CN101535482A - Genetically encoded fluorescent coumarin amino acids - Google Patents

Abstract

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine and related translation systems.

Description

The fluorescent coumarin amino acids of genetic coding

The cross reference of related application

The application requires the right of priority of following application: the U.S. Provisional Application sequence number 60/808,099 that on May 23rd, 2006 submitted to; With the U.S. Provisional Application sequence number of submitting on June 14th, 2,006 60/813,856; The content of two pieces of applications is included this paper by reference in full in.

To the following statement of making the right of invention of federal funding research and development

The present invention makes under the government of the contract number ER46051 of Ministry of Energy supports.Government enjoys certain right to the present invention.

Invention field

The invention belongs to the translation biochemical field.The present invention relates to preparation and utilize quadrature (orthogonal) tRNA, quadrature aminoacyl-tRNA synthetic enzyme and their pairing that alpha-non-natural amino acid is mixed proteinic composition and method.The invention still further relates to the protein that in cell, produces method of protein and produce in this way with this pairing.

Background of invention

Because highly sensitivity and operational safety, fluorescence has become one of most important detection signal in the biotechnology.Such as methods such as FRET (fluorescence resonance energy transfer) (FRET) or fluorescence polarization can real-time analysis biomolecules in conjunction with situation, motion or conformational change.Extremely help in the external and body of protein structure and function research (Hermanson (1996) publishes in BioconjugateTechniques (biological coupling technology), academic press (Academic Press): San Diego with the proteinic ability of fluorescent probe selective modification; And Tsien (1998) Annu.Rev.Biochem., 67:509-544).

The proteinic fluorescent method of the interior research of body at present often depends on and contains big fluorescin, for example the fusion construct of green fluorescent protein (GFP).Can be used for protein expression, location and bio-molecular interaction though confirmed this probe, its size conference causes tangible structure to be disturbed.GFP merges C-or the N-end also be confined to target protein, and to its environmental facies to insensitive (Tsien (1998) Annu.Rev.Biochem., 67:509-44).GFP also needs many transcripts to obtain appropriate signal, and it folds and the fluorophore maturation needs retardation time.

Thereby also can adopt chemical process to reduce structure as far as possible and disturb (Hermanson with various little, synthetic fluorophore selective modification protein, publish in Bioconjugate Techniques (biological coupling technology), the academic press: San Diego (1996); Martin etc., Nat.Biotech., 23:1308-1314 (2005); Keppler etc., Nat.Biotech., 21:86-89 (2003); Lin etc., J.Am.Chem.Sci., 128:4542-3 (2006)).Yet these technology are confined to reactive surfaces unique on the isolated protein usually can (for example, modify halfcystine with maleimide derivatives near residue; Referring to Hermanson, publish in Bioconjugate Techniques (biological coupling technology), (1996), and the academic press: the San Diego), it is not good to show regioselectivity, and having cytotoxicity maybe needs to introduce the dye-bond protein motif.

Proof can be utilized biosynthetic labelling method (Mendel etc., Annu.Rev.Biophys.Biomol.Struct. (1995) 24:435-462 of chemical wrong acidylate (misacylated) tRNA experimentally; But proteinic productive rate is limited and can only carry out external usually Hohsaka etc., FEBS Lett. (2004) 560:173-177).

Directly in live organism, the fluorescence amino acid of genetic coding is mixed proteinic definite site and can overcome the proteinic many limitation of fluorescent mark (Wang and Schultz, Angew.Chem.Int.Ed. (2005) 44:34-66; Wang etc., Annu.Rev.Biophys.Biomol.Struct., (2006) 35:225-249).Locus specificity mixes fluorescence amino acid can introduce interference as few as possible in host protein, can also carry out the fluorescence resonance ability with higher tolerance range and shift (FRET) detection (Truong and Ikura, Curr.Opin.Struct.Bio.2001,11:573-578).In addition, utilize fluorescence amino acid can detect the local environment of each amino acid position, find out mediation and the interactional residue of other cellular component by the amino acid whose position of fluorescence in the change protein.This is for research protein folding also very useful (Lakowicz, Principles of Fluorescence Spectroscopy (fluorescence spectroscopy principle), second edition; KluwerAcademic/Plenum Publishers: New York, 1999), (Lipman etc. in the unit molecule system particularly, Science (2003) 301:1233-1235), because protein molecule contains a plurality of tryptophan residues usually and also is a problem with fluorescent probe chemical specificity labelled protein.

Be research protein structure and function, this area needs the fluorescence alpha-non-natural amino acid to be mixed proteinic new departure.Now developed in protokaryon and eukaryote and will mix proteinic universal method in the various alpha-non-natural amino acid bodies locus specificity.These methods depend on quadrature protein translation component, and described component discerns suitable selector's codon (selector codon) thereby the polypeptide translate duration inserts the qualification position with required alpha-non-natural amino acid in vivo.These methods are utilized the quadrature tRNA (O-tRNA) of identification selection person's codon, and corresponding specificity quadrature aminoacyl-tRNA synthetic enzyme (O-RS) loads this O-tRNA with alpha-non-natural amino acid.These components not with the intravital any endogenous tRNA of host living beings, RS, amino acid or codon cross reaction (that is, it must be orthogonal).Utilize the different alpha-non-natural amino acid of this quadrature tRNA-RS pairing possibility a large amount of structures of genetic coding.

This area is generally known to utilize and is suitable for preparing the proteinic orthogonal translation system that contains one or more alpha-non-natural amino acids, for example produces the universal method of orthogonal translation system.For example, referring to international publication number WO 2002/086075, its " METHODS AND COMPOSITION FOR THEPRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNASYNTHETASE PAIRS (producing quadrature tRNA-aminoacyl-tRNA synthetic enzyme method of matching and composition) " by name; WO 2002/085923, its " IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS (mixing in the body of alpha-non-natural amino acid) " by name; WO2004/094593, its " EXPANDING THE EUKARYOTIC GENETICCODE (expansion eucaryon genetic code) " by name; The WO 2005/019415 that on July 7th, 2004 submitted to; The WO 2005/007870 that on July 7th, 2004 submitted to; The WO2005/007624 that on July 7th, 2004 submitted to; With the WO 2006/110182 that submitted on October 27th, 2005, its " ORTHOGONAL TRANSLATION COMPONENTS FOR THE IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS (mixing the orthogonal translation components of alpha-non-natural amino acid in the body) " by name.This paper is included in these applications separately by reference in full in.Other that mixes the orthogonal translation system of alpha-non-natural amino acid and their generation and using method discussed also can be referring to Wang and Schultz, " Expanding the Genetic Code (expansion genetic code) ", Chem.Commun. (Camb.) 1:1-11 (2002); Wang and Schultz " Expanding the GeneticCode (expansion genetic code) ", Angewandte Chemie Int.Ed., 44 (1): 34-66 (2005); Xie and Schultz, " An Expanding Genetic Code (expansion genetic code) ", Methods36 (3): 227-238 (2005); Xie and Schultz, " Adding Amino Acids to the GeneticRepertoire (amino acid is added hereditary storehouse) " Curr.Opinion in Chemical Biology9 (6): 548-554 (2005); Wang etc., " Expanding the Genetic Code (expansion genetic code) ", Annu.Rev.Biophys.Biomol.Struct., 35:225-249 (2006); Xie and Schultz, " A chemical toolkit for proteins-an expanded genetic code (genetic code of proteinic chemical tools case-expansion) ", Nat.Rev.Mol.Cell Biol., 7 (10): 775-782 (2006), their content is included this paper separately by reference in full in.

This area need be developed and the fluorescence alpha-non-natural amino acid can be mixed proteinic orthogonal translation components, and wherein said fluorescence alpha-non-natural amino acid is incorporated in specified location.Can understand after scanning hereinafter that the present invention described herein has satisfied these and other demand.

Summary of the invention

The invention provides in vivo (for example in host cell) to selector's codon, react and tonka bean camphor alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine is mixed the composition and the method for the polypeptide chain in the extension as the amber terminator codon.These compositions do not comprise and the interactional quadrature-tRNA of host cell translating mechanism (O-tRNA) and quadrature aminoacyl-tRNA synthetic enzyme (O-RS) pairing.That is, endogenous host cell aminoacyl-tRNA synthetic enzyme is not used amino acid (natural or non-natural) and is loaded O-tRNA (or the loading level is not obvious).Similarly, O-RS provided by the invention does not load endogenous tRNA with remarkable or detectable level with amino acid (natural or non-natural).These novel composition can produce and contain a large amount of protein of translating L-(umbelliferone-4-yl) ethyl glycine that mixes.These fluorescently-labeled protein have various application.

In some respects, the invention provides translation system.These systems comprise first quadrature aminoacyl-tRNA synthetic enzyme (O-RS), the first quadrature tRNA (O-tRNA) and first alpha-non-natural amino acid; be L-(umbelliferone-4-yl) ethyl glycine, a wherein said O-RS is with the described O-tRNA of the preferential aminoacylation of described first alpha-non-natural amino acid.

This translation system can be used the component derived from various sources.In one embodiment, a described O-RS is derived from Methanococcus jannaschii (Methanococcus jannaschii) aminoacyl-tRNA synthetic enzyme, for example wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase.The O-RS that is used for this system can comprise the conservative variant of aminoacid sequence shown in the SEQ ID NO:4 and this sequence.In some embodiments, described O-tRNA is amber inhibition type tRNA.In some embodiments, described O-tRNA comprises SEQ ID NO:1 or by its coding.

In some respects, translation system also comprises the nucleic acid of coding proteins of interest matter, and wherein said nucleic acid has at least one selector's codon of O-tRNA identification.

In some respects, translation system comprises second quadrature pairing (i.e. the 2nd O-RS and the 2nd O-tRNA) that utilizes second alpha-non-natural amino acid, and at least two different alpha-non-natural amino acids can mix in the different selected locations of polypeptide in this system now.In this duplex system, the 2nd O-RS is with being different from preferential aminoacylation the 2nd O-tRNA of second alpha-non-natural amino acid of first alpha-non-natural amino acid, and the 2nd O-tRNA identification is different from selector's codon of selector's codon that an O-tRNA discerned.

In some embodiments, translation system is arranged in host cell (comprising this host cell).Used host cell is not done concrete qualification, as long as O-RS and O-tRNA can keep its orthogonality in their host cell environment.Described host cell can be the eubacterium cell, as intestinal bacteria.Described host cell can contain the polynucleotide that one or more codings comprise the translation system component of O-RS or O-tRNA.In some embodiments, the polynucleotide of coding O-RS comprise nucleotide sequence shown in the SEQ ID NO:5.

The present invention also provides and is created in the method for protein that one or more alpha-non-natural amino acids are contained in the selected location.These methods are utilized above-mentioned translation system.These methods start from the step that the translation system that contains following component is provided usually: (i) first alpha-non-natural amino acid, i.e. L-(umbelliferone-4-yl) ethyl glycine; (ii) first quadrature aminoacyl-tRNA synthetic enzyme (O-RS); The (iii) first quadrature tRNA (O-tRNA), wherein said O-RS is with the described O-tRNA of the preferential aminoacylation of described alpha-non-natural amino acid; The (iv) nucleic acid of coded protein, wherein said nucleic acid contain at least one selector's codon of O-tRNA identification.This method reacts to selector's codon and described alpha-non-natural amino acid is mixed described proteinic selected location in described proteinic translation process then, thereby is created in the protein that described alpha-non-natural amino acid is contained in the selected location.

Can utilize all ingredients and step extensively to implement this method.The polynucleotide of coding O-RS are provided in some embodiments.In some embodiments, provide O-RS, wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase for example can be provided derived from Methanococcus jannaschii aminoacyl-tRNA synthetic enzyme.In some embodiments, this provides step to comprise provides the O-RS that contains aminoacid sequence shown in the SEQ ID NO:4 and conservative variant thereof.

In some embodiments of these methods; provide the step of translation system to comprise the amino acid binding pocket (binding pocket) of wild-type aminoacyl-tRNA synthetic enzyme is undergone mutation, select gained O-RS with the described O-tRNA of the preferential aminoacylation of described alpha-non-natural amino acid by site-directed mutagenesis.The described O-RS of selection is just being selected and born to described selection step from the aminoacyl-tRNA synthetic enzyme library of molecules that obtains after can comprising site-directed mutagenesis.In some embodiments, provide step that the polynucleotide of coding O-tRNA are provided, for example, O-tRNA is amber inhibition type tRNA, and perhaps O-tRNA comprises polynucleotide shown in the SEQ ID NO:1 or by its coding.In these methods, providing step also to comprise provides the nucleic acid that contains the used amber selector codon of translation system.

Also can improve these methods in protein, to mix an above alpha-non-natural amino acid.In those methods, the coupling second orthogonal translation system and first translation system, wherein second system has different amino acid and selector's codon specificity.For example; providing step to comprise provides the 2nd O-RS and the 2nd O-tRNA; wherein the 2nd O-RS is with being different from preferential aminoacylation the 2nd O-tRNA of second alpha-non-natural amino acid of first alpha-non-natural amino acid, and is different from selector's codon of selector's codon that an O-tRNA discerned in the 2nd O-tRNA identification nucleic acid.

Also can in the host cell environment, implement to produce the method for protein that contains alpha-non-natural amino acid.In these situations, the host cell that provides contains the nucleic acid that contains at least one selector's codon of alpha-non-natural amino acid, O-RS, O-tRNA and coded protein, can cause mixing of alpha-non-natural amino acid and cultivate this host cell.In some embodiments, provide step to comprise eubacterium host cell (for example, intestinal bacteria) is provided.In some embodiments, provide step to comprise the host cell of the polynucleotide that contain the O-RS that encodes is provided.For example, the polynucleotide of coding O-RS can comprise nucleotide sequence shown in the SEQ ID NO:5.In some embodiments, by providing cell extract to realize providing the step of translation system.

The present invention also provides and comprises nucleic acid and proteinic various composition.Except composition contains described nucleic acid or protein, the character of said composition is not done concrete restriction.The present composition can contain other component of any amount, any character.

For example; the invention provides the composition that contains the O-RS polypeptide; wherein said polypeptide contains aminoacid sequence shown in the SEQ IDNO:4 or its conservative variant, wherein said conservative variant polypeptide be at least with the efficient of alpha-non-natural amino acid aminoacylation association (cognate) quadrature tRNA (O-tRNA) aminoacyl-tRNA synthetic enzyme that contains this O-tRNA, this alpha-non-natural amino acid and contain aminoacid sequence shown in the SEQ ID NO:4 the viewed efficient of translation system 50%.The present invention also provides the polynucleotide of the above-mentioned any polypeptide of coding.In some embodiments, these polynucleotide can contain nucleotide sequence shown in the SEQ IDNO:5.In some embodiments, polypeptide is in cell.

The present invention also provides the polynucleotide compositions that contains nucleotide sequence shown in the SEQ ID NO:5.In some embodiments, the invention provides the carrier that contains described polynucleotide, as expression vector.In some embodiments, the invention provides the cell that contains above-mentioned carrier.

Definition

Before describing the present invention in detail, will be appreciated that the present invention is not limited to concrete biosystem, the present invention certainly has various variations.It is also understood that term used herein just in order to describe concrete embodiment, and nonrestrictive.Unless spell out in addition, singulative " ", " a kind of " and " being somebody's turn to do " of using in this specification sheets and the claims of enclosing also comprise plural object.Therefore, for example address the combination that " cell " comprises two or more cells; " polynucleotide " in fact comprise many parts of copies of these polynucleotide.

Except defined with the following rest part of specification sheets herein, all technology used herein and scientific terminology all with the conventional same meaning of understanding of one skilled in the art of the present invention.

Quadrature: term used herein " quadrature " refers to that corresponding molecule with the endogenous of certain cell or translation system compares, certain molecule (for example, quadrature tRNA (O-tRNA) and/or quadrature aminoacyl-tRNA synthetase (O-RS)) efficient that works with this cell endogenous component reduces, or can not work with the endogenous component of this cell.With regard to tRNA and aminoacyl-tRNA synthetic enzyme, just interdigital with compare with endogenous tRNA that endogenous tRNA synthetic enzyme works, quadrature tRNA can not reduce with the efficient that endogenous tRNA synthetic enzyme works or works; Perhaps with the endogenous tRNA synthetic enzyme that endogenous tRNA works compare, quadrature aminoacyl-tRNA synthetase and endogenous tRNA efficient inoperative or that work reduces, described efficient for example reduces, efficient less than 20%, efficient less than 10%, efficient less than 5%, or less than 1% efficient.The quadrature molecule lacks normally functioning endogenous complementary molecule in the cell.For example, tRNA compares with endogenous RS aminoacylation endogenous, and the efficient of quadrature tRNA reduces or even is 0 in any endogenous RS aminoacylation cell of cell.In another example, tRNA compares with endogenous RS aminoacylation endogenous, and the efficient of any endogenous tRNA reduces or even is 0 in the quadrature RS aminoacylation cells of interest.Can will be able to introduce cell with the second quadrature molecule that the first quadrature molecule works.For example, quadrature tRNA/RS pairing comprises the complementary component of introducing, with contrast, for example corresponding tRNA/RS endogenous pairing or the pairing of active quadrature (as tyrosyl quadrature tRNA/RS pairing) are compared, the efficient that they concur in cell is, for example 45% efficient, 50% efficient, 60% efficient, 70% efficient, 75% efficient, 80% efficient, 90% efficient, 95% efficient or 99% efficient, or higher.

Quadrature tyrosyl-tRNA: quadrature tyrosyl-tRNA used herein (tyrosyl-O-tRNA) be and the orthogonal tRNA of translation system interested, wherein said tRNA is: (1) is identical with natural tyrosyl-tRNA or similar substantially; (2) by natural or induced mutations derived from natural tyrosyl tRNA; (3) any method by the wild-type of having considered (1) or (2) or mutant tyrosyl tRNA sequence produces; (4) with wild-type or mutant tyrosyl tRNA homology; (5) with among Fig. 9 be called any exemplary tRNA homology of tyrosyl-t RNA synthetase substrate; Or be called the conservative variant of any exemplary tRNA of tyrosyl-t RNA synthetase substrate among (6) Fig. 9.Tyrosyl tRNA can be loaded with amino acid, or is in unsupported state.It is also understood that " tyrosyl-O-tRNA " optional associated (cognate) synthetic enzyme loads (aminoacylation) amino acid except that tyrosine, for example alpha-non-natural amino acid respectively.Should be understood that and in fact preferably utilize tyrosyl-O-tRNA of the present invention selector's codon is reacted and any amino acid (no matter natural or non-natural) can be mixed in the polypeptide of extension basically at translate duration.

Quadrature tyrosyl amino acid synthetase: (tyrosyl-O-RS) is with the enzyme of the preferential aminoacylation tyrosyl-O-tRNA of amino acid in translation system interested to quadrature tyrosyl amino acid synthetase used herein.The amino acid that tyrosyl-O-RS is loaded on tyrosyl-O-tRNA can be any amino acid, no matter is natural, non-natural or artificial, and is not limited to as herein described.Optional and the identical or homology of natural tyrosyl amino acid synthetase of this synthetic enzyme perhaps with among Fig. 9 is called the identical or homology of synthetic enzyme of O-RS.For example, described O-RS can be the conservative variant of tyrosyl-O-RS of Fig. 9, and/or with Fig. 9 in the sequence of O-RS have 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or above identical at least.

Related (cognate): term " association " refers to concur or has certain specific component, for example quadrature tRNA and quadrature aminoacyl-tRNA synthetase each other.These components also can be called " complementary " mutually.

Preferential aminoacylation: for orthogonal translation used herein system, when O-RS in certain expression system makes the efficient of O-tRNA band upper amino acid be higher than its efficient that makes any endogenous tRNA band upper amino acid, the related O-tRNA of this O-RS " preferential aminoacylation ".That is, exist in the system of serving as interpreter mol ratio about equally O-tRNA and during any given endogenous tRNA, the frequency that O-RS loads O-tRNA is higher than the frequency that it loads endogenous tRNA.When the O-tRNA of volumetric molar concentration and endogenous tRNA such as having in the system of serving as interpreter, preferably higher by the O-tRNA that O-RS loads with the relative proportion of the endogenous tRNA that loads by O-RS, preferably cause O-RS only to load or almost only load O-tRNA.When the O-tRNA of volumetric molar concentrations such as existence and O-RS, O-tRNA that O-RS loads and the relative proportion of endogenous tRNA are greater than 1:1, preferably at least about 2:1, more preferably 5:1, more preferably 10:1, more preferably 20:1, more preferably 50:1, more preferably 75:1, more preferably 95:1,98:1,99:1,100:1,500:1,1,000:1,5,000:1 or higher.

When (a) compares with endogenous tRNA, the preferential aminoacylation O-tRNA of O-RS compares with any natural amino acid aminoacylation O-tRNA with O-RS with (b), and aminoacylation when special, claims that O-RS " preferentially uses alpha-non-natural amino acid aminoacylation O-tRNA " to alpha-non-natural amino acid.That is, when non-natural that has equimolar amount in the translation system that comprises O-RS and O-tRNA and natural amino acid, the frequency that O-RS loads O-tRNA with alpha-non-natural amino acid is higher than uses the natural amino acid loading frequency.The O-tRNA that is loaded with alpha-non-natural amino acid is preferably higher with the relative proportion of the O-tRNA that is loaded with natural amino acid.O-RS preferably makes O-tRNA only load, and perhaps almost only is loaded with alpha-non-natural amino acid.When the natural and alpha-non-natural amino acid of volumetric molar concentration such as having in the system of serving as interpreter, make O-tRNA be with alpha-non-natural amino acid with make O-tRNA be with natural amino acid whose relative proportion greater than 1:1, preferably at least about 2:1, more preferably 5:1, more preferably 10:1, more preferably 20:1, more preferably 50:1, more preferably 75:1, more preferably 95:1,98:1,99:1,100:1,500:1,1,000:1,5,000:1 or higher.

Selector's codon: term " selector's codon " refers to be in the translation process codon of not discerned for endogenous tRNA that O-tRNA discerns.Selector's codon on the O-tRNA anticodon loop identification mRNA also mixes its amino acid, for example alpha-non-natural amino acid in this position of polypeptide.Selector's codon can comprise, nonsense codon for example, as terminator codon (as, amber, ochre and opal codon); Four bases or the above codon of four bases; Rare codon; Derived from codon natural or the non-natural base pair, or the like.

