino Acids by Vertebrate Cells
CN102307905A (en) * 2008-12-10 2012-01-04 斯克利普斯研究院 Production of carrier-peptide conjugates using chemically reactive unnatural amino acids
CN102307905B (en) * 2008-12-10 2015-11-25 斯克利普斯研究院 Chemical reactivity alpha-non-natural amino acid is utilized to produce carrier-peptide conjugate
CN104203971A (en) * 2012-01-20 2014-12-10 医药研究委员会 Polypeptides and methods
CN104203971B (en) * 2012-01-20 2019-08-09 医药研究委员会 Polypeptide and method
CN110959041A (en) * 2017-06-02 2020-04-03 Ambrx公司 Methods and compositions for enhancing the production of proteins containing unnatural amino acids
CN110959041B (en) * 2017-06-02 2023-09-29 Ambrx公司 Methods and compositions for promoting production of proteins containing unnatural amino acids
US11851662B2 (en) 2017-06-02 2023-12-26 Ambrx, Inc. Methods and compositions for promoting non-natural amino acid-containing protein production
CN108752452A (en) * 2018-06-12 2018-11-06 中国医学科学院北京协和医院 The application of SARS and its mutant
CN108752452B (en) * 2018-06-12 2022-03-08 中国医学科学院北京协和医院 SARS and its mutant application

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