Inhibition type tRNA: inhibition type tRNA reacts to terminator codon and mixes amino acid (that is, " even reading ") at the polypeptide translate duration to change the tRNA that messenger RNA(mRNA) in the given translation system (mRNA) is read usually.In some respects, selector's codon of the present invention is an inhibition type codon, for example, terminator codon (as, amber, ochre and opal codon), four base codons, rare codon etc.

Suppress active: term used herein " suppresses active " and (for example refers to tRNA generally, inhibition type tRNA) can the company of translation read in other situation, (for example can cause translation termination or mistranslation, the ability of codon frameshit) (for example, as the amber codon of selector's codon or four or with the codon of last base).The inhibition activity of inhibition type tRNA can be expressed as and the second inhibition type tRNA, or and contradistinction system, the contradistinction system that for example lacks O-RS is compared, and observed translation connects reads active per-cent.

The invention provides quantitative assay and suppress active the whole bag of tricks.Specific O-tRNA and O-RS are to selector's codon interested (for example, amber codon) inhibition per-cent refers to, in interested translation system, this the given expression test badge that contains selector's codon in coding is expressed the nucleic acid of test badge (for example, LacZ) per-cent that activity is compared with the positive control construction, described interested translation system comprises O-RS and O-tRNA, and described positive control does not have O-tRNA, O-RS and selector's codon.Therefore, for example, if observe the activity of the active positive control mark construction that does not contain selector's codon in given translation system is X (its unit is relevant with described mark test), the inhibition per-cent that then contains the test builds thing of selector's codon be with the essentially identical envrionment conditions of the expression of positive control mark under (except this test badge construction is expressed in the translation system that also contains O-tRNA and O-RS), the X per-cent that this test badge construction shows.The translation system of expressing this test badge generally also comprises the amino acid of O-RS and O-tRNA identification.Can choose wantonly by test badge and " background " or " feminine gender " contrasting marking construction relatively being proofreaied and correct the observed value that suppresses per-cent, described " background " or " feminine gender " contrasting marking construction contain the selector codon identical with test badge, but the system at its place does not contain the related amino acid of O-tRNA, O-RS and/or O-tRNA and/or O-RS identification.This negative control can be used for stdn and suppresses the percentage test value to compensate the influence of the background signal of mark in the interested translation system.

Can suppress efficient by many test determinations known in the art.For example, can adopt the test of beta-galactosidase enzymes report, as deutero-1acZ plasmid (this construction contains selector's codon in the 1acZ nucleotide sequence) being introduced the cell of suitable biology (for example, can utilize the biology of quadrature component) together with the plasmid that contains O-tRNA of the present invention.Also can introduce related synthetic enzyme (polynucleotide of this association synthetic enzyme of encoding in the time of can being polypeptide or expression).Cell grows to desired density in substratum, for example to OD ₆₀₀Be about 0.5, carry out the beta-galactosidase enzymes test, for example use the BetaFluor of Nova King Company (Novagen) ^TMThe beta-galactosidase enzymes test kit.Can with suppress percentage calculation be sample with respect to can relatively contrasting, for example, the active per-cent of the observed value of deutero-lacZ construction, this construction has corresponding sense codon but not selector's codon in desired location.

Translation system: term " translation system " refers to amino acid is mixed each component of the polypeptide chain (protein) in the extension.The component of translation system can comprise, for example rrna, tRNA, synthetic enzyme, mRNA etc.O-tRNA of the present invention and/or O-RS can add external or the interior translation system of body or its part, for example be present in non-eukaryotic cell, in bacterium (as intestinal bacteria), or be present in the eukaryotic cell, as yeast, mammalian cell, vegetable cell, alga cells, fungal cell, insect cell etc.

Alpha-non-natural amino acid: term used herein " alpha-non-natural amino acid " refers to not any amino acid at the row of 20 kinds of common natural amino acids or seleno-cysteine or pyrroles's Methionin (pyrrolysine), the amino acid of modification and/or amino acid analogue.For example, the present invention can use alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine (also to write umbelliferone-ethyl glycine; Referring to Fig. 1, structure 1).

Derived from: term used herein " derived from " refer to separate from specific molecular or biology, or certain component that makes with specific molecular or biological information.For example, the polypeptide derived from second polypeptide contains and the identical or substantially similar aminoacid sequence of this second amino acid sequence of polypeptide.With the polypeptide is example, can pass through, and for example mutagenesis of natural generation, artificial orientation's mutagenesis or artificial random mutagenesis obtain the deutero-kind.The mutagenesis of polypeptide of being used to derive can be to have a mind to directed or have a mind at random, or uses two kinds of methods with.The mutagenesis polypeptide can be at random (for example, polysaccharase distortion due to) to produce not homopolypeptide derived from first (polypeptide), can pass through suitable screening method, method evaluation polypeptides derived for example as herein described.The mutagenesis polypeptide need be operated the polynucleotide of this polypeptide of encoding usually.

Just selecting or selection markers: term used herein " is just being selected or selection markers " to refer to when having this mark that (for example expressed or activation etc.) can never have in the cell of feature and is being identified the cell with feature, for example having the cell of positive selective marker.

The negative selection or selection markers: term used herein " negative select or selection markers " refers to that when this mark of existence (for example expressed or activation etc.) can identify the cell (for example, comparing with the cell that has this characteristic or feature really) that does not contain selected characteristics or feature.

Reporter molecule: term used herein " reporter molecule " is meant the component that can be used for identifying and/or selecting the target component of system interested.For example, reporter molecule can comprise protein, if can give enzyme (as β-Nei Xiananmei, E.C. 2.3.1.28 (CAT) etc.), fluorescent screening mark (as green fluorescent protein (as GFP), YFP, EGFP, RFP etc.), cold light mark (as the Lampyridea luciferase protein), the avidity selection markers of antibiotics resistance or susceptibility, perhaps selectable plus or minus marker gene such as lacZ, β-gal/lacZ (beta-galactosidase enzymes), ADH (ethanol dehydrogenase), his3, ura3, leu2, lys2 etc.

Eukaryote: term used herein " eukaryote " refers to belong to the biology of Eucaryotae (KingdomEucarya).Eukaryote is different from prokaryotic organism because of its following feature usually: typical many cells tissue (but not all is many cells, yeast for example), there are the nuclear of film restriction and the organoid of other film restriction, linear genetic material (promptly, linear karyomit(e)), there is not operon, exists intron, courier (RNA) to add cap and poly-A mRNA, and other biochemical characteristics, as different Ribosome Structures.Eukaryote comprises, animal (as Mammals, insect, Reptilia, bird etc.) for example, ciliate, plant (as monocotyledons, dicotyledons, algae etc.), fungi, yeast, Flagellata, microsporidium, protozoon etc.

Prokaryotic organism: term used herein " prokaryotic organism " refers to belong to the biology of Prokaryota (being also referred to as prokaryotic organism (Procarya)).Prokaryotic organism are different from eukaryote because of its following feature usually: unicellular tissue, by sprouting or splitted monogony, lack the nuclear of film restriction and the organoid of other film restriction, ring chromosome, there is operon, do not exist intron, courier (RNA) to add cap and poly-A mRNA, and other biochemical characteristics, as different Ribosome Structures.Prokaryotic organism comprise eubacterium and archeobacteria (Archaea) (being sometimes referred to as " archeobacteria (Archaebacteria) ") suberathem.Sometimes be divided into cyanobacteria (blue-green algae) and mycoplasma different classes of in Prokaryota.

Bacterium: term used herein " bacterium " and " eubacterium " refer to be different from ArcheobacteriaProkaryotic organism.Similarly, archeobacteria refers to be different from eubacterial prokaryotic organism.Can distinguish eubacterium and archeobacteria according to many forms and biological chemistry standard.For example, can adopt the difference of ribosome-RNA(rRNA) sequence, RNA polymerase structure, whether the existence of intron, antibiotics sensitivity, whether the existence of whole cell peptidoglycan and other cell-wall component, the branch of membrane lipid and apparatus derivatorius not exist/do not exist histone and histone-like protein and certain biology are appointed as eubacterium or archeobacteria.

Eubacterial example comprises intestinal bacteria (Escherichia coli), thermus thermophilus (Thermusthermophilus), subtilis (Bacillus subtilis) and bacstearothermophilus (Bacillusstearothermophilus) etc.).The example of archeobacteria comprises Methanococcus jannaschii (Methanococcusjannaschii) (Mj); Mei Shi sarcina methanica (Methanosarcina mazei) (Mm); thermophilic alkali methagen (Methanobacteriurn thermoautotrophicum) (Mt); sea natural pond methane coccus (Methanococcus maripaludis); methane thermophile bacteria (Methanopyrus kandleri); salt bacterium (Halobacterium) (for example rich salt bacterium (Haloferax volcanii) of Wo Shi and salt bacterium kind NRC-1); glimmer ancient green-ball bacterium (Archaeoglobus fulgidus) (Af); fierce fireball bacterium (Pyrococcus furiosus) (Pf); extreme hyperthermophilic archaeon strain (Pyrococcus horikoshii) (Ph); aerobic fireball bacterium (Pyrobaculum aerophilum); deep-sea fireball bacterium (Pyrococcus abyssi); sulfolobus solfataricus (Sulfolobus solfataricus) (Ss); the ancient bacterium (Sulfolobus tokodaii) of super thermophilic spring; thermophilic spring is given birth to archeobacteria (Aeuropyrum pernix) (Ap); thermoplasma acidophilum (Thermoplasma acidophilum) and hot volcanic substance (Thermoplasmavolcanium).

Examples of conservative variations: with regard to translation components, term used herein " examples of conservative variations " refers to certain translation component, for example examples of conservative variations O-tRNA or examples of conservative variations O-RS, its performed function is similar to the basal component (for example O-tRNA or O-RS) similar to it, but compare with reference O-tRNA or O-RS variation is arranged in the sequence.For example, the examples of conservative variations of O-RS or this O-RS can be used alpha-non-natural amino acid, for example the related O-tRNA of L-(umbelliferone-4-yl) ethyl glycine alpha-non-natural amino acid aminoacylation.In this example, the aminoacid sequence of described O-RS and examples of conservative variations O-RS is inequality.Described examples of conservative variations for example can have place variation, the variation of two places, the variation of three places, variation everywhere or five places or more many places variation in sequence, if this examples of conservative variations still with related corresponding O-tRNA or O-RS complementation (for example, working with it).

In some embodiments, examples of conservative variations O-RS compares with its O-RS of deriving, and contains one or more conservative amino acid and replaces.In some embodiments, examples of conservative variations O-RS compares with its O-RS of deriving, and contains one or more conservative amino acid and replaces, and has kept the biologic activity of O-RS in addition; For example, examples of conservative variations O-RS has kept its biologic activity of parent O-RS molecule at least 10%, perhaps at least 20%, at least 30%, or at least 40% of deriving.In some preferred implementations, described examples of conservative variations O-RS has kept its biologic activity of parent O-RS molecule at least 50% of deriving.The conservative amino acid replacement of examples of conservative variations O-RS can appear in any structure territory of this O-RS, comprises the amino acid binding pocket.

Select or screening reagent: term used herein " select or screening reagent " refer to when existing can be from certain colony the reagent of some component of selection/screening.For example, select or screening reagent can be but is not limited to, as the polynucleotide of the light of nutrition, microbiotic, certain wavelength, antibody, expression etc.Selective reagents can be different because of for example concentration, intensity etc.

React: term used herein " reacts " and refers to the process that O-tRNA identification selection person codon of the present invention and mediation and this tRNA link coupled alpha-non-natural amino acid mix the polypeptide chain in the extension.

Coding: term used herein " coding " refers to any process of instructing second kind of molecule being different from this first molecule or sequence chain or sequence chain to produce with the information in poly macromole or the sequence chain.This term application used herein is extensive, can be used for each field.In some respects, the process of semiconservative DNA replication described in term " coding ", and wherein double chain DNA molecule chain is as template, by the DNA synthetic enzyme that the relies on DNA complementary sisters' chain of new synthetic of encoding.

On the other hand, term " Coding" refer to instruct any process that chemical property is different from second kind of molecule of this first molecule that produces with a kind of information of molecule.For example, dna molecular codified RNA molecule (transcription that for example, comprises the RNA polymerase that relies on DNA).The RNA molecule is the codified polypeptide also, for example translation process.When term " coding " when being used to describe translation process, its implication also extends to the codeword triplet of coded amino acid.In some respects, RNA molecule codified dna molecular, for example the reverse transcription process of the DNA synthetic enzyme by comprising dependenc RNA.On the other hand, dna molecular codified polypeptide will be appreciated that this situation comprises simultaneously with " coding " to transcribe and translation process.

The accompanying drawing summary

Fig. 1 is the synoptic diagram of chemosynthesis L-(umbelliferone-4-yl) ethyl glycine (structure 1).

Fig. 2 is the absorption and the emmission spectrum of L-(umbelliferone-4-yl) ethyl glycine.With pH7.4, two kinds of spectrum of 100mM sodium phosphate buffer record.Under this pH, L-(umbelliferone-4-yl) ethyl glycine exists phenol and two kinds of forms of phenates (ratio is about 2:1).The optical extinction coefficient of 360nM phenates form is 17000, and quantum yield (quantum yield) is 0.63.

Fig. 3 provides the structure (pdb encode 105M) of Physter macrocephalus myohaemoglobin.Marked the R-group of residue Ser4 and His37.

Fig. 4 provides the urea of wild-type and mutated form holomyarian red eggs white (holomyoglobin) to induce not folding circular dichroism analytical results.The mutant form of analyzing is that Ser4 → umbelliferone-ethyl glycine or His37 → umbelliferone-ethyl glycine mutant holomyarian red eggs are white.CD and fluorescence intensity by umbelliferone-ethyl glycine are monitored not folded situation.Two kinds of experiments are carried out among the pH 7.4 all at the 100mM sodium phosphate buffer, and the various concentration of urea as shown in the figure.Compare with the 0M urea, have the 5M urea to exist down, the myohaemoglobin mutant shows that fluorescence intensity increases by 30%.According to fully folding state, by the fluorescence intensity or the molar ellipticity calculating " folding mark " of stdn 5M urea.

The photo that the SDS-PAGE that Fig. 5 provides TAG4 mutant myohaemoglobin to express (shown in the black arrow) analyzes.Middle figure has been presented at and has not had 1mM L-(umbelliferone-4-yl) ethyl glycine to have down the coomassie of cumulative protein-painted SDS-PAGE separation case.Right figure shows that SDS-PAGE separates the fluoroscopic image of back wild-type and TAG4 mutant myohaemoglobin.Left figure shows painted molecular weight marker.

Fig. 6 provides the ESI-MS collection of illustrative plates of TAG4 mutant myohaemoglobin.Inset shows the figure (deconvoluted spectrum) that deconvolutes.Estimated molecular weight: 18512Da; Measured value: 18511Da.

Fig. 7 provides 60 μ M tonka bean camphor base amino acid L-(umbelliferone-4-yl) absorption spectrums of ethyl glycine in the 100mM sodium phosphate buffer.

Fig. 8 provides mutant MjTyrRS (CouRS-D8) structural models of avtive spot.Adopt wild-type MjTyrRS structure (pdb encode 1J1U) to produce this model.Substitute the tyrosine substrate with L-(umbelliferone-4-yl) ethyl glycine, it is as follows to suddenly change: Tyr32Glu, Leu65His, Ala67Gly, His70Gly, Phe108Tyr, Gln109His, Asp158Gly and Leu162Gly.Employing California, USA San Diego city acthar Le Si software company

Software (AccelrysSoftware,

, San Diego, CA USA), optimizes structure by energy minimization.

Fig. 9 provides various Nucleotide and aminoacid sequence.

Detailed Description Of The Invention

The invention provides and can be in Escherichia coli optant's codon (for example amber terminator codon TAG) be reacted and with the selective quadrature tRNA/ aminoacyl of introducing protein-tRNA synzyme pairing in Fluorescent amino acid L-(umbelliferone-4-yl) ethyl glycine (referring to Fig. 1, the structure 1) body. The invention provides with alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine specificity and load the new quadrature aminoacyl of the quadrature tRNA (O-tRNA) that is correlated with-tRNA synzyme (O-RS) polypeptide.

The umbelliferone molecule at first because of its fluorescence quantum yield is high, the Stoke displacement large (referring to Fig. 2), size little, obtain studying (Zinsli, J.Photochem (1974) 3:55-69) to pH (referring to Fig. 7) and solvent polarity are sensitive.

In some aspects, be proof (but not being restriction) the present invention, this paper content proof can partly be mixed alpha-non-natural amino acid model protein (referring to embodiment 4 and 5). Mix alpha-non-natural amino acid and need not be confined to any specific protein. Can understand that from the present invention it is favourable for various purposes that the non-natural Fluorescent amino acid is mixed interested specified protein.

This paper has also described exploitation and can work in eubacteria and optant's codon is reacted, thereby (for example mix the non-natural Fluorescent amino acid locus specificity, the method of novel quadrature tRNA/ aminoacyl alpha-non-natural amino acid L-shown in Figure 1 (umbelliferone-4-yl) ethyl glycine, structure 1)-tRNA synzyme pairing. In brief, we have identified the novel mutation body that selectively loads the Methanococcus jannaschii tyrosyl-tRNA synthetase of inhibition type RNA in e. coli host cell with alpha-non-natural amino acid.

The tRNA-synzyme pairing that can utilize these exploitations with non-natural Fluorescent amino acid locus specificity mix protein. Can make it by the polynucleotide sequence of engineered coding proteins of interest matter to contain and to send the optant's codon that mixes the alpha-non-natural amino acid signal, thereby the alpha-non-natural amino acid programming can be mixed any desired location.

Invention described herein is provided at eubacteria, for example in the Escherichia coli with L-(umbelliferone-4-yl) ethyl glycine alpha-non-natural amino acid genetic coding and the quadrature pairing of mixing protein, wherein said quadrature component not with the endogenous Escherichia coli component cross reaction of host cell translating mechanism, but can identify required alpha-non-natural amino acid and to optant's codon (for example, the amber nonsense codon TAG) reacts and it is mixed protein. Quadrature component provided by the invention comprises derived from the quadrature aminoacyl of Methanococcus jannaschii tyrosyl tRNA-synzyme-tRNA synzyme, and saltant tyrosyl tRNACUA amber mortifier, and the two is used as the quadrature pairing in the eubacteria host cell.

The invention provides and identify and produce other quadrature tRNA-aminoacyl-tRNA synzyme pairing, for example composition and the method for O-tRNA/O-RS pairing, described pairing can be used for L-(umbelliferone-4-yl) ethyl glycine alpha-non-natural amino acid is mixed protein. O-tRNA/O-RS pairing energy mediation L-(umbelliferone-4-yl) ethyl glycine of the present invention mixes the protein of polynucleotide encoding, and wherein said polynucleotides comprise optant's codon of described O-tRNA identification. Optant's codon on the anticodon loop of the described O-tRNA identification mRNA, and then alpha-non-natural amino acid mixed this site in the polypeptide. Quadrature aminoacyl of the present invention-tRNA synzyme is usually only with its O-tRNA of the preferential aminoacylation of a kind of specific alpha-non-natural amino acid (or loading).

Quadrature tRNA/ aminoacyl-tRNA synzyme technology

Understand the activity relevant with quadrature aminoacyl-tRNA synzyme pairing with quadrature tRNA and help to understand new compositions of the present invention and method. For in genetic code, adding extra alpha-non-natural amino acid, need to contain aminoacyl-tRNA synzyme and the suitably mating first month of the lunar year pair of tRNA, they can effectively work in host's translating mechanism, but be " quadrature " for described translation system, this means that it can not rely on the endogenous synzyme of translation system and tRNA and works. The required feature of quadrature pairing comprises decode or identifying can't help the tRNA of only a kind of specific cryptosystem (for example optant's codon) that any endogenous tRNA decodes, and can only be with the aminoacyl of its cognate tRNA of the preferential aminoacylation of a kind of specific alpha-non-natural amino acid (or " loading ")-tRNA synzyme. The endogenous synzyme is aminoacylation O-tRNA (or aminoacylation namely loads not good) not usually. For example; in the escherichia coli host system; quadrature pairing comprises not with the aminoacyl of any endogenous tRNA (for example, having 40 kinds in the Escherichia coli) cross reaction-tRNA synzyme with not by the quadrature tRNA of any endogenous synzyme (for example, having 21 kinds in the Escherichia coli) aminoacylation.

The generic principles that is suitable for preparing the orthogonal translation system of the protein that contains one or more alpha-non-natural amino acids known in the art for example prepares the universal method of orthogonal translation system. For example, can be referring to international publication number WO 2002/086075, " METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS " by name (producing the method and composition of quadrature tRNA-aminoacyl-tRNA synzyme pairing); WO 2002/085923, " IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS " (mixing in the body of alpha-non-natural amino acid) by name; WO 2004/094593, " EXPANDING THE EUKARYOTIC GENETIC CODE " by name (expansion eucaryon genetic code); The WO 2005/019415 that on July 7th, 2004 submitted to; The WO 2005/007870 that on July 7th, 2004 submitted to; The WO 2005/007624 that on July 7th, 2004 submitted to; The WO 2006/110182 that on October 27th, 2005 submitted to, the U.S. utility application sequence number 11/715 that " ORTHOGONAL TRANSLATION COMPONENTS FOR THE IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS " by name (being used for mixing in the body orthogonal translation components of alpha-non-natural amino acid) and on March 7th, 2007 submit to, 672, " SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS (in the eubacteria host cell, expressing the system of orthogonal translation components) " by name. This paper is included in these applications separately by reference in full in. The discussion of mixing the orthogonal translation system of alpha-non-natural amino acid and their generation and using method also can be referring to Wang and Schultz, " Expanding the Genetic Code (expansion genetic code) ", Chem. Commun. (Camb.) 1:1-11 (2002); Wang and Schultz, " Expanding the Genetic Code (expansion genetic code) ", Angewandte Chemie Int.Ed., 44 (1): 34-66 (2005); Xie and Schultz, " An Expanding Genetic Code (genetic code of expansion) ", Methods 36 (3): 227-238 (2005); Xie and Schultz, " Adding Amino Acids to the Genetic Repertoire (amino acid is added hereditary storehouse) ", Curr.Opinion in Chemical Biology 9 (6): 548-554 (2005); Wang etc., " Expanding the Genetic Code (expansion genetic code) ", Annu.Rev.Biophys.Biomol.Struct., 35:225-249 (2006); Xie and Schultz, " A chemical toolkit for proteins-an expanded genetic code (genetic code of the chemical tools case of protein-expansion) ", Nat.Rev.Mol.Cell Biol., 7 (10): 775-782 (2006); The content of these documents is included this paper by reference in full in.

The orthogonal translation system

It (can be prokaryotic that the orthogonal translation system generally comprises the cell that contains quadrature tRNA (O-tRNA), quadrature aminoacyl tRNA synthetase (O-RS) and alpha-non-natural amino acid; Escherichia coli for example), wherein said O-RS is with the described O-tRNA of described alpha-non-natural amino acid aminoacylation. Quadrature pairing of the present invention can comprise O-tRNA, such as inhibition type tRNA, frameshit tRNA etc. and related O-RS. Orthogonal system of the present invention usually comprises and is in the host cell environment or without the O-tRNA/O-RS pairing of host cell. Except multicomponent system, the present invention also provides novel one pack system, for example, and the polynucleotides (for example, SEQ ID NO:5) of novel quadrature aminoacyl-tRNA synzyme polypeptide (for example, SEQ ID NO:4) and encoding those polypeptides.

React and when loading amino acid, usually claim this quadrature pairing " inhibition " this optant's codon when quadrature pairing identification selection person's codon and to this optant's codon. That is, the optant's codon that is not translated (for example, cell) endogenetic mechanisms identification of system is not loaded usually, thereby has blocked the polypeptide generation, otherwise can be from this polypeptide of translated nucleic acid. In the quadrature pair system, O-RS is with specific alpha-non-natural amino acid aminoacylation O-tRNA. The O-tRNA identification selection person codon that loads also suppresses translation blocking-up due to optant's codon.

In some respects, with comprise polynucleotide sequence shown in this paper sequence table or compared by the suppression efficiency of the O-tRNA of its coding, O-tRNA identification selection person codon of the present invention and in the presence of related synzyme, optant's codon reacted and have at least about, for example, 45%, 50%, 60%, 75%, 80% or 90% or higher suppression efficiency.

In some embodiments, the suppression efficiency of coupling O-RS and O-tRNA is the pact of suppression efficiency that lacks the O-tRNA of O-RS, for example 5 times, 10 times, 15 times, 20 times or 25 times or higher. In some respects, the suppression efficiency of coupling O-RS and O-tRNA be quadrature synzyme shown in this paper sequence table pairing suppression efficiency at least about, for example 35%, 40%, 45%, 50%, 60%, 75%, 80% or 90% or higher.

Host cell utilizes O-tRNA/O-RS pairing that alpha-non-natural amino acid is mixed polypeptide chain in the extension, and for example via the nucleic acid of the polynucleotides that comprise the polypeptide of interest of encode, wherein said polynucleotides comprise optant's codon that described O-tRNA identifies. Some preferred aspect, cell can comprise one or more other O-tRNA/O-RS pairing, wherein said other O-RS is with different described other O-tRNA of alpha-non-natural amino acid loading. For example, one of O-tRNA can identify four base codons, and other O-tRNA can identify terminator codon. Perhaps, a plurality of different terminator codons or a plurality of four different base codons can be used for same code nucleic acid.

It should be noted that in some embodiments, can have a plurality of O-tRNA/O-RS pairings in cell or other translation system, thereby a plurality of alpha-non-natural amino acids can be mixed polypeptide. For example; cell also can comprise extra different O-tRNA/O-RS pairings and the second alpha-non-natural amino acid; wherein this extra O-tRNA identifies second optant's codon, and this extra O-RS is with this extra O-tRNA of the preferential aminoacylation of this second alpha-non-natural amino acid. For example, comprise cell (the wherein said O-tRNA identification of O-tRNA/O-RS pairing, amber optant codon for example) also can comprise the pairing of the second quadrature, the different optant's codon of the 2nd O-tRNA identification wherein, such as opal codon, four base codons etc. Different quadratures matches preferably derived from separate sources, thereby helps to identify different optant's codons.

In some embodiments, system comprises such as cells such as Bacillus coli cells, these cells contain quadrature tRNA (O-tRNA), quadrature aminoacyl tRNA synthetase (O-RS), alpha-non-natural amino acid and comprise the nucleic acid of the polynucleotides of the polypeptide of interest of encoding, and wherein said polynucleotides comprise optant's codon of described O-tRNA identification. Translation system can also be cell free system, for example transcribes/translation system with various commercially available the getting " external " of O-tRNA/O-RS pairing described herein and alpha-non-natural amino acid combination.

O-tRNA and/or O-RS can be natural generations, perhaps can be, for example natural tRNA and/or RS derive through sudden change, for example, and tRNA library and/or the RS library by producing various biologies and/or adopt various available sudden change schemes. For example, a kind of scheme that produces quadrature tRNA/ aminoacyl-tRNA synzyme pairing comprise with, for example derive from source except host cell or allos (for host cell) the tRNA/ synzyme pairing in a plurality of sources and input host cell. The characteristic of candidate's allos synzyme comprises, does not for example load any host cell tRNA, and the characteristic of candidate's allos tRNA comprises, for example not by any host cell synzyme institute aminoacylation. In addition, allos tRNA is quadratures for all host cell synzyme. The first scheme that produces the quadrature pairing comprises the mutant library that produces for screening and/or select O-tRNA or O-RS. But also these schemes of coupling.

Quadrature tRNA (O-tRNA)

Quadrature tRNA of the present invention (O-tRNA) preferably in body for example or external mediation alpha-non-natural amino acid is mixed in the protein, described protein is by the polynucleotide encoding that contains optant's codon, and this optant's codon can be identified by this O-tRNA. In some embodiments, with comprise this paper sequence table in polynucleotide sequence shown in the O-tRNA sequence or compared by the O-tRNA of its coding, O-tRNA of the present invention reacts to optant's codon and has at least 45%, 50%, 60%, 75%, 80%, 90% or higher suppression efficiency in the presence of related synzyme.

Can be by multiple test determination suppression efficiency known in the art. For example, can adopt the beta galactosidase reporter assay, for example, the lacZ plasmid that will derive (this construction contains optant's codon in the lacZ nucleotide sequence) and the plasmid that contains O-tRNA of the present invention import in the suitable biology cell of (as utilizing the biology of quadrature component) together. Also can import related synzyme (polynucleotides of the related synzyme of codified when polypeptide or expression). Cell is cultivated desired density in culture medium, such as OD₆₀₀, adopt for example BetaFluor of Nova King Company (Novagen) at about 0.5 o'clock^TMThe beta galactosidase detection kit is carried out the beta galactosidase test. Inhibition percentage may be calculated sample with respect to the active percentage that can relatively contrast (as the observed value of the lacZ construction of deriving, described construction contains corresponding sense codon rather than optant's codon in desired location).

The example of O-tRNA of the present invention is seen this paper sequence table, for example referring to Fig. 9 and SEQ ID NO:1. This paper content also provides the guidance that designs other O-tRNA of equal value. In RNA molecule (for example O-RS mRNA or O-tRNA molecule), to compare with given sequence (or vice versa for coding DNA) or its complementary series, thymidine (T) is replaced by uracil (U). Also can exist other modification of base with a large amount of generation function equivalence molecules.

The present invention also comprises the O-tRNA examples of conservative variations corresponding to the concrete O-tRNA of this paper. For example, the O-tRNA examples of conservative variations comprises that function and concrete O-tRNA (for example in this paper sequence table) are similar, and has kept the L-shape structure of tRNA because suitable self is complementary but does not have the sequence identical with for example sequence table or those (sequences) shown in Figure 9 and those molecules that preferably neither wild type tRNA molecule.

The composition that contains O-tRNA also can comprise quadrature aminoacyl-tRNA synzyme (O-RS), and wherein said O-RS is with the preferential aminoacylation O-tRNA of alpha-non-natural amino acid. In some embodiments, the composition that contains O-tRNA also can comprise (for example external or body in) translation system. Also can have one or more combination in the nucleic acid of the polynucleotides that contain the polypeptide of interest of encoding or these materials in the cell, wherein said polynucleotides contain optant's codon that O-tRNA can identify.

The method that produces quadrature tRNA (O-tRNA) also is one of feature of the present invention. The quadrature tRNA (O-tRNA) that produces by the method also is one of feature of the present invention. In some embodiments of the present invention, can produce O-tRNA by making up mutant library. Can adopt various induced-mutation technique known in the art to make up mutant tRNA library. For example, can pass through any combination results mutant tRNA of mutation site-specific, random order point mutation, homologous recombination, DNA reorganization or other recurrence mutagenesis (recursive mutagenesis) method, chimera structure or these technology, for example O-tRNA shown in the SEQ ID NO:1.

Also other sudden change can be introduced ad-hoc location, for example one or more non-conservative positions, conservative position, one or more random site or the block position of the two in the required ring of tRNA or zone (such as other zone or its combination of anticodon loop, acceptor stem, D arm or ring, variable loop, TPC arm or ring, tRNA molecule). Sudden change among the tRNA generally includes the anticodon loop sudden change that makes each member in the saltant tRNA library with can identification selection person's codon. The method also can comprise to O-tRNA and adds additional sequences. Compare with parent material (for example multiple tRNA sequence), O-tRNA has improved the orthogonality to required biology usually, keeps simultaneously it to the affinity of required RS.

The optional similitude (and/or homology of inferring) of analyzing tRNA and/or aminoacyl-tRNA synzyme sequence that comprises of these methods is to determine potential candidate O-tRNA, O-RS and/or its pairing of demonstration and particular organisms quadrature. Can utilize computer program known in the art and as herein described to carry out this analysis, for example available BLAST and pileup program. In one embodiment, for screening is used for colibacillary possible orthogonal translation components, can select not show with the eubacteria biology synzyme and/or the tRNA of approaching sequence similarity.

Usually can pass through, for example the negative cell mass of selecting the first species is to obtain O-tRNA, and wherein said cell contains certain member of multiple potential O-tRNA. Negative selection can be removed the cell that contains by the potential O-tRNA library member of cell endogenous aminoacyl-tRNA synzyme (RS) aminoacylation. So just can obtain the quadrature tRNA storehouse of the first species cell.

Some embodiment is introduced the coding negative selectable marker with optant's codon in negative the selection (for example can give enzyme such as the beta lactamase of antibiotic resistance; Can obtain to detect the enzyme of product such as beta galactosidase, chloramphenicol acetyltransferase (CAT), described product for example is toxic product, such as barnase) the nonessential position (for example, still can produce functional barnase) etc. of polynucleotides. Also choosing wantonly at selective reagent (such as antibiotic, such as ampicillin) exists lower cultured cell group to screen/select. In one embodiment, the concentration of selective reagent is different.

For example, for detecting the activity of inhibition type tRNA, can utilize the selective system that suppresses in the body based on optant's codon, for example nonsense (as stopping) or frameshift mutation be introduced in the polynucleotides (such as the gene (bla) of coding beta-lactamase) of coding negative selectable marker. For example, be structured in certain position (as, A184) contain the polynucleotides variant of optant's codon, such as the bla variant. With these polynucleotides transformants, such as bacterium. Take the quadrature tRNA that can not effectively be loaded by endogenous Escherichia coli synzyme as example, antibiotic resistance (for example amicillin resistance) should be about or less than the antibiotic resistance of the bacterium of not using Plasmid Transformation. If tRNA is not quadrature, perhaps can load allos synzyme co expression in this system of this tRNA, should can be observed higher levels of antibiotic (such as ampicillin) resistance. Select the cell that those can not be grown on antibiotic concentration and the cell LB agar plate about equally with Plasmid Transformation not, such as bacterium.

Take toxic product (such as ribalgilase or barnase) as example; as the member among the multiple potential tRNA during by endogenous host (such as Escherichia coli) synzyme (not being quadrature with host such as Escherichia coli synzyme namely) aminoacylation; optant's codon is suppressed, and the toxicity polynucleotide products that produces causes cell death. And the cell that contains quadrature tRNA or non-functional tRNA can be survived.

Then, in one embodiment, to just selecting with the tRNA storehouse of required biological quadrature, wherein optant's codon is placed positive selected marker (for example mark of drug resistance gene (such as the beta lactamase gene) coding). Following cell is just selected: contain coding and the tRNA storehouse member's of this cell quadrature polynucleotides or comprise this member polynucleotides, contain the polynucleotides of the positive selected marker of encoding and contain the polynucleotides of the related RS that encodes. In some embodiments, second group of cell contains the cell that can not remove by bearing selection. These polynucleotides are at intracellular expression, and cell is having growth in the presence of the selective reagent (such as ampicillin). Then select and to react and insert amino acid whose tRNA by the related synzyme aminoacylation of co expression and to this optant's codon. With one or more contain non-functional tRNA or can not by synzyme interested effectively the cell of the tRNA of identification compare, these cells show that usually suppression efficiency raises. Contain non-functional tRNA or can not by synzyme interested effectively the cell of the tRNA of identification to this antibiotic sensitive. The tRNA that therefore can retain in twice selection is: the substrate that (i) is not endogenous host (such as Escherichia coli) synzyme; (ii) can be by interested synzyme aminoacylation; And (iii) can in translation process, work.

Therefore, depend on the residing environment of screening, same mark can be the plus or minus mark. That is, if in order to screen this mark, then it is positive mark, if but in order to resist this mark then it is negative flag.

In the said method, screening changes the stringency of selecting such as the stringency of just selecting, bear selection or positive and negative selection optional comprising. For example, because barnase is the high protein of toxicity, can be incorporated into by the optant's codon with different numbers in the barnase gene and/or with inducible promoters and controls the negative stringency of selecting. In another embodiment, the concentration (such as the concentration of ampicillin) of selection or screening reagent can be different. Aspect more of the present invention, because required activity is lower in former the wheel, stringency can be different. Therefore, former applicable lower stringency screening criteria, then several applicable more rigorous standards of selecting of taking turns of taking turns. In some embodiments, negatively select, just select or positive and negative selection can be repeatedly. Also can use a plurality of different negative selectable markers, positive selected marker or positive negative selectable marker. In some embodiments, positive selected marker can be identical with negative selectable marker.

Selection/the screening technique of other types also can be used for the present invention with the preparation orthogonal translation components, such as O-tRNA, O-RS with can react and load the O-tRNA/O-RS pairing of alpha-non-natural amino acid optant's codon. For example, negative selectable marker, positive selected marker or positive negative selectable marker can comprise mark fluorescigenic or energy catalytic luminescence reaction in the presence of suitable reactants. In another embodiment, can pass through the product of fluorescence-activated cell sorting (FACS) or luminous certification mark. Mark is optional can to comprise affine selection markers. Also referring to Francisco, J.A. etc., (1993), " Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragement on the external surface (outside colibacillary preparation and the fluorescence-activated cell sorting of surface expression functional antibodies fragment) "Proc Natl Acad Sci USA.，90：10444-8。

As seen other method of preparation restructuring quadrature tRNA for example is called the international publication WO 2002/086075 of " METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS (method and composition of preparation quadrature tRNA-aminoacyl-tRNA synzyme pairing) "; The WO 2004/094593 of " EXPANDING THE EUKARYOTIC GENETIC CODE (expansion the eukaryotic genetic code) " by name; With the WO 2005/019415 that submitted on July 7th, 2004. Also referring to Forster etc., (2003), " Programming peptidomimetic synthetases by translating genetic codes designed de novo (by the translation genetic code peptide analog synthesis enzyme of programming of design from the beginning) "PNAS, 100 (11): 6353-6357; With Feng etc., (2003), " Expanding tRNA recognition of a tRNA synthetase by a single amino acid change (changing the tRNA identification that enlarges the tRNA synzyme by monamino acid) ",PNAS，100 (10)：5676-5681。

Quadrature aminoacyl-tRNA synzyme (O-RS)

O-RS of the present invention can be with the preferential aminoacylation O-tRNA of alpha-non-natural amino acid in external or body. Can O-RS of the present invention be offered translation system by the polypeptide that contains O-RS and/or the polynucleotides of encoding O-RS or its part, such as cell. For example, exemplary O-RS comprises amino acid sequence or its conservative variant shown in the SEQ ID NO:4. In another embodiment, O-RS or its part are polynucleotide sequence or its complementary polynucleotide sequential codings that is contained the amino acid sequence shown in this paper sequence table and the embodiment by coding. Can be referring to, polynucleotides shown in the SEQ ID NO:5 for example.

The quadrature aminoacyl that evaluation can be used with O-tRNA-tRNA synzyme (O-RS), for example the method for O-RS also is one of feature of the present invention. For example, a kind of method comprises the cell mass of selection (as just selecting) the first species, wherein said cell contains separately: 1) one of member of multiple aminoacyl-tRNA synzyme (RS) (for example, described multiple RS can comprise saltant RS, derived from the RS of the species outside the first species, or saltant RS and derived from the RS of the species outside the first species the two); 2) quadrature tRNA (O-tRNA) (for example, one or more species); And 3) codes selection (for example just selecting) mark and contain the polynucleotides of at least one optant's codon. Compare with the cell that the member who lacks described multiple RS or number of members are few, select or screening shows those cells that suppression efficiency improves. Available method known in the art and as herein described is measured suppression efficiency. The cell that suppression efficiency improves comprises the active RS of energy aminoacylation O-tRNA. The level of the RS aminoacylation that second group of tRNA of the level of the active RS aminoacylation of first group of tRNA of the first species (external or body in) and the second species lived is made comparisons. Measure the aminoacylation level by detectable substance (for example, the alpha-non-natural amino acid of mark). Usually select to compare with first group of tRNA the more effectively active RS of second group of tRNA of aminoacylation, thus obtain can with effective (optimization) quadrature aminoacyl of O-tRNA coupling-tRNA synzyme. The O-RS that adopts this method to identify also is one of feature of the present invention.

Can measure aminoacylation with many tests. These tests can be carried out in external or body. For example, as seen external aminoacylation tests for example Hoben and Soll, (1985), Methods Enzymol., 113:55-59. Also but coupling reporter molecule and orthogonal translation components and the reporter molecule that detects in the cell are measured aminoacylation, and described cellular expression contains the polynucleotides of at least one optant's codon and certain protein of encoding. Also referring to the WO 2002/085923 of " IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS (mixing in the body of alpha-non-natural amino acid) " by name and the WO 2004/094593 of " EXPANDING THE EUKARYOTIC GENETIC CODE (expansion the eukaryotic genetic code) " by name.

The O-RS that also can further transform evaluation to be changing the substrate specificity of this synzyme, thereby so that O-tRNA only loads required alpha-non-natural amino acid, rather than any 20 common seed amino acids. Generation comprises the method that alpha-non-natural amino acid has the quadrature aminoacyl tRNA-synzyme of substrate specificity, make synzyme at its avtive spot, edit machine-processed site, undergo mutation etc. in different loci at it such as the different structure territory by the combination synzyme, and selective method for application. Also can adopt the scheme of just selecting to bear again first selection. In just selecting, suppress to introduce the positive mark one or more non-critical positions optant's codon so that cell under positive selection pressure, surviving. When having at the same time natural and alpha-non-natural amino acid to exist, so the cell coding of survival makes quadrature inhibition type tRNA with the active synzyme of natural or alpha-non-natural amino acid. In negative the selection, the optant's codon that suppresses one or more non-critical positions of introducing negative marker makes synzyme lose the natural amino acid specificity. The cell coding of surviving through positive and negative selection is only used the synzyme of alpha-non-natural amino acid aminoacylation (loading) quadrature inhibition type tRNA. Then by for example DNA reorganization or these synzyme of the further mutagenesis of other recurrence method of mutagenesis.

Can adopt various induced-mutation technique known in the art to produce saltant O-RS library. For example, available mutation site-specific, random point mutation, homologous recombination, DNA reorganization or other recurrence method of mutagenesis, chimeric construct or their combination prepare saltant RS. For example, can other prepare saltant RS library such as less, that diversity is lower " Ya Wenku " from two or more. The present invention also comprises the chimeric library of RS. It should be noted that the tRNA synzyme library that can make up various biologies (for example microorganism, such as eubacteria or archeobacteria), for example have natural multifarious library (referring to, such as the U.S. Patent number 6,238,884 of authorizing Short etc.; Authorize the U.S. Patent number 5,756,316 of Schallenberger etc.; Authorize the U.S. Patent number 5,783,431 of Petersen etc.; Authorize the U.S. Patent number 5,824,485 of Thompson etc.; Authorize the U.S. Patent number 5,958,672 of Short etc.), and optional the structure and the pairing of screening quadrature.

In case synzyme experience positive and negative selection/screening technique, just further these synzyme of mutagenesis. The nucleic acid of separable coding O-RS for example; Produce one group of polynucleotides (for example by random mutagenesis, site-specific mutagenesis, restructuring or their any combination) of encoding mutant O-RS from this nucleic acid; Can repeat the combination of each step or each step until obtain can be with the sudden change O-RS of the preferential aminoacylation O-tRNA of alpha-non-natural amino acid. Aspect more of the present invention, these steps can be carried out repeatedly, and for example at least twice.

The inventive method also can adopt the selection/screening severity of other level to produce O-tRNA, O-RS or its pairing.Can change the selection or the screening severity of one or two step in the O-RS production method.This comprises, for example, changes the consumption of selection/screening reagent etc.Also can additionally carry out several just the wheel and/or negative the selection.Select or screen also to comprise following one or more changes: amino acid perviousness, translation efficiency, translation fidelity of reproduction (translational fidelity) etc.One or more changes produce one or more transgenations in the proteinic biology according to matching with quadrature tRNA-tRNA synthetic enzyme usually.

Other the conventional details that produces O-RS and change the synthetic enzyme substrate specificity is seen the WO 2002/086075 of " METHODS AND COMPOSITIONS FOR THE PRODUCTION OFORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS; (producing quadrature tRNA-aminoacyl tRNA synthetase method of matching and composition) " by name and the WO 2004/094593 of " EXPANDING THE EUKARYOTIC GENETIC CODE; (expansion the eukaryotic genetic code) " by name.Also can be referring to Wang and Schultz, " Expanding theGenetic Code (expansion genetic code) ", Angewandte Chemie Int.Ed., 44 (1): 34-66 (2005), its content is included in full with way of reference.

Source and host living beings

Orthogonal translation components of the present invention (O-tRNA and O-RS) can derive from any biology (or biological combination), can be used for host's translation system of any other species, as long as described O-tRNA/O-RS component and host system can work with orthogonal manner.O-tRNA and O-RS in certain quadrature pairing need not to derive from same biology.In some respects, the quadrature component derives from ancient bacterium (that is the archeobacteria) gene that is used for the eubacterium host system.

For example, the quadrature O-tRNA bacterium biology from ancient times of can deriving, archeobacteria for example is as Methanococcus jannaschii, thermophilic alkali methagen; The salt bacterium is as rich salt bacterium of Wo Shi and salt bacterium kind NRC-1; Glimmer ancient green-ball bacterium, fierce fireball bacterium, extreme hyperthermophilic archaeon strain, thermophilic spring given birth to archeobacteria, extra large natural pond methane coccus, methane and had a liking for high hot bacterium (Methanopyrus kandleri), Mei Shi sarcina methanica (Mm), superhigh temperature resistant hot pin bacterium (Pyrobaculum aerophilum), deep-sea fireball bacterium, sulfolobus solfataricus (Ss), super hyperthermophilic archaeon strain (Sulfolobus tokodaii), thermoplasma acidophilum, hot volcanic substance etc.; Or eubacterium, as intestinal bacteria, thermus thermophilus, Bacillus subtillis (Bacillus subtilis), bacstearothermophilus etc.; And quadrature O-RS can derive from following biology (or biological combination), and archeobacteria for example is as Methanococcus jannaschii, thermophilic alkali methagen; The salt bacterium is as rich salt bacterium of Wo Shi and salt bacterium kind NRC-1; Glimmer ancient green-ball bacterium, fierce fireball bacterium, extreme hyperthermophilic archaeon strain, thermophilic spring given birth to archeobacteria, extra large natural pond methane coccus, methane and had a liking for high hot bacterium, Mei Shi sarcina methanica, superhigh temperature resistant hot pin bacterium, deep-sea fireball bacterium, sulfolobus solfataricus, super hyperthermophilic archaeon strain, thermoplasma acidophilum, hot volcanic substance etc.; Or eubacterium, as intestinal bacteria, thermus thermophilus, Bacillus subtillis, bacstearothermophilus etc.In one embodiment, the eukaryote source, for example plant, algae, protozoon, fungi, yeast, animal (for example Mammals, insect, arthropods etc.) etc. also can be used as the source of O-tRNA and O-RS.

Each component of O-tRNA/O-RS paired can derive from same biology or different biological.In one embodiment, the O-tRNA/O-RS pairing can be from same biology.Perhaps, O-tRNA/O-RS paired O-tRNA can be from different biological with O-RS.

O-tRNA, O-RS or O-tRNA/O-RS pairing can be in vivo or external selection or screening and/or can be used for cell, eubacterium cell for example, thus produce the polypeptide that contains alpha-non-natural amino acid.Used eubacterium cell is such as but not limited to intestinal bacteria, thermus thermophilus, Bacillus subtillis, bacstearothermophilus etc.The eubacterium cell composition that contains translation components of the present invention also is one of feature of the present invention.

Another kind of biological in a kind of biology, screening O-tRNA and/or O-RS to be used for, also can be referring to the international application published WO2004/094593 of " the EXPANDING THE EUKARYOTICGENETIC CODE (expansion the eukaryotic genetic code) " by name that submitted on April 16th, 2004.

Though the orthogonal translation system (for example, contain O-RS, O-tRNA and alpha-non-natural amino acid) can utilize the host cell generation of cultivation to contain the protein of alpha-non-natural amino acid, but be not that requirement orthogonal translation of the present invention system needs complete, great-hearted host cell.For example, in the presence of cell extract, the orthogonal translation system can utilize cell free system.In fact, using acellular in-vitro transcription/translation system to produce protein is recognized techniques.Utilize orthogonal translation system components as herein described, these vitro system are applicable to produce the protein contain alpha-non-natural amino acid also to belong to scope of the present invention.

Selector's codon

Selector's codon of the present invention has enlarged the genetic code subframe of protein biosynthesis mechanism.For example, selector's codon comprises, as the codon of three base codons of uniqueness, nonsense codon (as terminator codon (as amber codon (UAG) or opal codon (UGA))), non-natural codon, at least four bases, rare codon etc.Many selector's codons can be introduced required gene, for example more than one, more than two, three with first-class.Utilize different selector's codons, can utilize many to quadrature tRNA/ synthetic enzyme pairing, thereby can utilize these different selector's codons to mix a plurality of alpha-non-natural amino acids simultaneously locus specificity, for example comprise at least one alpha-non-natural amino acid.

In one embodiment, these methods comprise that utilization mixes alpha-non-natural amino acid as selector's codon of terminator codon in the body in cell.For example, preparation can be discerned the O-tRNA of terminator codon, and by O-RS this O-tRNA of alpha-non-natural amino acid aminoacylation.Host's natural aminoacyl-this O-tRNA of tRNA synthetic enzyme nonrecognition.Can adopt conventional site-directed mutagenesis terminator codon to be introduced interested site in the polynucleotide of coding polypeptide of interest.Can referring to, Sayers for example, J.R. etc., (1988), " 5 ', 3 ' Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis (5 ', 3 ' exonuclease in the thiophosphatephosphorothioate oligonucleotide directed mutagenesis) ", Nucleic Acids Res, 791-802.When for example in vivo during the nucleic acid of coupling O-RS, O-tRNA and coding polypeptide of interest, can react and mix alpha-non-natural amino acid terminator codon, thereby obtain to contain the polypeptide of alpha-non-natural amino acid at specific site.In an embodiment of the invention, the terminator codon as selector's codon is amber codon UAG and/or umber codon.In one embodiment, UAG and UGA are used as 22 amino acid of genetic code codified of selector's codon, keep the ochre nonsense codon simultaneously, UAA, and it is the abundantest termination signal.

Mix alpha-non-natural amino acid in the body and can significantly not disturb host cell.For example, at non-eukaryotic cell, in intestinal bacteria, (it can be in conjunction with the UAG codon with releasing hormone 1 (RF1) because the inhibition efficient of UAG codon depends on O-tRNA (as amber inhibition type tRNA), and then cause that the peptide in the extension discharges from rrna) between competition, therefore can pass through, for example improve the expression level of O-tRNA (as inhibition type tRNA), or utilize the RF1 deficient strain to regulate and suppress efficient.In eukaryotic cell, because the inhibition efficient of UAG codon depends on O-tRNA (as amber inhibition type tRNA) and eucaryon releasing hormone (as eRF) (but its combination termination codon, and then cause that the peptide in the extension discharges from rrna) between competition, therefore can pass through, for example improve the expression level of O-tRNA (as inhibition type tRNA) and regulate inhibition efficient.In addition, also can have other compound, reductive agent for example is as dithiothreitol (DTT) (DTT).

Alpha-non-natural amino acid also can be encoded by rare codon.For example, when the arginine density loss in the external protein synthesis reaction, confirm that seldom used arginine codon AGG can utilize the synthetic tRNA of L-Ala acidylate and effectively insert ala.Can be referring to Ma etc., (1993), Biochemistry, 32:7939.In this case, synthetic tRNA can with the natural tRNA that exists as submember in the intestinal bacteria ^ArgCompetition.In addition, some biologies can not utilize all codeword triplets.Can in in-vitro transcription/translation extract, utilize the sub-AGA of not designated pin of micrococcus luteus (Micrococcus luteus) to insert amino acid.Can referring to, for example Kowal and Oliver, (1997), Nucl.Acid.Res., 25:4685.Can produce each component of the present invention to utilize these rare codons in the body.

Selector's codon also can comprise the extension codon, and the codon of four or more base for example is as four, five, six or the codon of polybase base more.The example of four base codons comprises, for example AGGA, CUAG, UAGA, CCCU etc.The example of five base codons comprises, for example AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC etc.The inventive method can comprise the extension codon that use suppresses based on frameshit.The codon of four or more base can insert same protein with for example one or more alpha-non-natural amino acids.In other embodiments, for example at least a four base codons of anticodon loop decodable code, at least a five base codons or at least a hexabasic basic codon or more.Because four possible base codons have 256 kinds, so the codon of available four or more base multiple alpha-non-natural amino acid of encoding in the same cell.Also can referring to, Anderson etc., (2002), " Exploring the Limits of Codon and AnticodonSize (research of codon and anticodon size limit) ", Chemistry and Biology, 9:237-244 and Magliery, (2001), " Expanding the Genetic Code:Selection ofEfficient Suppressors of Four-base Codons and Identification of ＂ Shifty ＂ Four-base Codons with a Library Approach in Escherichia coli (amplification genetic code: in intestinal bacteria, select effective four base codon inhibitor and identify " variation " four base codons) " with the library method, J.Mol.Biol., 307:755-769.

For example, adopted the external biological synthetic method to utilize four base codons that alpha-non-natural amino acid is mixed protein.Referring to, Ma etc. for example, (1993), Biochemistry, 32:7939 and Hohsaka etc., (1999), J.Am.Chem.Soc., 121:34.The frameshit inhibition type tRNA of coupling CGGG and AGGU and two kinds of chemical acylation mixes Streptavidin simultaneously at external NBD derivative with 2-naphthyl L-Ala and Methionin.Can referring to, Hohsaka etc. for example, (1999), J.Am.Chem.Soc., 121:12194.In the research, Moore etc. have detected the tRNA that contains the NCUA anticodon in vivo ^LeuDerivative suppresses the ability of UAGN codon (N can be U, A, G or C), finds to contain the tRNA of UCUA anticodon ^LeuThe tetrad UAGA that can decode, its efficient is 13 to 26%, and decoding hardly in 0 or-1 frame.Can be referring to Moore etc., (2000), J.Mol.Biol., 298:195.In one embodiment, the present invention can utilize the extension codon based on rare codon or nonsense codon, and they can be reduced in other missense of not expecting the site and even read the inhibition of (missense readthrough) and frameshit.Four base codons have been used as selector's codon in various orthogonal systems.Can referring to, for example, WO 2005/019415; WO 2005/007870 and WO 2005/07624.Also can be referring to Wang and Schultz, " Expanding the Genetic Code (expansion genetic code) ", AngewandteChemie Int.Ed., 44 (1): 34-66 (2005), its content mode is by reference included in full.Though following example utilizes amber selector codon, but make it to comprise four base O-tRNA and the modified synthetic enzyme that comprises the sudden change similar by the example that improves this paper, also can use four bases or the codon of polybase base more with the sudden change of each alpha-non-natural amino acid O-RS of description before.

For giving fixed system, selector's codon also can comprise that natural base codon that endogenous system in the natural three base codons need not (or seldom using).For example, this comprises that shortage can discern the system of the tRNA of these natural three base codons, and/or this three bases codon is the system of rare codon.

The optional non-natural base pair that comprises of selector's codon.These non-natural base pairs have further enlarged existing hereditary character set (genetic alphabet).A kind of extra base pair can increase to 125 from 64 with the number of codeword triplet.The characteristic of the 3rd base pair comprises: stable and optionally base pairing, utilize polysaccharase with high fidelity effectively enzymatic mix DNA, the extension of remaining valid of the synthetic back of newborn non-natural base pair primer.The description that is applicable to the non-natural base pair of these method and compositions comprises: Hirao etc. for example, (2002), " An unnatural base pair for incorporating amino acidanalogues into protein (amino acid analogue is mixed proteinic non-natural base pair) ", Nature Biotechnology, 20:177-182.Also can be referring to Wu, Y. etc., (2002), J.Am.Chem.Soc., 124:14626-14630.Hereinafter listed other relevant publication.

For using in the body, non-natural nucleoside is that film is permeable, but and phosphorylation form corresponding phosphotriester.In addition, the genetic information of increase is stablized and is not destroyed by cellular enzymes.Benner and the work that other people are previous have utilized and has been different from classical Watson-Crick paired hydrogen bond pattern, and wherein the most noticeable example is different-C: different-G matches (iso-C:iso-G pair).Referring to, Switzer etc. for example, (1989), J.Am.Chem.Soc., 111:8322; Piccirilli etc., (1990), Nature, 343:33; Kool, (2000), Curr.Opin.Chem.Biol., 4:602.These bases usually have to a certain degree mispairing with natural base, thereby can not duplicate by enzymatic.Kool and colleague's proof, hydrophobic packing interaction (hydrophobic packing interaction) the alternative hydrogen bond between base forms to drive base pair.Referring to Kool, (2000), Curr.Opin.Chem.Biol., 4:602; Guckian and Kool, (1998), Angew.Chem.hit.Ed.Engl., 36:2825.In the process of being devoted to develop the non-natural base pair that meets above all requirements, Schultz, Romesberg and colleague are systematically synthetic and studied the hydrophobic base of a series of non-naturals.Find that PICS:PICS self pairing is more stable than natural base pair, and the Klenow fragment (KF) of available e. coli dna polymerase I is mixed DNA effectively.Can referring to, McMinn etc. for example, (1999), J.Am.Chem.Soc., 121:11586; With Ogawa etc., (2000), J.Am.Chem.Soc., 122:3274.With regard to biological function, synthetic 3MN:3MN self paired efficient of KF and selectivity are enough.Can referring to, Ogawa etc. for example, (2000), J.Am.Chem.Soc., 122:8803.Yet these two kinds of bases all work as the chain terminator that further duplicates.Recently developed and can be used for duplicating PICS self paired mutant DNA polymerases.In addition, reproducible 7AI self pairing.Can referring to, Tae etc. for example, (2001), J.Am.Chem.Soc., 123:7439.Also developed with can forming after Cu (II) combines and stablized paired metal base (metallobase) Dipis:Py.Can referring to, Meggers etc., (2000), J.Am.Chem.Soc., 122:10714.Because extend codon and non-natural codon in itself with natural codon quadrature, so the inventive method can utilize this characteristic to produce their quadrature tRNA.

Also can utilize the translation bypath system that alpha-non-natural amino acid is mixed required polypeptide.In the translation bypath system, a big sequence can be inserted gene, but this sequence is not translated into protein.This sequence contains the structure that can be used as prompting, thereby can induce rrna to skip the downstream translation that this sequence is recovered this insertion sequence then.

Alpha-non-natural amino acid

Alpha-non-natural amino acid used herein refers to any amino acid except that the a-amino acid of seleno-cysteine and/or pyrroles's Methionin and following 20 kinds of genetic codings, amino acid or the amino acid analogue modified: L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, Xie Ansuan.The universal architecture of a-amino acid is suc as formula shown in the I:

I

Alpha-non-natural amino acid generally has any structure shown in the formula I, and wherein the R group is any substituting group used in 20 kinds of natural amino acids.The structure of 20 kinds of natural amino acids can referring to, for example " biological chemistry " (Biochemistry), L.Stryer compiles, the third edition, 1988, Man in the good fortune (Freemanand Company), New York.Notice that alpha-non-natural amino acid of the present invention can be the natural compounds except that above 20 kinds of a-amino acids.

Since alpha-non-natural amino acid of the present invention and natural amino acid usually difference at side chain, so the generation type of amido linkage is identical in the mode of alpha-non-natural amino acid and other amino acid (for example natural or non-natural) formation amido linkage and the natural protein.Yet the side chain of alpha-non-natural amino acid is different from the side chain of natural amino acid.

This paper is interested especially to be that non-natural coumarin amino acids L-(umbelliferone-4-yl) ethyl glycine (is also write umbelliferone-ethyl glycine; Referring to Fig. 1, structure 1).Except umbelliferone-ethyl glycine alpha-non-natural amino acid, other alpha-non-natural amino acid can be mixed interested polypeptide simultaneously, for example suitable the 2nd O-RS/O-tRNA pairing and quadrature pairing provided by the invention of coupling.Known many other such alpha-non-natural amino acids and suitable quadrature pairing.The reference of quoting referring to this paper content and this paper.For example, referring to Wang and Schultz, " Expanding theGenetic Code (expansion genetic code) ", Angewandte Chemie Int.Ed., 44 (1): 34-66 (2005); Xie and Schultz, " An Expanding Genetic Code (expansion genetic code) ", Methods 36 (3): 227-238 (2005); Xie and Schultz, " Adding Amino Acids to theGenetic Repertoire (amino acid is added hereditary storehouse) ", Curr.Opinion in ChemicalBiology 9 (6): 548-554 (2005); With Wang etc., " Expanding the Genetic Code (expansion genetic code) ", Annu.Rev.Biophys.Biomol.Struct., 35:225-249 (2006); These documents content is separately included this paper in as a reference by reference in full.

Though mainly interested among the embodiment described herein is L-(umbelliferone-4-yl) ethyl glycine (Fig. 1 is shown in the structure 1), does not really want strictness of the present invention is limited to this structure.In fact, be not difficult to prepare variously being easy to obtain and the analogue of structurally associated, these analogues keep main fluorescent characteristic, and aminoacyl of the present invention-tRNA synthetic enzyme (for example, O-RS shown in the SEQ ID NO:4) these analogues of specific recognition also.These relevant fluorescence analogues belong to scope of the present invention.

In other alpha-non-natural amino acid; for example the R among the formula I can choose wantonly contain alkyl-, aryl-, acyl group-, hydrazine, cyano group-, halo-, hydrazides, thiazolinyl, ether, boronate (borate), boric acid ester group (boronate), phosphorus, phosphono, phosphine, ketenes, imines, ester, azanol, amine etc., or any combination of above-mentioned group.Interested other alpha-non-natural amino acid includes but not limited to: contain the amino acid that light can activate linking agent, the amino acid of spin-mark, fluorescence amino acid, melts combine amino acid, metallic amino acid, radioactivity amino acid, the amino acid that contains new functional group, can with the covalently or non-covalently interactional amino acid of other molecule, the amino acid of photolocking (photocaged) and/or photic isomery (photoisomerizable), the amino acid that contains vitamin H or vitamin H analogue, the amino acid of ketone group containing, glycosylated amino acid, the sugar moieties that links to each other with amino acid side chain, the amino acid that contains polyoxyethylene glycol or polyethers, the amino acid that heavy atom replaces, the amino acid that chemical process can be cut or light can cut, compare the amino acid that side chain prolongs with natural amino acid (as polyethers or long chain hydrocarbon, as above about 5, about 10 carbon atoms etc.), carbon containing connects the amino acid of sugar, the amino acid that contains the amino acid of amino thioic acid sulfoacid and contain one or more toxicity parts.

The present invention provides the alpha-non-natural amino acid that has with universal architecture shown in the following formula IV on the other hand:

IV

Alpha-non-natural amino acid with this structure generally can be any structure, wherein R ₁Be the used substituting groups of one of 20 kinds of natural amino acids (for example, tyrosine or phenylalanine), R ₂It is substituting group.Therefore, this type of alpha-non-natural amino acid can be regarded the natural amino acid derivative as.

Except containing Fig. 1, outside the alpha-non-natural amino acid of tonka bean camphor side chain, alpha-non-natural amino acid also can be chosen wantonly and contain modified skeleton structure, for example the structure shown in formula II and the III shown in the structure 1:

Wherein, Z generally comprises OH, NH ₂, SH, NH-R ' or S-R '; X and Y can be identical or different, generally comprise S or O, and R and R ' can choose wantonly identical or different, are selected from composition and the hydrogen identical with the R group of alpha-non-natural amino acid shown in the above-mentioned formula I usually.For example, alpha-non-natural amino acid of the present invention is also chosen wantonly and is being comprised substituting group in amino or the carboxyl shown in formula II and the III.The alpha-non-natural amino acid of the type includes but not limited to: for example contain alpha-hydroxy acid, α-thioic acid sulfoacid, alpha-amino group carbothioic acid ester of the corresponding side chain of 20 kinds of common amino acids or non-natural side chain etc.In addition, the substituting group on the α carbon is also optional to comprise L, D or α-α-two substituted amino acids, as D-L-glutamic acid, D-L-Ala, D-methyl-O-tyrosine, aminobutyric acid etc.Other alternative structure comprises annular amino acid, and as proline analogs and 3,4,6,7,8 and 9 yuan of loop proline analogues, β and γ amino acid are as beta Alanine and the γ-An Jidingsuan that replaces.

In some respects, the present invention uses the alpha-non-natural amino acid of L-configuration.Yet the present invention is not limited only to use the alpha-non-natural amino acid of L-configuration.The D-enantiomorph of also having considered these alpha-non-natural amino acids can be used for the present invention.

Not strict tonka bean camphor alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine that is confined to of alpha-non-natural amino acid that the present invention is used.Those skilled in the art will know that the various non-natural analogues of being not difficult to obtain natural amino acid.For example, but be not limited to be not difficult to prepare non-natural (amino acid) derived from tyrosine.The tyrosine analogue comprises; the tyrosine that replaces of the tyrosine that replaces of the tyrosine of para-orientation, ortho position and a position for example, wherein the tyrosine of this replacement contains alkynyl, ethanoyl, benzoyl, amino, hydrazine, azanol, sulfydryl, carboxyl, sec.-propyl, methyl, C ₆-C ₂₀Straight or branched hydrocarbon, saturated or undersaturated hydrocarbon, O-methyl, polyether group, nitro etc.In addition, also comprise polysubstituted aromatic ring.Glutamine analogue of the present invention includes but not limited to: the glutamine derivative that the derivative of Alpha-hydroxy derivative, γ-replacement, annular derivant and acid amides replace.The example of phenylalanine analogues includes but not limited to: the phenylalanine that a phenylalanine that the phenylalanine of para-orientation, ortho position replace and a position replace, wherein said substituting group contains alkynyl, hydroxyl, methoxyl group, methyl, allyl group, aldehyde radical, nitro, sulfydryl or ketone group etc.The object lesson of alpha-non-natural amino acid includes but not limited to: right-ethylenebis dithiocarbamate carbonyl-L-phenylalanine; right-(3-oxobutanoyl)-L-phenylalanine; 1,5-dansyl-L-Ala; 7-amino-coumarin amino acids; 7-hydroxyl-coumarin amino acids; nitrobenzyl-Serine; O-(2-nitrobenzyl)-L-tyrosine; right-carboxymethyl-the L-phenylalanine; right-cyano group-the L-phenylalanine; between-cyano group-L-phenylalanine; biphenyl alanine; 3-amino-L-tyrosine; the bipyridyl L-Ala; right-(2-amino-1-hydroxyethyl)-L-phenylalanine; right-isopropylthio carbonyl-L-phenylalanine; 3-nitro-L-tyrosine and right-nitro-L-phenylalanine.Also comprise right-alkynes propoxy-phenylalanine; 3; 4-dihydroxyl-L-phenylalanine (DHP); 3; 4; 6-trihydroxy--L-phenylalanine; 3; 4,5-trihydroxy--L-phenylalanine; 4-nitro-phenylalanine; right-ethanoyl-the L-phenylalanine; O-methyl-L-tyrosine; L-3-(2-naphthyl) L-Ala; the 3-methylphenylalanine; O-4-allyl group-L-tyrosine; 4-propyl group-L-tyrosine; 3-nitro-tyrosine; 3-sulfydryl-tyrosine; three-O-ethanoyl-GlcNAc β-Serine; L-DOPA (Dopa); fluoridize phenylalanine; sec.-propyl-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-iodo-phenylalanine; right-the bromobenzene L-Ala; right-amino-L-phenylalanine and sec.-propyl-L-phenylalanine etc.The reference that this paper quotes has disclosed the structure of various alpha-non-natural amino acids.Also can be referring to international publication WO 2004/094593, " EXPANDING THE EUKARYOTICGENETIC CODE (expansion eukaryotic cell genetic code) " by name; With the WO 2006/110182 that submitted on October 27th, 2005, " ORTHOGONAL TRANSLATION COMPONENTSFOR THE IN VIVO INCORPORATION OF UNNATURAL AMINOACIDS (mixing the orthogonal translation components of alpha-non-natural amino acid in the body) " by name.

The chemosynthesis of alpha-non-natural amino acid

Above-mentioned many alpha-non-natural amino acids can from, for example the A Erde Ritchie company (Aldrich) in the Sigma company (Sigma) of the U.S. or Wisconsin, USA Milwaukee city etc. buys.Those alpha-non-natural amino acids of can not commercialization buying can be chosen wantonly as described in the various publications or adopt standard method well known by persons skilled in the art synthetic.Organic synthesis technology can referring to, for example Fessendon and Fessendon, Organic Chemistry (organic chemistry), (1982, second edition, the special press (WillardGrant Press) of Mary Willard Glan, Boston, Massachusetts); March, and Advanced Organic Chemistry (senior organic chemistry) (third edition, 1985, prestige is stood father and son company (Wiley and Sons), New York); And the Advanced Organic Chemistry (senior organic chemistry) of Carey and Sundberg (third edition, A and B part, 1990, Pu Lainamu press (Plenum Press), New York).Other is described alpha-non-natural amino acid synthetic publication and comprises: the International Patent Application WO 2002/085923 of " mixing in the body of alpha-non-natural amino acid " by name; Matsoukas etc., (1995) J.Med.Chem., 38,4660-4669; King and Kidd, (1949) " A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates " (from novel method of the synthetic glutamine of phthaloyl intermediate and γ-dipeptides) .J.Chem.Soc., 3315-3319; Friedman and Chatterrji, (1959) " Synthesis of Derivatives of Glutamine asModel Substrates for Anti-tumor Agents (synthetic glutamine derivative is as the simulation substrate of antitumor drug) " .J.Am.Chem.Soc.81,3750-3752; Craig etc., (1988) " AbsoluteConfiguration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl] amino] quinoline (Chloroquine) (7-chloro-4[[4-(diethylamino)-1-methyl butyl] amino] absolute configuration of quinoline (chloroquine) enantiomorph) " .J.Org.Chem.53,1167-1170; Azoulay etc., (1991) " Glutamine analogues as PotentialAntimalarials (the glutamine analogue is as the potential antimalarial drug) ", Eur.J.Med.Chem.26:201-205; Koskinen and Rapoport (1989) " Synthesis of 4-Substituted Prolines asConformationally Constrained Amino Acid Analogues (proline(Pro) that synthetic 4-replaces is as the amino acid analogue of conformation constraint) " .J.Org.Chem.54:1859-1866; Christie and Rapoport, (1985) " Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through Amino AcidDecarbonylation and Iminium Ion Cyclization (, being applied to) " .J.Org.Chem.1989:1859-1866 by amino acid decarbonylation base and always synthetic (+)-apovincamine of imido ion cyclisation from the pure piperidines acid esters of altheine synthesizing optical; Barton etc., (1987) " Synthesis of Novel α-Amino-Acids and Derivatives Using Radical Chemistry:Synthesis of L-andD-α-Amino-Adipic Acids, L-α-aminopimelic Acid and AppropriateUnsaturated Derivatives (adopting synthetic new a-amino acid and the derivative of free-radical chemistry method: synthesize L-and D-alpha amino acid, L-alpha-amino group pimelic acid and suitable unsaturated derivative) " .Tetrahedron Lett.43:4297-4308; And Subasinghe etc., (1992) " Quisqualic acidanalogues:synthesis of beta-heterocyclic 2-aminopropanoic acid derivativesand their activity at a novel quisqualate-sensitized site (Quisqualic Acid analogue: β-heterocycle 2-alanine derivatives synthetic and in the activity at Quisqualic Acid sensitization position) " .J.Med.Chem.35:4602-7.Also can be referring to the International Patent Application WO 2004/058946 of " ProteinArrays (protein array) " by name submitted on December 22nd, 2003.

The cellular uptake of alpha-non-natural amino acid

The cellular uptake alpha-non-natural amino acid normally designs and selects alpha-non-natural amino acid, for example is used for mixing one of problem that protein should consider.For example, the high charge density of a-amino acid points out these compounds can not see through cell.Natural amino acid is gone into cell by collection (effect) picked-up of protein transport system, and these systems often show the amino acid specificity that degree is different.Can carry out rapid screening assesses cell and will absorb which kind of alpha-non-natural amino acid (if any).Can be referring to, the toxicity test among the international publication WO 2004/058946 of " Protein Arrays (protein array) " by name for example submitted on December 22nd, 2003; Liu and Schultz, (1999), " Progress toward the evolution of anorganism with an expanded genetic code (progress that contains the biogenetic derivation of the genetic code that increases) ", PNAS, 96:4780-4785.Adopt various analysis of experiments picked-up situations though be not difficult, the another kind of method that design is suitable for the alpha-non-natural amino acid of cellular uptake approach provides biosynthetic pathway, thereby can produce amino acid in vivo.

The biosynthesizing of alpha-non-natural amino acid

Existing many biosynthetic pathways produce amino acid and other compound in the cell.Though for example in the cell, may there be the biosynthetic means of specific alpha-non-natural amino acid in occurring in nature, the invention provides this method.For example, by adding new enzyme or modifying existing host cell approach and can in host cell, choose the biosynthetic pathway that produces alpha-non-natural amino acid wantonly.Other new enzyme is optional to be the enzyme of natural generation or the enzyme of artificial exploitation.For example, biosynthesizing p-Aminophenylalanine (shown in the embodiment among the WO 2002/085923) relies on the mixture that adds other biological known enzyme.Can be by these genes being introduced cell with the plasmid transfection cell of the gene that contains these enzymes.When these genes were expressed in cell, they provided the enzymatic pathway of synthetic required compound.Can choose the example of the enzyme type of adding wantonly and see following examples.Other enzyme sequence is seen for example Genbank.Also can be in the same manner with the optional cell that adds of the enzyme of manually developing.Manipulation cell mechanism and resource are in order to produce alpha-non-natural amino acid in this way.

In fact, thereby can adopt the whole bag of tricks to produce new enzyme in vivo or the external biosynthetic pathway that is used for, improve existing approach, produce alpha-non-natural amino acid.The many existing method of exploitation enzyme and other biosynthetic pathway component is applicable to that the present invention is to produce alpha-non-natural amino acid (perhaps, in fact, be used to develop synthetic enzyme and make it to have new substrate specificity or other interested activity).For example, can choose wantonly and adopt DNA to reorganize the new enzyme and/or the approach of these enzymes developed, thereby can in external or body, produce alpha-non-natural amino acid (or producing new synthetic enzyme).Can be referring to, Stemmer for example, (1994), " Rapid evolution of a protein in vitro by DNA shuffling (by DNA reorganization at external tachytely protein) ", Nature, 370 (4): 389-391; Stemmer, (1994), " DNAshuffling by random fragmentation and reassembly:In vitro recombinationfor molecular evolution (DNA by random fracture and assembling reorganizes: the vitro recombination of molecular evolution) ", Proc.Natl.Acad.Sci.USA., 91:10747-10751.Methods involving is reorganized related (for example homologous) gene family, thereby can open the enzyme of desired characteristic fast.The example of this " family gene reorganization " method is seen Crameri etc., (1998), " DNA shuffling of a family ofgenes from diverse species accelerates directed evolution (orthogenesis has been quickened in the DNA reorganization of different sorts gene family) ", Nature, 391 (6664): 288-291.Also can adopt and be called " brachymemma that increases progressively that produces hybrid enzyme " DNA recombination method (ITCHY) to produce new enzyme (no matter being biosynthetic pathway component or synthetic enzyme), Ostermeier etc. for example, (1999), " Acombinatorial approach to hybrid enzymes independent of DNA homology (not relying on the combined method of the hybrid enzyme of dna homology) ", Nature Biotech is described in the 17:1205.This method also can be used for producing as one or more the enzyme of recombination method substrate or library of other approach variant in the external or body.Also can referring to, Ostermeier etc., (1999), " CombinatorialProtein Engineering by Incremental Truncation (employing increases progressively the combined protein matter engineering of brachymemma) ", Proc.Natl.Acad.Sci.USA, 96:3562-67; Ostermeier etc., (1999), " Incremental Truncation as a Strategy in the Engineering of NovelBiocatalysts (as the brachymemma that increases progressively of the method for engineered true tumor catalyzer) ", Biologicaland Medicinal Chemistry, 7:2139-44.The library that another kind method adopts index set mutagenesis (exponential ensemble mutagenesis) to produce enzyme or other approach variant, it is catalysis and the relevant biosynthesizing reaction of generation alpha-non-natural amino acid (or new synthetic enzyme) for example.In the method, the little group residue in picked at random (randomized) sequence interested abreast is to identify the amino acid that can produce functional protein in variant position.Be applicable to that the present invention sees Delegrave and Youvan, (1993), Biotechnology Research, 11:1548-1552 with the example that produces new enzyme and then produce these class methods of alpha-non-natural amino acid (or new synthetic enzyme).In other method, utilize to mix or degenerate oligonucleotide at random or half random mutagenesis can be used for engineered enzyme and/or pathway components, for example adopt as the described general mutafacient system of following document: Arkin and Youvan, (1992), " Optimizing nucleotide mixtures to encode specific subsets of amino acids forsemi-random mutagenesis " (optimizing the specific amino acids subgroup that mixture of ribonucleotides is encoded and is used for half random mutagenesis), Biotechnology 10:297-300; Or Reidhaar-Olson etc., (1991), " Random mutagenesis of protein sequences using oligonucleotide cassettes " (with oligonucleotide box random mutagenesis protein sequence), Methods Enzymol., 208:564-86.Utilize polynucleotide to reassembly and the another kind of method (often being called " nonrandom " mutagenesis) of site saturation mutagenesis can be used for producing enzyme and/or pathway component, screen the ability that they exercise one or more synthetic enzyme or biosynthetic pathway function (for example producing alpha-non-natural amino acid in vivo) then.Can be referring to, Short for example, " NON-STOCHASTIC GENERATION OF GENETIC VACCINES ANDENZYMES (the nonrandom generation of genetic vaccine and enzyme) ", WO 00/46344.

The alternative method of this mutation method comprises the whole genome of reorganization biology and the offspring's that selection obtains particular approach function (often being called " full genome reorganization ").This method is applicable to the present invention, for example can produce the biology (intestinal bacteria or other cell) of alpha-non-natural amino acid (or its intermediate) by genome reorganization and selection.For example, the method that following publication instructed can be applicable to the approach design, thereby can develop approach existing and/or new in the cell and then produce alpha-non-natural amino acid: Patnaik etc. in vivo, (2002), " Genome shuffling of lactobacillus for improved acid tolerance (the lactobacillus genome is reorganized and improved sour tolerance) ", Nature Biotechnology, 20 (7): 707-712; With Zhang etc., (2002), " Genome shuffling leads to rapid phenotypicimprovement in bacteria (genome reorganization causes the bacterium phenotype to be improved fast) ", Nature, February 7,415:644-646.

Other can be used for technology biological and metabolic pathway engineering transformation (for example for producing required compound) and also is applicable to the generation alpha-non-natural amino acid.Instruct the example of the publication of the useful engineered method in path to comprise: Nakamura and White, (2003), " Metabolic engineering for the microbialproduction of 1; 3propanediol (microorganisms producing 1; metabolic engineering of 3 propylene glycol) ", Curr.Opin.Biotechnol., 14 (5): 454-9; Berry etc., (2002), " Application of MetabolicEngineering to improve both the production and use of Biotech Indigo (using production and purposes that metabolic engineering improves Biotech Indigo) ", J.Industrial Microbiology andBiotechnology, 28:127-133; Banta etc., (2002), " Optimizing an artificialmetabolic pathway:Engineering the cofactor specificity of Corynebacterium2; 5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis (optimize artificial pathways metabolism: the engineered vitamin C bio synthetic coryneform bacteria 2 that is used for; the cofactor specificity of 5-diketo-D-glyconic acid reductase enzyme) ", Biochemistry, 41 (20): 6226-36; Selivonova etc., (2001), " Rapid Evolution of Novel Traits in Microorganisms (tachytely of new features in the microorganism) ", Applied and Environmental Microbiology, 67:3645 and many other publications.

No matter adopt any method, the concentration of the alpha-non-natural amino acid that produces with the engineered biosynthetic pathway of the present invention is enough to biosynthesizing protein effectively, n cell content for example, but should not reach the amino acid whose concentration of other cell of remarkably influenced or exhaust the degree of cell resource.The concentration that is produced in vivo is generally about 10mM to about 0.05mM in this way., can choose wantonly and adopt the generation of selecting in the body with further optimization alpha-non-natural amino acid in case thereby cell by engineered required enzyme and the alpha-non-natural amino acid of particular approach that produced, is grown with cell for ribosomal protein is synthetic.

Mix the quadrature component of alpha-non-natural amino acid

Can be thereby the invention provides preparation quadrature component in vivo to selector's codon, react as amber terminator codon, nonsense codon, four bases or polybase base codon etc. and alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine (referring to Fig. 1, structure 1) is mixed method and composition in the polypeptide chain of extension.For example, the invention provides quadrature-tRNA (O-tRNA), quadrature aminoacyl-tRNA synthetic enzyme (O-RS) and pairing thereof.These pairings can be used for alpha-non-natural amino acid is mixed in the polypeptide chain of extension.

Composition of the present invention comprises quadrature aminoacyl-tRNA synthetic enzyme (O-RS), the described O-RS preferential aminoacylation O-tRNA of umbelliferone-ethyl glycine.In some embodiments, O-RS comprises aminoacid sequence and the conservative variant thereof that contains SEQ ID NO:4.In some embodiments of the present invention; O-RS surpasses any endogenous tRNA with the preferential aminoacylation O-tRNA of specific alpha-non-natural amino acid; wherein said O-RS has preference (bias) to O-tRNA; the O-tRNA that wherein is loaded with alpha-non-natural amino acid and the ratio of the endogenous tRNA that is loaded with identical alpha-non-natural amino acid are greater than 1:1, and more preferably O-RS exclusively or almost exclusively loads O-tRNA.

The composition that contains O-RS also can be chosen wantonly and contain quadrature tRNA (O-tRNA), this O-tRNA identification selection person codon.With (for example comprise this paper sequence table, SEQ ID NO:1) compares with the listed polynucleotide sequence of embodiment or by the inhibition efficient of the O-tRNA of its coding, in the presence of related synthetic enzyme, O-tRNA of the present invention selector's codon is reacted and have usually at least about, for example 45%, 50%, 60%, 75%, 80%, 90% or higher inhibition efficient.In one embodiment, O-RS is not have O-RS to have the inhibition efficient height of O-tRNA down, for example 5 times, 10 times, 15 times, 20 times, 25 times or higher in conjunction with the inhibition efficient of O-tRNA at least.In some respects, O-RS is derived from 45% of the inhibition efficient of the quadrature tyrosyl-tRNA synthetase of Methanococcus jannaschii with the inhibition efficient of O-tRNA at least.

The composition that comprises O-tRNA also can be chosen wantonly and comprise cell (eubacterium cell for example is as Bacillus coli cells etc., or eukaryotic cell, as yeast cell) and/or translation system.

The present invention also provides the cell that comprises translation system (as eubacterium cell or yeast cell), and described translation system comprises quadrature-tRNA (O-tRNA); Quadrature aminoacyl-tRNA synthetic enzyme (O-RS) and umbelliferone-ethyl glycine alpha-non-natural amino acid.O-RS surpasses any endogenous tRNA with the preferential aminoacylation O-tRNA of alpha-non-natural amino acid usually; this O-RS has preference to O-tRNA; the O-tRNA that is loaded with alpha-non-natural amino acid and the ratio of the endogenous tRNA that is loaded with identical alpha-non-natural amino acid are greater than 1:1, and more preferably this O-RS exclusively or almost exclusively loads O-tRNA.O-tRNA discerns first selector's codon, and O-RS can be with the preferential aminoacylation O-tRNA of alpha-non-natural amino acid.In one embodiment, O-tRNA contains polynucleotide sequence shown in the SEQ ID NO:1 or its complementary polynucleotide sequence, perhaps by its coding.In one embodiment, this O-RS contains aminoacid sequence shown in the SEQ ID NO:4 and its conservative variant.

Cell of the present invention also can be chosen wantonly and comprise other the different O-tRNA/O-RS pairing and second alpha-non-natural amino acid; for example; this O-tRNA discerns second selector's codon; this O-RS is with the corresponding O-tRNA of the preferential aminoacylation of second alpha-non-natural amino acid, and wherein said second amino acid is different from first alpha-non-natural amino acid.Cell of the present invention can be chosen the nucleic acid that comprises the polynucleotide that contain the polypeptide of interest of encoding wantonly, and described polynucleotide contain selector's codon that described O-tRNA can discern.

In some embodiments, cell of the present invention is eubacterium cell (as intestinal bacteria), described cell contains quadrature-tRNA (O-tRNA), quadrature aminoacyl-tRNA synthetic enzyme (O-RS), alpha-non-natural amino acid and contains the nucleic acid of the polynucleotide of the polypeptide of interest of encoding, and wherein said polynucleotide contain selector's codon that this O-tRNA can discern.In some embodiments of the present invention, described O-RS is with the described O-tRNA of the preferential aminoacylation of described alpha-non-natural amino acid, and its efficient is higher than the efficient of any endogenous tRNA of this O-RS aminoacylation.

In some embodiments of the present invention, O-tRNA of the present invention contains polynucleotide sequence or its complementary polynucleotide sequence shown in this paper sequence table (for example, SEQ ID NO:1) or the embodiment, or by these sequence encodings.In some embodiments of the present invention, O-RS contains aminoacid sequence shown in the ordered list or its conservative variant.In one embodiment, O-RS or its part are by polynucleotide sequence or its complementary polynucleotide sequence coding of aminoacid sequence shown in coding this paper sequence table and the embodiment.

O-tRNA of the present invention and/or O-RS can be derived from various biologies (as eucaryon and/or non-eukaryotes).

Polynucleotide also are features of the present invention.Polynucleotide of the present invention (for example, SEQ ID NO:5) comprises artificial (producing) polynucleotide as synthetical and non-natural, described polynucleotide sequence contains the nucleotide sequence of polypeptide shown in the coding this paper sequence table, and/or with this polynucleotide sequence complementation.Polynucleotide of the present invention also can be included under the highly rigorous condition nucleic acid that can hybridize with above-mentioned polynucleotide on the nucleic acid of basic total length.Polynucleotide of the present invention comprise that also the sequence with natural tRNA or corresponding encoded nucleic acid has, for example at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or the polynucleotide of higher homogeny (but polynucleotide of the present invention are not natural tRNA or respective coding nucleic acid), described tRNA identification selection person codon is as four base codons.Have with above-mentioned any polynucleotide sequence and/or the polynucleotide that contain the conservative variant of above-mentioned any sequence, for example at least 80%, at least 90%, at least 95%, at least 98% or the artificial polynucleotide of higher homogeny be also included within the polynucleotide of the present invention.

The carrier that contains polynucleotide of the present invention also is a feature of the present invention.For example, carrier of the present invention can be plasmid, clay, phage, virus, expression vector etc.The cell that contains carrier of the present invention also is a feature of the present invention.

The preparation method of O-tRNA/O-RS pairing component also is a feature of the present invention.Component with these method preparations also is a feature of the present invention.For example, generation is at least a comprises preparation mutant tRNA library with the method orthogonal tRNA of cell (O-tRNA); Make the anticodon loop sudden change of each member in the mutant tRNA library make that it can identification selection person's codon, potential O-tRNA is provided library by this, and first cell mass of first species is born selection, described cell contains the member in potential O-tRNA library.Negative selection can be removed the cell that contains potential O-tRNA library member, and this potential O-tRNA can be by the endogenous of cell aminoacyl-tRNA synthetic enzyme (RS) aminoacylation.The storehouse with the orthogonal tRNA of the cell of first species is provided thus, thereby a kind of O-tRNA is provided at least.The O-tRNA that produces with the inventive method also is provided.

In some embodiments, these methods also comprise second cell mass of just selecting first species, and wherein said cell contains member, related aminoacyl-tRNA synthetic enzyme and the positive selectable marker with the orthogonal tRNA of the cell of first species storehouse.Adopt and just selecting optional those cells that contain energy associated aminoacyl-tRNA synthetic enzyme aminoacylation and in the presence of positive selectable marker, can show the tRNA library member of required reaction of selecting or filter out, O-tRNA is provided by this.In some embodiments, second cell mass contains not by the negative cell of selecting removal.

The authentication method that can load quadrature-aminoacyl-tRNA synthetic enzyme of O-tRNA with alpha-non-natural amino acid also is provided.For example, this method comprises that the cell mass to first species screens, wherein said cell contains separately: 1) member of multiple aminoacyl-tRNA synthetic enzyme (RS) (for example, described multiple RS can comprise mutant RS, derived from the RS of the species beyond first species or mutant RS and derived from the RS of the species beyond first species the two); 2) quadrature-tRNA (O-tRNA) (as derived from one or more species); With 3) coding positive selectable marker and comprise the polynucleotide of at least one selector's codon.

Select or screening and the member who does not contain described multiple RS or the less cell of contained number of members are compared and shown in the cell (as host cell) and suppress those cells that efficient improves.The cell of these selection/screenings contains the active RS of energy aminoacylation O-tRNA.Quadrature aminoacyl-tRNA the synthetic enzyme that identifies with these methods also is a feature of the present invention.

Being created in the selected location in cell (eubacterium cell for example is as Bacillus coli cells etc., or yeast cell), to contain the method for protein of alpha-non-natural amino acid also be feature of the present invention.For example, a kind of method is included in culturing cell in the suitable substratum, wherein said cell contains nucleic acid, and this nucleic acid contains at least one selector's codon and a kind of albumen of encoding, thereby alpha-non-natural amino acid is provided and in the translation process of the nucleic acid that contains described at least one selector's codon, this alpha-non-natural amino acid is mixed this proteinic specific position, thereby produce this protein.Cell also contains: the also quadrature-tRNA (O-tRNA) of identification selection person's codon works in cell; And with quadrature aminoacyl-tRNA synthetic enzyme (O-RS) of the preferential aminoacylation O-tRNA of alpha-non-natural amino acid.The protein of Chan Shenging also is feature of the present invention in this way.

The present invention also provides and contains proteinic composition, and wherein said protein comprises umbelliferone-ethyl glycine.In some embodiments, aminoacid sequence that protein comprises and known protein have at least 75% homogeny as treatment albumen, diagnosis albumen and industrial enzymes or its a part of aminoacid sequence.Composition is chosen wantonly and is comprised pharmaceutically acceptable carrier.

Nucleic acid and peptide sequence and variant

As described herein, the invention provides for example polynucleotide sequence of O-tRNA and O-RS of encoding, polypeptid acid sequence, O-RS for example, and the composition, the system and method that for example comprise described polynucleotide or peptide sequence.This paper has disclosed the example of described sequence, as amino acid and the nucleotide sequence (seeing Fig. 9, for example SEQ ID NO:4 and 5) of O-tRNA and O-RS.Yet those skilled in the art should know that the present invention is not limited to this paper, for example those sequences that disclose in embodiment and the sequence table.Those skilled in the art should know that the present invention also provides the many correlated serieses with function described herein, the polynucleotide and the polypeptide of the conservative variant of the disclosed O-RS of this paper that for example encodes.

Embodiment 3-5 has described structure and the analysis that can use the quadrature synthetic enzyme kind (O-RS) of umbelliferone-ethyl glycine alpha-non-natural amino acid aminoacylation O-tRNA.These embodiment describe the structure and the analysis of the O-RS kind that can mix alpha-non-natural amino acid umbelliferone-ethyl glycine.

The invention provides polypeptide (O-RS) and polynucleotide,, be used to separate aminoacyl-tRNA synthetic enzyme clone's oligonucleotide etc. as the polynucleotide of O-tRNA, coding O-RS or its all part.Polynucleotide of the present invention comprise coding proteins of interest of the present invention or polypeptide and contain those polynucleotide of one or more selector's codons.In addition, polynucleotide of the present invention comprise, for example contain the polynucleotide of nucleotide sequence shown in the SEQ ID NO:5; Polynucleotide with its polynucleotide sequence complementation or its polynucleotide sequence of encoding.Polynucleotide of the present invention comprise that also coding contains any polynucleotide of the O-RS aminoacid sequence of SEQ ID NO:4.Similarly, under the rigorous condition of height, can also be polynucleotide of the present invention with the artificial nucleic acid of above-mentioned polynucleotide hybridization (and not being natural polynucleotide sequence) on the nucleic acid of basic total length.In one embodiment, composition comprises polypeptide of the present invention and vehicle (as damping fluid, water, pharmaceutically acceptable vehicle etc.).The present invention also provide can with the antibody or the antiserum(antisera) of polypeptide generation specific immune response of the present invention.Artificial polynucleotide are artificial rather than the polynucleotide of natural generation.

Polynucleotide of the present invention also comprise artificial polynucleotide, promptly have as at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or higher homogeny (but not being natural tRNA) with natural tRNA.Polynucleotide also comprise with natural tRNA having as at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or the artificial polynucleotide of higher homogeny (but be not 100% identical).

In some embodiments, carrier (as plasmid, clay, phage, virus etc.) contains polynucleotide of the present invention.In one embodiment, described carrier is an expression vector.In another embodiment, described expression vector comprises the promotor that links to each other with one or more polynucleotide operability of the present invention.In another embodiment, cell comprises the carrier that contains polynucleotide of the present invention.

Those skilled in the art also know the multiple variant that the present invention includes disclosed sequence.For example, the conservative variant of the disclosed sequence of generation function identical sequence also belongs to the present invention.The variant of nucleic acid polynucleotide sequence also belongs to the present invention, described variant and at least a disclosed sequence hybridization.This paper discloses unique subsequence of sequence, for example can also belong to the present invention by unique subsequence that the standard sequence comparison techniques is measured.

The conservative property variation

Because the degeneracy of genetic code, " the reticent replacement " (that is, the replacement in the nucleotide sequence does not cause coded polypeptide to change) is the feature that every nucleotide sequence implied of encoding amino acid sequence.Similarly, " conservative amino acid replacements " that also being not difficult to identify has one or a few amino acids to be had the height similar characteristics in the aminoacid sequence different aminoacids replaces is because it is similar to the construction height of disclosure.This conservative property variation of each sequence that discloses is one of feature of the present invention.

" the conservative property variation " of concrete nucleotide sequence refer to encode those nucleic acid of identical or essentially identical aminoacid sequence, perhaps, if nucleic acid encoding amino acid sequence not then refers to substantially the same sequence.Various replacements, disappearance or insertion that the technician knows change, adds or delete in the coded sequence amino acid or small part amino acid (generally be lower than 5%, more common be lower than 4%, 2% or 1%) are " conservative property are modified variation ", and these changes cause amino acid whose disappearance, insertion or replaced by the similar amino acid of chemical property.Therefore, " the conservative property variation " of the cited peptide sequence of the present invention comprises the amino acid of the small part (be usually less than 5%, more common is to be lower than 2% or 1%) with this peptide sequence of aminoacid replacement of same conservative property replacement group.At last, add that not change the coded active sequence of certain nucleic acid molecule (for example adding no function sequence) be that the conservative property of basic nucleic acid makes a variation.

Well knownly provide functionally similar amino acid whose conservative property replacement table, one of them amino-acid residue is replaced by the amino-acid residue that another has similar chemical property (for example aromatic side chains or positively charged side chain), does not therefore change the functional performance of this peptide molecule basically.Below exemplify the similar natural amino acid of many group chemical propertys, wherein, the replacement in each group i.e. " conservative property replacement ".

Conservative amino acid replaces

Nonpolar and/or aliphatic lateral chain	Polarity, uncharged side chain	Aromatic side chains	Positively charged side chain	Electronegative side chain
					Glycine L-Ala Xie Ansuan leucine Isoleucine proline(Pro)	Serine threonine halfcystine methionine(Met) arginine glutamine	Phenylalanine tyrosine tryptophane	Methionin arginine Histidine	Aspartic acid L-glutamic acid

Nucleic acid hybridization

Can adopt relatively hybridization to identify nucleic acid of the present invention, comprise the examples of conservative variations of nucleic acid of the present invention, this comparison hybridizing method is the preferred method of difference nucleic acid of the present invention.In addition, under height, hypervelocity and super hypervelocity preciseness condition, can be feature of the present invention with the target nucleic acid of nucleic acid hybridization shown in the SEQ ID NO:5.The example of this nucleic acid comprises with certain given nucleotide sequence to be compared, and contains the nucleic acid that one or a little silence or conservative property nucleic acid replace.

When the hybridization degree of test nucleic acid and probe is and at least 50% of the complementary target hybridization degree of Perfect Matchings, be half of the signal to noise ratio signal to noise ratio that to be this probe and target at least hybridize under the complementary target bonded condition of the probe of Perfect Matchings and Perfect Matchings when high, can claim this test nucleic acid and probe nucleic acid specific hybrid; Under this condition, the complementary target nucleic acid bonded signal to noise ratio of the probe of Perfect Matchings and Perfect Matchings be at least hybridize viewed signal to noise ratio with any target nucleic acid that do not match about 5-10 doubly.

When nucleic acid combination (generally in solution), claim its " hybridization ".Nucleic acid is because of the various physics and chemistry power that fully characterize, for example hydrogen bond, solvent repulsion, base stacking etc. and hybridize.The extensive guide of nucleic acid hybridization is seen Tijssen, (1993), Laboratory Techniques in Biochemistry and MolecularBiology--Hybridization with Nucleic Acid Probes (laboratory technique of Biochemistry and Molecular Biology---nucleic acid probe hybridization), first part's chapter 2, " Overview of principlesof hybridization and the strategy of nucleic acid probe assays (hybridization principle summary and nucleic acid probe test method) ", (Ai Ersaifu company (Elsevier), New York); And Current Protocolsin Molecular Biology (up-to-date molecular biology method), volumes such as Ausubel, " CurrentProtocols " (fresh approach), Green publishes partnership, and (Greene Publishing Associates is Inc.) with John Wei Li father and son (John Wiley of company; Sons) joint venture, (augmenting in 2006); Hames and Higgins, (1995), Gene Probes 1 (gene probe 1) and Gene Probes 2 (gene probe 2), IRL press, Oxford University Press, Oxford, England, they provide synthetic, mark, detection and quantitative assay DNA and RNA, comprise the details of oligonucleotide.

The example of preciseness hybridization conditions that has the complementary nucleic acid hybridization of complementary residue more than 100 on Southern or northern trace filter membrane is: contain 50% formalin of 1mg heparin, 42 ℃ of hybridization are spent the night.The example of preciseness wash conditions is 65 ℃, and (description of SSC damping fluid can be referring to Sambrook etc. with 0.2 * SSC washing 15 minutes; Sambrook etc., Molecular Cloning-ALaboratory Manual (molecular cloning-laboratory manual), (third edition), 1-3 volume, cold spring harbor laboratory, cold spring port, New York, 2001).Usually hang down the preciseness washing earlier and remove the background probe signals, carry out high preciseness washing again.Exemplary low preciseness washing was 40 ℃, with 2 * SSC washing 15 minutes.Signal to noise ratio is that 5 times of (or higher) ordinary representations of the irrelevant observed signal to noise ratio of probe detect specific hybrid in the concrete cross experiment.

(for example, Southern and northern hybridization) " preciseness hybridization wash conditions " depends on sequence, and different with different environmental parameters in the nucleic acid hybridization experiment.The extensive guide of nucleic acid hybridization is seen Tijssen, (1993), the laboratory technique of Biochemistry and Molecular Biology---nucleic acid probe hybridization, first part's chapter 2, " hybridization principle summary and nucleic acid probe test method ", (Ai Ersaifu company, New York); Hames and Higgins, (1995), gene probe 1, IRL press, Oxford University Press, Oxford, England and Hames and Higgins, (1995), gene probe 2, IRL press, Oxford University Press, Oxford, England.Be not difficult to determine by rule of thumb to be fit to the preciseness hybridization and the wash conditions of any test nucleic acid.For example, when definite preciseness hybridization and wash conditions, can promote gradually hybridization and wash conditions (for example, the temperature by raise hybridization or washing, reduce salt concn in hybridization or the washing, increase the detergent concentration in hybridization or the washing and/or increase hybridization or wash in organic solvent such as the concentration of formalin) until meeting selected standard.For example, in height preciseness hybridization and wash conditions, promote hybridization and wash conditions gradually and be at least this probe and the viewed signal to noise ratio of target hybridization that do not match 5 times until the complementary target bonded signal to noise ratio of probe and Perfect Matchings.

The condition of " very rigorous " selected equals the pyrolysis chain temperature (T of concrete probe _m).T _mTemperature (under the ionic strength and pH of regulation) when being the probe hybridization of 50% cycle tests and Perfect Matchings.Be purpose of the present invention, the hybridization of " highly rigorous " and wash conditions are typically chosen under the ionic strength of regulation and pH than the T of concrete sequence _mLow 5 ℃ approximately.

The hybridization and the wash conditions of " superelevation is rigorous " refer to, the preciseness that increases hybridization and wash conditions is any target nucleic acid that do not match 10 times of observed signal to noise ratio when hybridizing until the complementary target nucleic acid bonded signal to noise ratio of probe and Perfect Matchings at least.Target nucleic acid is 1/2 o'clock of complementary target nucleic acid of Perfect Matchings with the signal to noise ratio of probe hybridization under this condition at least, can claim it to combine with probe under superelevation preciseness condition.

Similarly, hybridization that can be by promoting relevant cross experiment gradually and/or wash conditions is determined even higher preciseness level.For example, the preciseness that promotes the condition of hybridization and washing is that any target nucleic acid that do not match is hybridized 10,20,50,100 or 500 times of signal to noise ratio or higher until the complementary target nucleic acid bonded signal to noise ratio of probe and Perfect Matchings at least.Target nucleic acid is 1/2 o'clock of complementary target nucleic acid of Perfect Matchings with the signal to noise ratio of probe hybridization under this condition at least, can claim it to combine with probe under super hypervelocity preciseness condition.

If the nucleic acid encoded polypeptide of not hybridizing each other under the preciseness condition is basic identical, then these nucleic acid are still basic identical.For example, can this thing happens when utilizing maximum code degeneracy that genetic code allows to produce the copy of nucleic acid.

Unique subsequence

In some respects, the invention provides the nucleic acid that contains unique subsequence in the nucleic acid that is selected from O-tRNA that this paper discloses and O-RS sequence.With compare corresponding to the nucleic acid of any known O-tRNA or O-RS nucleotide sequence, described unique subsequence is unique.Can adopt, the BLAST that default parameters for example is set carries out the nucleic acid comparison.Any unique subsequence can be used as, and for example probe is identified nucleic acid of the present invention or associated nucleic acid.

Similarly, the present invention includes the polypeptide that contains unique subsequence in the O-RS polypeptide of sequence that is selected from this paper disclosure.With compare corresponding to any known peptide polypeptide of sequence, unique subsequence of this paper is unique.

The present invention also provides the target nucleic acid that can hybridize with the oligonucleotides coding of uniqueness under the preciseness condition, described oligonucleotide coding is selected from the unique subsequence in the O-RS polypeptide of sequence, wherein with corresponding to any contrast polypeptide (for example, parental generation sequence by sudden change acquisition synthetic enzyme of the present invention) polypeptide is compared, and described unique subsequence is unique.Can measure unique sequences as mentioned above.

Sequence comparison, homogeny and homology

For two or more nucleic acid or peptide sequence, term " identical " or " homogeny per-cent " refer to when coming comparison and the maximum correspondence of comparison with sequence comparison algorithm (or other algorithm of technician's available) hereinafter described or by visual observations, these two or more sequences or subsequence are identical, perhaps have the amino-acid residue of particular percentile or Nucleotide identical.

For two nucleic acid or polypeptide (for example, the DNA of coding O-tRNA or O-RS, or the aminoacid sequence of O-RS), term " basic identical " refers to when utilizing sequence comparison algorithm or come comparison and comparison during maximum correspondence by visual observations, and two or more sequences or subsequence have an appointment 60% at least, about 80%, about 90-95%, about 98%, about 99% or more Nucleotide or amino-acid residue are identical.The sequence of this " basic identical " is commonly referred to be " homologous ", no matter and actual ancestors.Preferably at the sequence area that is about 50 residues at least, more preferably exist in the zone at least about 100 residues " basic identical property ", two sequences to be compared is preferably at least about 150 residues or basic identical on total length.

When protein and/or protein sequence (natural or artificially) during derived from same ancestors' albumen or protein sequence, they are " homologous ".Similarly, when nucleic acid and/or nucleotide sequence (natural or artificially) during derived from same ancestors' nucleic acid or nucleotide sequence, they are " homologous ".For example, can modify any natural acid by any available mutafacient system and make it to contain one or more selector's codons.When the expression of nucleic acid of this mutagenesis, its encoded polypeptides contains one or more alpha-non-natural amino acids.Certainly, this mutation method also can change one or more standard cipher, thereby has also changed the one or more standard amino acids in the gained mutant protein.Usually can infer homology from the sequence similarity between two or more nucleic acid or protein (or its sequence).Can be used for confirming that accurate similarity per-cent is different because of nucleic acid and the protein studied between the sequence of homology, confirm homology but conventional utilization is low to moderate 25% sequence similarity.Also available higher levels of sequence similarity, for example 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or highlyer confirm homology.This paper has described the method (for example, adopting the BLASTP and the BLASTN of parameter acquiescence) of measuring sequence similarity per-cent, and these methods are well-known.

Compare and homology mensuration for sequence, usually a sequence is compared with cycle tests as reference sequence.When adopting sequence comparison algorithm, with cycle tests and reference sequence input computer, can specify the subsequence coordinate if desired, and specified sequence algorithm routine parameter.According to specified program parameter, sequence comparison algorithm can calculate the sequence homogeny per-cent of cycle tests than reference sequence then.

Available following method compares the best comparison of sequence, local homology's algorithm of Smith and Waterman for example, Adv.Appl.Math., 2:482, (1981); The homology alignment algorithm of Needleman and Wunsch, J., Mol.Biol., 48:443, (1970); The similarity retrieval method of Pearson and Lipman, Proc.Nat ' l.Acad.Sci.USA, 85:2444, (1988); Computer is carried out these algorithms (GAP in the Wisconsin heredity software package, BESTFIT, FASTA and TFASTA, genetics computer set (Genetics Computer Group), 575 Science Dr., Madison, the state of Wisconsin); Perhaps visual observations (generally can be referring to Current Protocols in MolecularBiology (up-to-date molecular biology method), volumes such as Ausubel, " Current Protocols (fresh approach) ", Green publishes the joint venture of partnership and John Wei Li father and son company, augments in 2006).

An example that is applicable to the algorithm of measuring sequence homogeny and sequence similarity per-cent is Altschul etc., J.Mol.Biol., 215:403-410, (1990) described BLAST algorithm.The software that carries out the BLAST analysis is by NCBI (referring to the NCBI network address) open to the public.This algorithm comprises: at first long identify higher assessment sub-sequence pairing (HSP) for the short word string of W by identifying in search sequence, meet when the word string of equal length is compared in these word strings and the database sequence or satisfy some on the occasion of the threshold value T that marks.T be called contiguous word string scoring threshold value (Altschul etc., (1990), J.Mol.Biol., 215:403-410).These original contiguous word strings choose (hit) to look for the longer HSP that contains them as the seed that starts retrieval.These word strings are chosen along the two-way extension of each bar sequence, so that accumulation comparison scoring increase.For nucleotide sequence, with parameter M (the award scoring of a pair of coupling residue; Permanent in 0) and the N (point penalty of mispairing residue; Permanent in 0) calculate and accumulate scoring.For aminoacid sequence, then calculate the accumulation scoring with rating matrix.The word string that stops all directions when following situation occurring is chosen extension: accumulation comparison scoring is from its maximum decline X that reaches; The accumulation of comparing because of one or more negative scoring residues causes the accumulation scoring to be reduced to below 0 or 0; Perhaps arrive the end of each sequence.The sensitivity and the speed of BLAST algorithm parameter W, T and X decision comparison.Blast program (being used for nucleotide sequence) acquiescence 11 is that expected value (E), 100 is cutoff value, M=5, N=-4 for word length (W), 10, and carries out two strands relatively.For aminoacid sequence, blast program acquiescence 3 for word length (W), 10 for expected value (E) and adopt the BLOSUM62 rating matrix (referring to Henikoff and Henikoff, (1989), Proc.Natl.Acad.Sci.USA, 89:10915).

Except sequence of calculation homogeny per-cent, the BLAST algorithm also can to the similarity between two sequences carry out statistical analysis (referring to, for example Karlin and Altschul, Proc.Nat ' l.Acad.Sci.USA, 90:5873-5787, (1993)).One of similarity measurement that the BLAST algorithm provides is minimum total probability (P (N)), and the probability of coupling at random takes place between two Nucleotide of its indication or the aminoacid sequence.For example,, be more preferably less than approximately 0.01, most preferably, can think that then this nucleic acid is similar to reference nucleic acid less than about 0.001 if minimum total probability is less than about 0.1 in the comparison of test nucleic acid and reference nucleic acid.

Mutagenesis and other Protocols in Molecular Biology

Can adopt Protocols in Molecular Biology operation of the present invention and be used for polynucleotide of the present invention and polypeptide.Describe molecular biological general teaching material and comprise Berger and Kimmel, Guide to MolecularCloning Techniques (molecule clone technology guide), Methods in Enzymology (Enzymology method), the 152nd volume, (the Academic Press of academic press company, Inc.), San Diego, California; Sambrook etc., Molecular Cloning-A Laboratory Manual (molecular cloning-laboratory manual), (third edition), the 1-3 volume, cold spring harbor laboratory, cold spring port, New York, 2001 and CurrentProtocols in Molecular Biology (up-to-date molecular biology method), volumes such as Ausubel; " Current Protocols in Molecular Biology (molecular biology fresh approach) ", Green publishes the joint venture of partnership and John Wei Li father and son company, (augmenting in 2006).These teaching materials have been described application, the promotor of mutagenesis, carrier and have been related to, and produce the protein that contains alpha-non-natural amino acid, quadrature tRNA, quadrature synthetic enzyme and paired thereof many other relevant problems thereby for example produce the gene that contains selector's codon.

The present invention can adopt various types of mutagenesis, for example so that selector's codon of the alpha-non-natural amino acid in the sudden change of tRNA molecule, generation tRNA library, generation synthetic enzyme library, insertion coding proteins of interest matter or the polypeptide.They include but not limited to: site-directed mutagenesis, random point mutagenesis, homologous recombination, DNA reorganization or other recurrence mutafacient system, chimeric construct, utilization contain the template mutagenesis of uridylic, mutagenesis that oligonucleotide instructs, DNA mutagenesis that thiophosphatephosphorothioate is modified, utilize the mutagenesis of breach duplex DNA etc., or their any combination.Other suitable method comprises a mispairing reparation, utilizes the mutagenesis of rectification of defects host strain, restricted selection and restricted purifying, deletion mutagenesis, the synthetic mutagenesis of total gene, double-strand break reparation etc.The present invention also comprises, for example relates to the mutagenesis of chimeric construct thing.In one embodiment, available natural molecule or through changing or the Given information of the natural molecule of sudden change, sequence for example, sequence comparison, physical property, crystalline structure wait and instruct mutagenesis.

Can utilize polynucleotide of the present invention or contain the construction of polynucleotide of the present invention, carrier for example of the present invention (for example can be cloning vector or expression vector) comes genetic modification (for example transform, transduction or transfection) host cell.For example, can and treat that the coding region operability of derived protein is connected in the genetic expression controlling elements that can work with quadrature tRNA, quadrature tRNA synthetic enzyme in required host cell.Typical carrier contains transcribes with translation termination, transcribes with translation initiation sequence and the promotor that is used to regulate concrete target nucleic acid expression.These carriers can randomly comprise the genetic expression box, described expression cassette contains at least one independently terminator sequence, allow this expression cassette in eukaryotic cell or prokaryotic cell prokaryocyte or the sequence (for example, shuttle vectors) of duplicating in the two and the selective marker that is applicable to protokaryon and eukaryotic system.Carrier is applicable at prokaryotic cell prokaryocyte, eukaryotic cell or preferably duplicates and/or integrate in the two.Referring to Giliman and Smith, Gene, 8:81, (1979); Roberts etc., Nature, 328:731, (1987); Schneider etc., Protein Expr.Purif., 6435:10, (1995); Berger and Kimmel, molecule clone technology guide, Enzymology method, the 152nd volume, academic press company, San Diego, California; Sambrook etc., molecular cloning-laboratory manual, (third edition), 1-3 volume, cold spring harbor laboratory, cold spring port, New York, 2001; With up-to-date molecular biology method, volumes such as Ausubel; Fresh approach, Green publishes the joint venture of partnership and John Wei Li father and son company, (augmenting in 2006).For example, carrier can be plasmid, bacterium, virus, naked polynucleotide or the polynucleotide form of puting together.Can be by standard method with carrier transfered cell and/or microorganism, described method comprises electroporation (From etc., Proc.Natl.Acad.Sci.USA, 82,5824, (1985)), viral vector infection utilizes the short grained high speed bullet that contains nucleic acid in globule or particulate matrix or on its surface to penetrate (Klein etc., Nature, 327:70-73, (1987)) etc.

Developed and in intestinal bacteria, to have reacted and the alpha-non-natural amino acid locus specificity is mixed proteinic efficient and general simple substance grain system amber terminator codon (UAG).In this new system, the pairing of Methanococcus jannaschii inhibition type tRNAtyr (CUA) and tyrosyl-tRNA synthetase is by compatible with most of coli expression carriers single plasmid-encoded.Be structured in the monocistron tRNA operon under the control of proK promotor and terminator, be used to optimize secondary structure and tRNA processing.The synthetic enzyme glnS promotor of introducing mutant form has significantly improved inhibition efficient and fidelity of reproduction.Utilize the tRNA gene of multiple copied and the specific sudden change (D286R) on this synthetic enzyme also can improve and suppress efficient (Kobayashi etc., (2003), " Structural basis for orthogonal tRNA specificities of tyrosyl-tRNAsynthetases for genetic code expansion (the specific architecture basics of quadrature tRNA that is used for the tyrosyl-tRNA synthetase of genetic code expansion) ", Nat.Struct.Biol., 10 (6): 425-432).The versatility of mixing efficiently and accurately also prove this optimization system of several different alpha-non-natural amino acids, the unique applications of described alpha-non-natural amino acid in research protein function and structure is confirmed.

ATCC provides bacterium and the bacteriophage catalogue that can be used for cloning, the The ATCC Catalogue of Bacteria and Bacteriophage (ATCC bacterium and bacteriophage catalogue) that publishes of ATCC for example, (1996), volumes such as Gherna.Order-checking, clone's other basic skills and the thinking of molecular biological others and basic theory also can be referring to Sambrook etc., molecular cloning-laboratory manual, (third edition), 1-3 volume, cold spring harbor laboratory, cold spring port, New York, 2001; Up-to-date molecular biology method, volumes such as Ausubel; Fresh approach, Green publishes the joint venture of partnership and John Wei Li father and son company, (augmenting in 2006); With Watson etc., (1992), Recombinant DNA (recombinant DNA), second edition, Scientific Beauty state books company (Scientific American Books), New York.In addition, can order any basically nucleic acid (with the nucleic acid of in fact any mark to various commercial source customizations or standard, standard or off-gauge no matter), for example the Midland of Texas's Midland is examined and determine reagent company (Midland Certified Reagent Company), (the The Great American Gene Company of the big U.S. genome company in Lei Mengna city, California, Ramona, CA), the Expression Genetics Inc. in Chicago, Illinois city (ExpressGen Inc.), the operon technology company in my Mi Da city, California (Operon Technologies Inc.) and many other companies.

Can be utilized as and for example screen step, activate promotor or select the activity of transformant and do the host cell of suitable improved conventional nutritional medium culturing engineering transformation.The optional genetically modified organism of cultivating into of these cells.Other available reference, for example cellular segregation and cultivation are (for example, be used for separate nucleic acid subsequently) document comprise: Freshney, (1994), Culture of Animal Cells, a Manual ofBasic Technique (animal cell culture, the basic fundamental handbook), the third edition, Wei Lilisi company (Wiley-Liss), New York and the reference of being quoted thereof; Payne etc., (1992), Plant Cell andTissue Culture in Liquid Systems (vegetable cell in the liquid system and tissue culture), John Wei Li father and son company, New York, New York; Gamborg and Phillips compile, (1995), Plant Cell, Tissue and Organ Culture (vegetable cell, tissue and organ culture); FundamentalMethods (basic skills), Springer Lab Manual (Springer laboratory manual), S-V company (Springer-Verlag) (Bai Linhai Dare fort New York); Atlas and Parks compile, The Handbook ofMicrobiological Media (microbiological culture media handbook), (1993), CRC press (CRCPress), Berkeley village, Florida State.

Protein of interest matter and polypeptide

Being created in the method for protein that specific position contains alpha-non-natural amino acid in cell also is one of feature of the present invention.For example, a kind of method comprises that wherein said cell comprises the nucleic acid that contains at least one selector's codon and coded protein with suitable culture medium culturing cell; With alpha-non-natural amino acid is provided; Wherein said cell also comprises: the quadrature-tRNA (O-tRNA) that has function and identification selection person's codon in cell; With quadrature aminoacyl-tRNA synthetic enzyme (O-RS) with the preferential aminoacylation O-tRNA of alpha-non-natural amino acid.The protein that this method produced also is one of feature of the present invention.

In some embodiments, compare with any endogenous tRNA in the expression system, O-RS is partial to the O-tRNA of aminoacylation association.When O-tRNA and O-RS with etc. volumetric molar concentration when existing, the relative proportion of O-tRNA that O-RS loaded and endogenous tRNA is greater than 1:1, preferably at least about 2:1, more preferably 5:1, more preferably 10:1, more preferably 20:1, more preferably 50:1, more preferably 75:1, more preferably 95:1,98:1,99:1,100:1,500:1,1,000:1,5,000:1 or higher.

The present invention also provides and comprises proteinic composition, and described protein contains alpha-non-natural amino acid.In some embodiments, the aminoacid sequence that contains of described protein and the sequence of human cytokines, diagnostic albumen, industrial enzymes or its part have at least 75% identical.

The composition of composition of the present invention and the inventive method preparation is optional to be in the cell.O-tRNA/O-RS pairing of the present invention or each component can be used for the translating mechanism of host system then, thereby alpha-non-natural amino acid is mixed protein.On April 16th, 2004 submitted to, the international publication number WO 2004/094593 of " EXPANDINGTHE EUKARYOTIC GENETIC CODE (expansion eucaryon genetic code) " by name and the international publication number WO 2002/085923 of " IN VIVO INCORPORATION OF UNNATURALAMINO ACIDS (mixing in the body of alpha-non-natural amino acid) " by name have described this method, and these two pieces of documents are included this paper by reference in.For example, when the host is introduced in O-tRNA/O-RS pairing, for example during Bacillus coli cells, this pairing reacts to selector's codon and with alpha-non-natural amino acid, for example umbelliferone-ethyl glycine mixes protein in vivo.The alpha-non-natural amino acid of adding system can be a synthesizing amino acid, phenylalanine or tyrosine derivative that for example can exogenous adding growth medium.Composition of the present invention is optional to be external translating system, or system in the body.

Cell of the present invention can synthesize the protein that comprises alpha-non-natural amino acid in a large number.In some respects, composition is optional to be comprised, 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 protein that contains alpha-non-natural amino acid, perhaps the body internal protein production method content (this paper provides the details of recombinant protein preparation and purifying) that can reach more.On the other hand, (for example for example be included in the composition in cell pyrolysis liquid, damping fluid, pharmacy damping fluid or other liquid suspension, volume is the about 100L of about 1nL-) protein concn optional be, for example whenever rise to few 10 micrograms of protein, whenever rise to few 50 micrograms of protein, whenever rise to few 75 micrograms of protein, whenever rise to few 100 micrograms of protein, whenever rise to few 200 micrograms of protein, whenever rise to few 250 micrograms of protein, whenever rise to few 500 micrograms of protein, whenever rise to few 1 milligram of protein or whenever rise to few 10 milligrams of protein or higher.The protein that preparation a large amount of (for example, usually can getable amounts greater than other method of employing such as external translation) comprises at least one alpha-non-natural amino acid in cell is one of feature of the present invention.

Can mix alpha-non-natural amino acid, for example change protein structure and/or function, as change the accessibility of size, acidity, nucleophilicity, hydrogen bond, hydrophobicity, proteolytic enzyme target position, to the target (as being used for protein arrays) of certain part, mix marker or reactive group etc.The proteinic catalysis or the physical property that contain alpha-non-natural amino acid can improve, even obtain brand-new catalysis or physical property.For example, can choose wantonly and improve following characteristic by alpha-non-natural amino acid being mixed protein: toxicity, bio distribution, structural performance, spectral response curve, chemistry and/or photochemical properties, catalytic capability, transformation period (for example serum half-life), with the response capacity (as covalently or non-covalently) of other molecule etc.Comprise the proteinic composition that contains at least one alpha-non-natural amino acid and can be used as, for example new therapeutic agent, diagnostic reagent, katalaze enzyme, industrial enzymes, conjugated protein (as antibody) and be used for for example studying protein structure and function.Referring to for example Dougherty, (2000), " Unnatural Amino Acids as Probes of ProteinStructure and Function (as the alpha-non-natural amino acid of protein structure and function probe "), Current Opinion in Chemical Biology, 4:645-652.

Aspect more of the present invention, composition comprises and at least aly contains at least one, the protein 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 acids.Described alpha-non-natural amino acid can be identical or different, for example can contain 1,2,3,4,5,6,7,8,9 or 10 or how different alpha-non-natural amino acids on 1,2,3,4,5,6,7,8,9 or 10 or how different site in the protein.On the other hand, composition comprises protein, and the concrete amino acid of at least one that exists in this protein (but not being whole) is alpha-non-natural amino acid.For the given protein that contains a plurality of alpha-non-natural amino acids, described alpha-non-natural amino acid can identical or different (can comprise two or more dissimilar alpha-non-natural amino acids as protein, perhaps can comprise two identical alpha-non-natural amino acids).For the given protein that contains two above alpha-non-natural amino acids, described alpha-non-natural amino acid can be identical, difference, or the combination of the alpha-non-natural amino acid of a plurality of same types alpha-non-natural amino acid different with at least one.

Utilize this paper composition and method can prepare any basically protein (or its part) (, for example containing the nucleic acid of one or more selector's codons) that contains alpha-non-natural amino acid with any respective coding nucleic acid.Do not attempt to identify hundreds of known protein matter, can modify in them any and make it to contain one or more alpha-non-natural amino acids, thereby for example make in the relevant translation system and comprise one or more suitable selector's codons by improving any available mutation method.The consensus sequence storehouse of known protein matter comprises GenBankEMBL, DDBJ and NCBI.Be not difficult to identify other storehouse by the retrieval internet.

These protein usually with any available protein (for example, human cytokines, diagnostic albumen, industrial enzymes or its part etc.) have, 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 higher homogeny, these protein contain one or more alpha-non-natural amino acids simultaneously.Therapeutic, diagnostic and other proteinic example that can modifiedly comprise one or more alpha-non-natural amino acids referring to but be not limited to international publication number WO 2004/094593 and " the IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS. (mix in the body of alpha-non-natural amino acid " by name of " the Expanding the Eukaryotic Genetic Code (expansion the eukaryotic genetic code) " by name that submitted on April 16th, 2004) WO2002/085923.The therapeutic that can modifiedly comprise one or more alpha-non-natural amino acids, diagnostic and other proteinic example include but not limited to: α-1 antitrypsin for example, angiostatin, AHF (Antihemolytic factor), antibody (other details of antibody sees below), lipophorin, take off auxilliary albumen, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide (Atrial peptides), the C-X-C chemokine is (as T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), thyrocalcitonin, the CC chemokine is (as MCP-1, MCP-2, monocyte chemoattractant protein-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, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine (as, epithelium neutrophilia activation peptide-78 (epithelial Neutrophil Activating Peptide-78), GRO α/MGSA, GRO β, GRO γ, MIP-1 α, MIP-1 δ, MCP-1), Urogastron (EGF), erythropoietin (" EPO "), spalling toxin A and B, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, Hedgehog albumen is (as Sonic, Indian, Desert), oxyphorase, pHGF (HGF), r-hirudin, human serum albumin, Regular Insulin, rhIGF-1 (IGF), Interferon, rabbit (as, IFN-α, IFN-β, IFN-γ), interleukin (as, 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, neurotrophic factor (Neurturin), neutrophil supressor (NIF), tumour-inhibiting protein M, bone morphogenic protein, Triiodothyronine, PD-ECSF, PDGF, peptide hormone (as, human growth hormone), the multi-effect nutrient factor, A albumen, G albumen, the thermal source exotoxin A, B and C, Relaxin, 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, the somatotropin inhibitor, somatotropin, streptokinase, superantigen, i.e. staphyloentero-toxin (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 a lot of other albumen.

Can adopt the class protein of composition that can mix alpha-non-natural amino acid in vivo described herein and method preparation to comprise transcriptional regulatory agent or its part.Exemplary transcriptional regulatory agent comprises gene and the transcription regulatory protein of regulating cell growth, differentiation, regulation and control etc.The transcriptional regulatory agent is present in prokaryotic organism, virus and eukaryote (comprising fungi, plant, yeast), insect and the animal (comprising Mammals), thereby the wide range of therapeutic target spot is provided.Should be appreciated that and express and transcriptional activator is transcribed by many mechanism regulation, for example by express with receptors bind, the reaction of stimulus signal transductory cascade, regulative transcription factor, combine, unwind with the protein bound that combines promotor and enhanser, DNA with promotor and enhanser, preceding-the mRNA montage, RNA polyadenylation and RNA degrade.

Class protein of the present invention (for example, the protein that contains one or more alpha-non-natural amino acids) comprise biologically active proteins, cytokine for example, inflammatory molecule, somatomedin, their acceptor and oncoprotein are as interleukin (as IL-1, IL-2, IL-8 etc.), Interferon, rabbit, 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 yalin (hyalurin)/CD44; Signal transducers and corresponding oncoprotein, for example Mos, Ras, Raf and Met; Transcriptional activator and repressor, for example p53, Tat, Fos, Myc, Jun, Myb, Rel and steroid hormone receptor, those acceptors of oestrogenic hormon, progesterone, testosterone, aldosterone for example, ldl receptor part and Kendall compound.

The present invention also provides enzyme (for example industrial enzymes) or its part that contains at least one alpha-non-natural amino acid.The example of enzyme includes but not limited to: Ntn hydrolase; amino acid racemase; acyltransferase; the dehalogenation enzyme; dioxygenase; diaryl propane peroxidase (diarylpropane peroxidases); epimerase; epoxide hydrolase; esterase; isomerase; kinases; glucose isomerase; Glycosylase; glycosyltransferase; haloperoxidase (haloperoxidases); monooxygenase (for example p450); lipase; lignin peroxidase; Nitrile hydratase; nitrilase; proteolytic enzyme; Phosphoric acid esterase; subtilisin; transaminase and ribozyme.

But (referring to, Sigma Biological Science Co., Ltd (SigmaBioSciences) for example) buied in many these protein commercializations, (referring to, Genbank for example) that corresponding proteins matter sequence and gene and many variants thereof are normally known.Can modify any of these albumen by inserting one or more alpha-non-natural amino acids according to the present invention, thereby (for example) changes one or more the interested treatments of these protein, diagnosis or enzymic activity.The treatment correlation properties comprise serum half-life, preserve transformation period, stability, immunogenicity, therapeutic activity, detectability (for example, by add reporter group (as marker or mark binding site) in alpha-non-natural amino acid), LD ₅₀Or the reduction of other negative effects, enter intravital ability (as oral availability) etc. by gi tract.The example of diagnostic feature comprises preserves transformation period, stability, diagnosis activity, detectability etc.The example of involved enzyme characteristic comprises preserves transformation period, stability, enzymic activity, throughput etc.

Also can adopt the inventive method and composition to modify various other protein and make it to comprise one or more alpha-non-natural amino acids.For example, the present invention can comprise with alpha-non-natural amino acid and replace one or more natural amino acids in one or more vaccine protein matter, for example the protein in following source: infectious fungi, plant as aspergillus (Aspergillus), candiyeast (Candida); Bacterium is in particular as the intestinal bacteria of pathogenic bacteria model, and medically important bacterium, as Staphylococcus (Staphylococci) (as, streptococcus aureus) or streptococcus (Streptococci) (as, streptococcus pneumoniae); Protozoon, as Sporozoa (as, plasmodium (Plasmodia)), rhizopod (rhizopods) (as, inner amoeba (Entamoeba)) and Flagellata (trypanosome (Trypanosoma), leishmania (Leishmania), trichomonas (Trichomonas), giardia lamblia (Giardia) etc.); Virus, (example comprises poxvirus, as vaccinia virus (vaccinia) as (+) RNA viruses; Picornavirus is as poliovirus (polio); Togavirus is as rubella virus (rubella); Flavivirus (Flaviviruses) is as HCV; And coronavirus), (-) RNA viruses is (as rhabdovirus (Rhabdovirus), as VSV; Paramyxovirus is as RSV; Orthomyxovirus is as influenza virus; Bunyavirus and arenvirus), dsDNA virus (as reovirus), RNA is to the virus of DNA, i.e. retrovirus is as HIV and HTLV and some DNA virus to RNA, as hepatitis B virus.

The albumen that agricultural is relevant also is the suitable target position that alpha-non-natural amino acid is modified, for example pest-resistant albumen (as Cry albumen), starch and lipid produce enzyme, plant and insect toxins, toxin tolerance proteins, mycotoxins and separate toxalbumin, plant-growth enzyme (as ribulose 1,5-bisphosphate carboxylase/oxygenase, " RUBISCO "), lipoxygenase (LOX) and phosphoenolpyruvic acid (PEP) carboxylase.

In some embodiments, polypeptide of interest in the inventive method and/or the composition or protein (or its part) are by nucleic acid encoding.Described nucleic acid contain usually 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, ten or more a plurality of selector's codon.

Can adopt those skilled in the art to know the gene at coding proteins of interest matter of the method mutagenesis described in " mutagenesis and other Protocols in Molecular Biology " or polypeptide, mix alpha-non-natural amino acid thereby make it to contain (for example) one or more selector's codons with this paper.For example, but the nucleic acid of mutagenesis proteins of interest matter makes it to contain one or more selector's codons to insert one or more alpha-non-natural amino acids.The present invention includes any proteinic this variant (for example mutant) form, for example contain at least one alpha-non-natural amino acid.Similarly, the present invention also comprises corresponding nucleic acids, promptly has any nucleic acid of one or more selector's codons of the one or more alpha-non-natural amino acids of coding.

For preparation contains the protein of alpha-non-natural amino acid, can utilize the host cell and the biology that are fit to mix in vivo alpha-non-natural amino acid by quadrature tRNA/RS pairing.One or more carriers of available expression quadrature tRNA, quadrature tRNA synthetic enzyme and the carrier that coding is treated derived protein come genetic modification (for example transform, transduction or transfection) host cell.Each component can be positioned at identical carrier or be positioned at different carriers separately, perhaps can two component be positioned at a carrier and the 3rd component is positioned at second carrier.Carrier can be the form of plasmid, bacterium, virus, naked polynucleotide or coupling polynucleotide for example.

Measure polypeptide by immunoreactivity

(for example, be example with synthetic protein in this paper translation system, polypeptide contains alpha-non-natural amino acid because polypeptide of the present invention provides various new peptide sequences; Perhaps, be example with new synthetic enzyme, then be the new sequence of standard amino acid), these polypeptide also provide the new texture feature that can discern in immunoassay for example.Generation can and can be one of feature of the present invention with this serum bonded polypeptide with polypeptid specificity bonded antiserum(antisera) of the present invention.Term used herein " antibody " includes but not limited to: basically by one or more immunoglobulin gene encoded polypeptides, or the fragment of its energy specificity combination and discriminance analysis thing (antigen).Example comprises polyclone, mono-clonal, mosaic type and single-chain antibody etc.Term used herein " antibody " also comprises immunoglobulin fragment, comprises Fab fragment and the fragment that is produced by expression library (comprising phage display library).The structure of antibody and term can referring to, Paul for example, Fundamental Immunology (basic immunology), the 4th edition, 1999, Lei Wen press (Raven Press), New York.

For generation is used for the antiserum(antisera) of immunoassay, can generation as described herein and one or more immunogenic polypeptides of purifying.For example, can in reconstitution cell, produce recombinant protein.Employing standard mouse immune scheme (about the antibody that is used to measure specific immune response produce, the standard declaration of immunoassay form and condition can referring to, for example Harlow and Lane, (1988), Antibodies, A Laboratory Manual (antibody, laboratory manual), cold spring port press, New York), with the inbred strain of immunogenic protein and standard adjuvant (as freund's adjuvant) immune mouse (this type of mouse makes experimental result circulation ratio higher being used for measure because of its actual hereditary homogeny).Other details of protein, antibody, antiserum(antisera) etc. is the international publication number WO 2004/094593 of " EXPANDING THE EUKARYOTIC GENETIC CODE (expansion the eukaryotic genetic code) " by name as seen; The WO2002/085923 of " IN VIVO INCORPORATIONOF UNNATURAL AMINO ACIDS (mixing in the body of alpha-non-natural amino acid) " by name; The WO2004/035605 of " GLYCOPROTEIN SYNTHESIS (glycoprotein is synthetic) " by name and the WO2004/058946 of " PROTEIN ARRAYS (protein array) " by name.

O-tRNA and O-RS and O-tRNA/O-RS paired are used

Composition of the present invention and optional being present in the cell of composition for preparing with the inventive method.O-tRNA/O-RS pairing of the present invention or each component can be used for the translating mechanism of host system then, thereby alpha-non-natural amino acid is mixed protein.The international publication number WO 2002/085923 of people such as Schultz " mixing in the body of alpha-non-natural amino acid " by name has described this method, and this piece document is included this paper by reference in.For example, when the host is introduced in the O-tRNA/O-RS pairing, for example when Bacillus coli cells or yeast, this pairing is to selector's codon, for example the amber nonsense codon reacts and alpha-non-natural amino acid that can exogenous adding growth medium mixes protein in vivo, for example in myohaemoglobin test proteins or the treatment albumen.Composition of the present invention can be chosen wantonly and be in external translating system, or in the interior system of cell paste.Containing the proteinic of alpha-non-natural amino acid has wide range of applications.For example mix the target that proteinic non-natural partly can be used as extensive modification, as with other protein, crosslinked with small molecules such as marker or dyestuff and/or biomolecules.By these modifications, mix alpha-non-natural amino acid and can produce improved treatment albumen, can be used for changing or improving the catalysis of enzyme.In some respects, mix alpha-non-natural amino acid in the protein and be modified with subsequently help study proteinic structure, with other protein interactions, or the like.

Test kit

Test kit also is one of feature of the present invention.For example, be provided in cell, producing the proteinic test kit that contains at least one alpha-non-natural amino acid, at least one container is housed in the described test kit, and this container contains the polynucleotide sequence of the O-tRNA that encodes and/or polynucleotide sequence and/or the O-RS of O-tRNA and/or coding O-RS.In one embodiment, described test kit also comprises alpha-non-natural amino acid umbelliferone-ethyl glycine.In another embodiment, described test kit also comprises the operation instruction material of preparation protein and/or host cell.

Embodiment

To provide following examples just in order illustrating, not really want to limit the present invention.The technician will be appreciated that and can change various nonessential parameters and can not depart from the scope of the present invention.Will be appreciated that, embodiment as herein described and embodiment just for the purpose of illustration, those skilled in the art use for reference these embodiment and embodiment and can make various improvement or change and still belong in the scope of the application's design and the authority and the claims of enclosing.

Embodiment 1

Universal method

All chemical reagent are not further purified promptly and use all available from commercial source.The biological spin of Brooker company (Bruker BioSpin GmbH, Rheinstetten/Karlsruhe, Bruker AMX-400 multinuclear spectrophotometer record Germany) with this Teng/Karlsruhe of TUV ¹H NMR collection of illustrative plates, report is than the chemical shift of tetramethylsilane.(Scripps Center for Mass Spectrometry, La Jolla CA) obtain proteomic image at the Scripps mass spectrum center in La Jolla, California city.

Plasmid and clone

Plasmid pBK-lib5 coding Methanococcus jannaschii tyrosyl-t RNA synthetase (MjTyrRS) mutant library, wherein picked at random (randomized) residue Tyr32, Leu65, Phe108, Gln109, Asp158 and Leu162; In addition, make His70 be mutated into Gly, Ala67 is mutated into Ala or Gly.Plasmid pREP (2)/YC MjtRNA that encodes _CUA ^Tyr, be E.C. 2.3.1.28 (CAT) gene of TAG codon at residue 112 places, the GFP gene under the control of T7 promotor is the mutant t7 rna polymerase of TAG codon at residue 1 and 107 places, and Tet ^rMark.Plasmid pLWJ17B3 is coded in the MjtRNA under lpp promotor and the control of rrnC terminator _CUA ^Tyr, the barnase gene (three amber codons that contain residue 2,44 and 65 places) under the control of ara promotor, and Amp ^rMark.Plasmid pBAD/JYAMB-4TAG-Myo coding contains the mutant Physter macrocephalus myoglobin gene of pectinose promotor and rrnB terminator, contains the MjtRNA of lpp promotor and rrnC terminator ^Tyr _CUAAnd tetracyclin resistance mark.Utilize coli strain -Fis, F ^-McrA Δ (mrr-hsdRMS-mcrBC) 80lacZ Δ M15 Δ lacX74 recA1 endA1 araD139 Δ (ara-leu) 7697 galU galK λ rpsL (Str ^R) nupG, the protein that contains alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine that fis::Tn7 (DHFR) institute expresses.

Embodiment 2

Synthetic alpha-non-natural amino acid L-(umbelliferone-4-yl) ethyl glycine

Non-natural coumarin amino acids L-(umbelliferone-4-yl) ethyl glycine (is also write umbelliferone-ethyl glycine; Referring to Fig. 1, structure 1) synthetic is at first N-α-Cbz-L-L-glutamic acid α-benzyl ester to be transformed into side chain beta-ketoester (keto ester), makes itself and resorcin reaction (von Pechmann reaction then in methylsulfonic acid; Brun etc., Angew.Chem., Int.Ed., (2004) 43:3432-3436) thus obtain amino acid L-(umbelliferone-4-yl) ethyl glycine.These steps of Xiang Shuing hereinafter.

Step 1-synthesizes ethyl malonic acid magnesium

The Virahol that adds 5 volumes in the 1.5M magnesium chloride in stirring and the aqueous solution of 3M ethyl malonic acid potassium.After the filtration, vacuum-drying ethyl malonic acid magnesium.

Step 2-synthesizes (2S)-2-benzyloxycarbonyl amino-5-oxo-pimelic acid 1-benzyl ester 7-ethyl ester (Fig. 1, structure 3)

(5g 13.46mmol) is dissolved among the anhydrous THF of 50ml with Z-Glu-Obzl (N-α-Cbz-L-L-glutamic acid α-benzyl ester) under the room temperature.Slowly add N, N '-carbonyl dimidazoles (1.1 equivalent) at room temperature stirred the mixture 2 hours then.Add ethyl malonic acid magnesium (0.55 equivalent), stir the mixture under the room temperature and spend the night.Use the extracted with diethyl ether product, use 10% NaHCO ₃, water and salt water washing.(ethyl acetate: hexane 50:50) purifying contains 3 residue, and the concentrated acquisition of Rotary Evaporators productive rate is 40% white solid by flash chromatography on silica gel. ¹H-NMR(400MHz，CDCl3)：δ?1.26(t，J＝7.2，3H)，1.9-2.01(m，1H)，2.1-2.28(m，1H)，2.5-2.7(m，2H)，3.37(s，2H)，4.18(q，J＝7.2，2H)，4.41(s，1H)，5.11(s，2H)，5.18(s，2H)，5.44(d，J＝8，1H)，7.25-7.41(m，10H)。C ₂₄H ₂NO ₇(M+H ⁺) LC-MS (ESI) calculated value be 442.18, measured value is 442.0.

Step 3-synthesizes L-(umbelliferone-4-yl) ethyl glycine (Fig. 1, structure 1)

(2S)-2 benzyloxycarbonyl amino-5-oxo that previous step is rapid-pimelic acid 1-benzyl ester 7-ethyl ester (2.35g, 5mmol) slowly add methylsulfonic acid (20ml) preparation Resorcinol (3g, 25mmol) in, stirred 2 hours under the room temperature.In mixture, add 5 volume ether then, be cooled to-30 ℃.Ether washing precipitate with cold is dissolved in the water it, filters and freeze-drying.Reversed-phase HPLC purifying L-(umbelliferone-4-yl) ethyl glycine acquisition productive rate is 50% brown solid (YMCAA12S052503WT post, 12ml/ minute flow velocity, the 10%-90% CH of water preparation ₃CN, 0.1%TFA (w/v), 12 minutes altogether). ¹H-NMR(DMSO)：δ?2.06(s，2H)，2.70-2.90(m，2H)，3.82-4.0(m，1H)，6.06(s，1H)，6.75(dd，J＝8.8，2.4，1H)，6.82(d，J＝2.4，1H)，7.62(d，J＝8.8，1H)，8.17(s，3H)。C ₁₃H ₁₄NO ₅(M+H ⁺) LC-MS (ESI) calculated value be 264.08Da, measured value is 264.0Da.

Embodiment 3

Heredity selects that umbelliferone-ethyl glycine is had specific mutant synthetic enzyme

For in intestinal bacteria, umbelliferone-ethyl glycine selectivity being mixed the appointment site in the protein, developed and to have reacted and umbelliferone-ethyl glycine is had specific mutant Methanococcus jannaschii tyrosyl amber inhibition type tRNA (MjtRNA TAG selector codon ^Tyr _CUA)/tyrosyl-tRNA synthetase (MjTyrRS) pairing.

Library construction

For holding the bigger tonka bean camphor side chain of alpha-non-natural amino acid umbelliferone-ethyl glycine, preparation MjTyrRS library pBK-lib5, wherein wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase (Fig. 9; SEQ ID NO:2 and 3) His70 is mutated into Gly, and Ala67 is fixed as Ala or Gly to increase the avtive spot size.6 residue: Tyr-32, Leu-65, Phe-108, Gln-109, Asp-158 and Leu-162 (Kobayashi etc., Nat.Struct.Biol., (2003) 10:425-432 near the picked at random bonded tyrosine; Zhang etc., Protein Sci., (2005) 14:1340-1349).The pBK-lib5 library is by 2 * 10 ⁹Individual TyrRS independent cloning constitutes, and it adopts standard pcr to make up.

Just/negative the selection

Subsequently, several positive and negatives of taking turns being carried out in this library selects.In just selecting, when using pBK-lib and MjtRNA ^Tyr _CUAWhen the cell of cotransfection is grown in the presence of 1mM alpha-non-natural amino acid (UAA) and paraxin are arranged, cell survival depends on allows the inhibition (Xie and Schultz, Methods (2005) 36:227-238) of the amber codon in site to introducing E.C. 2.3.1.28 (CAT) gene.The clone that will just select is transformed into and contains MjtRNA then ^Tyr _CUAWith coding toxicity barnase albumen and in the cell of the gene of allowing 3 amber mutations of site introducing.Make these cells not have in the presence of the UAA growth utilize the amino acid whose any clone of endogenous (the negative selection) to remove.These positive and negatives hereinafter have been described in detail in detail to be selected.

Just select-contain the intestinal bacteria DH10B of pREP (2)/YC plasmid as the host strain of just selecting.With pBK-lib5 library transformant, in SOC, reclaimed 1 hour, added on the GMML-agar plate of kantlex, paraxin, tsiklomitsin and umbelliferone-ethyl glycine that concentration is respectively 50 μ g/mL, 60 μ g/mL, 15 μ g/mL and 1mM earlier with containing (GMML) washed twice of leucic glyceryl basal culture medium (glycerol minimal media with leucine), being applied to again.Dull and stereotyped 60 hours of 37 ℃ of incubations scrape the cell of survival, extract plasmid DNA and by the gel electrophoresis purifying.

The negative selection-pBK-lib5 DNA is transformed into contains negative electricity-competent cell of selecting plasmid pLWJ17B3 then, in SOC, reclaimed 1 hour, be applied to then on the LB-agar plate that contains 0.2% pectinose, 50 μ g/mL Ampicillin Trihydrates and 50 μ g/mL kantlex.Dull and stereotyped 8-12 hour of 37 ℃ of incubations, extract survival clone's pBK-lib5 DNA as mentioned above then.

Subsequently this library is carried out just taking turns selecting, then bear and select and last is just taken turns and selects (with the paraxin of 70 μ g/mL).In this stage, select 96 independent clones, they are suspended among the 50 μ L GMML of 96-orifice plate, duplicate point sample is on two groups of GMML flat boards.It is 60,80,100 and 120 μ g/mL paraxin and 1mM umbelliferone-ethyl glycine that one group of GMML-agar plate has been added tsiklomitsin (15 μ g/mL), kantlex (50 μ g/mL) and concentration.Another group is dull and stereotyped identical, but does not contain umbelliferone-ethyl glycine, and used chloramphenicol concentration is 0,20,40 and 60 μ g/mL.37 ℃ of incubations are after 60 hours, and when finding to have 1mM umbelliferone-ethyl glycine to exist, one is cloned in paraxin is can survive 100 μ g/mL under, and when not having umbelliferone-ethyl glycine to exist, and are can survive under the 20 μ g/mL at paraxin.Therefore, 3 take turns just select to take turns negative the selection with 2 after, be to survive under the 100 μ g/mL at paraxin when this library has been created in 1mM umbelliferone-ethyl glycine and exists, and when not having umbelliferone-ethyl glycine to exist, be a clone that can survive under the 20 μ g/mL only at paraxin.

This clone (being called CouRS-D8) finds to contain following 8 sudden change: Tyr32Glu, Leu65His, Ala67Gly, His70Gly, Phe108Tyr, Gln109His, Asp158Gly and Leu162Gly (referring to Fig. 9 and SEQ ID NO:4 and 5) through order-checking.Mutant synthetic enzyme peptide sequence among Fig. 9 shown in the SEQ ID NO:4 is represented the position that suddenlys change with boldface letter.Following table has further been summed up the mutant synthetase protein.

4 residues are mutated into glycine, and most probable produces enough spaces to hold umbelliferone-ethyl glycine residue.Also undergo mutation by hydrogen bond and bonded tyrosine bonded Tyr32 and Asp158 in wild-type enzyme, this is consistent (Kobayashi etc., Nat.Struct.Biol., (2003) 10:425-432 with loss of activity at tyrosine; Zhang etc., Protein Sci., (2005) 14:1340-1349).

Fig. 8 shows the predict model of mutant MjTyrRS (CouRS-D8) avtive spot.Utilize wild-type MjTyrRS structure (pdb password 1J1U) to produce model.The tyrosine substrate is substituted by L-(umbelliferone-4-yl) ethyl glycine, shows following sudden change: Tyr32Glu, Leu65His, Ala67Gly, His70Gly, Phe108Tyr, Gln109His, Asp158Gly and Leu162Gly.Utilize then California, USA San Diego city acthar Le Si software company (AccelrysSoftware,

, San Diego, CA, USA)

Software is optimized structure by energy minimization.

Embodiment 4

The expression and the sign that contain the mutant myohaemoglobin model protein of umbelliferone-ethyl glycine

Mix proteinic efficient and fidelity of reproduction for measuring umbelliferone-ethyl glycine, replace and contain the terminal His of C- ₆The amber selector codon of position four (Ser-4) or position 37 (His-37) in the Physter macrocephalus myohaemoglobin (referring to Fig. 3) of label.The synthetic enzyme CouRS-D8 and the MjtRNA that are selecting ^Tyr _CUAExist down, at minimum medium or added in the substratum of 1mM umbelliferone-ethyl glycine and cultivated intestinal bacteria with marking protein.Marking protein contrasts as negative in the presence of umbelliferone-ethyl glycine not having.This proteinic generation and be analyzed as follows describe in detail.

Produce mutant protein

The pBK carrier of plasmid pBAD/JYAMB-4TAG-Myo or pBAD/JYAMB-37TAG-Myo and expression CouRS-D8 independently is transfected into jointly

-Fis Bacillus coli cells.Amplifying cells in the 2YT substratum (5mL) of having added kantlex (50 μ g/mL) and tsiklomitsin (15 μ g/mL).Utilize initial nutrient solution (1mL) inoculation to add the 100mL liquid GMML or the 2YT of suitable microbiotic and 1mM umbelliferone-ethyl glycine.Then, 37 ℃ with cell cultures to OD ₆₀₀Be 0.5, express by adding 0.2% pectinose induced protein.After another 4-12 hour, centrifugal collecting cell.Under natural condition, by Ni-NTA affinitive layer purification TAG4 and TAG37 myohaemoglobin mutant.Make the sample desalination then.The output of TAG4 and TAG37 mutant myohaemoglobin is 2mg/L in each substratum.As a comparison, the output of wild-type myohaemoglobin is 5mg/L under simulated condition.

Proteomic image is analyzed

TAG4 mutant myohaemoglobin to purifying carries out the ESI mass spectroscopy.The ESI-mass spectroscopy of mutant myohaemoglobin obtains actual measurement molecular-weight average M _AvgBe 18511Da, with calculating molecular weight M _AvgBe 18512Da consistent (referring to Fig. 6).Because do not find in the ESI-mass spectrum when the convolution threshold value of utilizing 5% corresponding to the mixing of the material of natural amino acid, therefore to mix purity minimum be 95% to the umbelliferone-ethyl glycine that obtains from the signal to noise ratio of protein spectrum.Detection threshold so is set, makes and to detect 5% the material that abundance surpasses main peak.Because only observe main peak, so the purity of mutein 95%.

Split analysing protein by SDS-PAGE

The protein of analyzing purifying by SDS-PAGE shows that full length protein is only having expression in the presence of umbelliferone-ethyl glycine (referring to Fig. 5).In addition, after the SDS-PAGE fractionation, the TAG4 mutant myohaemoglobin that mixes the umbelliferone-ethyl glycine of genetic coding shows the fluorescence band, and the wild-type myohaemoglobin of the umbelliferone-ethyl glycine of shortage genetic coding does not show fluorescence.Fluorescence imaging utilize Piscataway city, New Jersey molecule power/

Health care life science company

Fluorescent scanning instrument (Molecular Dynamics/

Healthcare LifeSciences, Piscataway NJ) obtains.

Embodiment 5

Utilize the probe of the umbelliferone-ethyl glycine of genetic coding as protein configuration

Be one of the potential application of proof umbelliferone-ethyl glycine, with the probe of this amino acid as the urea dependency sex change of holomyarian red eggs white (holomyoglobin).Because tonka bean camphor fluorescence is to the solvent polarity sensitivity, its fluorescence intensity should be not folding relevant with near the part protein umbelliferone-ethyl glycine.The myohaemoglobin structure constitutes (Delli Castelli etc., Protein Sci., (2002) 11:2273-2276) by 8 spirals (A is to H) that link to each other with corner by becate.With the His37 among Ser4 among the spiral A and the spiral C (referring to Fig. 3; The main solvent contact of two residues, can be obviously with other near residue interact) separately independent mutation become selector's codon TAG, thereby having specificity quadrature O-tRNA/O-RS pairing to have that genetic coding mixes umbelliferone-ethyl glycine in the lower body.

As shown in Figure 4, induce folding curve identical in fact by the red proteic urea of wild-type, Ser4 → umbelliferone-ethyl glycine and His37 → umbelliferone-ethyl glycine holomyarian of circular dichroism monitoring, prompting is introduced not obvious interferencing protein stability of spiral A or spiral C with umbelliferone-ethyl glycine.When urea concentration is 2M, the fluorescence intensity of the umbelliferone-ethyl glycine of position 4 increased by 30% (keeping this level when the 2M-5M urea) during the holomyarian red eggs were white, pointing out proteinic this zone is unordered (Zhang etc., Protein Sci., (2005) 14:1340-1349).On the contrary, when urea was 2M, it is little that the mutant myohaemoglobin that contains umbelliferone-ethyl glycine at residue 37 places shows that its fluorescence intensity changes, but urea during for 3M the similar fluorescence of experience increase.Consistent with this result is, NMR in the past studies show that when urea concentration is higher than 2.2M, spiral A and B mainly are unordered, shown in the disappearance of the NOE of and medium range short-and-medium as this zone, and spiral C, D and F fold (Delli Castelli etc. subsequently when urea concentration is higher than 3.0M, Protein Sci., (2002) 11:2273-2276).Therefore, the circular dichroism that changes with average overall on the report entire structure is opposite, the locus specificity probe of amino acid umbelliferone-ethyl glycine protein conformation change seemingly.

Also tested the susceptibility of umbelliferone-ethyl glycine, as shown in Figure 7 to pH.In the 100mM sodium phosphate buffer, detect the absorption spectrum of 60 μ M tonka bean camphor base amino acid umbelliferone-ethyl glycines.The absorbancy of 360nM raises with pH to be increased.Calculating pKa from these detected values is 7.8.

Umbelliferone-ethyl glycine is to the susceptibility (Zinsli of pH (referring to Fig. 7) and solvent polarity, J.Photochem (1974) 3:55-69) make it become the useful external and intracorporeal probe (Brun etc. of many biological studies, Angew.Chem., Int.Ed., (2004) 43:3432-3436).For example, the conformational change in available amino acid umbelliferone-ethyl glycine monitoring bio interaction of molecules or the protein, or the topology characteristic of membrane bound protein.In addition, because the pKa of umbelliferone-ethyl glycine is 7.8, this pKa can systematically change (Sun etc., Bioorg.Med.Chem.Lett. by the substituted cumarin ring, (1998) 8:3107-3110), it should be the useful probe of organoid pH and pH dependent cell process.In addition, the umbelliferone of excited state is intensive light acid (photo-acid) (Cohen and Huppert, Phys.Chem.A (2001) 105:7157-7164), can promote the research of proton transport process in the protein.

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Though described some details of foregoing invention for purpose clear and that understand, will be appreciated that those skilled in the art can make various changes and do not depart from the scope of the present invention by reading this paper content form and details.Show separately for all purposes as each part publication, patent, patent application and/or other file and to include this paper by reference in that all publications that the application quotes, patent, patent application and/or other file are included this paper in by reference in full for all purposes.

Claims (40)

1. translation system, it comprises:

(a) first alpha-non-natural amino acid, i.e. L-(umbelliferone-4-yl) ethyl glycine alpha-non-natural amino acid;

(b) first quadrature aminoacyl-tRNA synthetic enzyme (O-RS); With

(c) the first quadrature tRNA (O-tRNA);

A wherein said O-RS is with the described O-tRNA of the preferential aminoacylation of described first alpha-non-natural amino acid.

2. translation system as claimed in claim 1 is characterized in that, a described O-RS is derived from Methanococcus jannaschii (Methanococcus jannaschii) aminoacyl-tRNA synthetic enzyme.

3. translation system as claimed in claim 1 is characterized in that, a described O-RS is derived from wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase.

4. translation system as claimed in claim 1 is characterized in that, a described O-RS comprises aminoacid sequence or its conservative variant shown in the SEQ ID NO:4.

5. translation system as claimed in claim 1 is characterized in that, a described O-tRNA is amber suppressor tRNA.

6. translation system as claimed in claim 1 is characterized in that, a described O-tRNA comprises polynucleotide sequence shown in the SEQ ID NO:1 or by its coding.

7. translation system as claimed in claim 1 also comprises the nucleic acid of coding proteins of interest matter, and described nucleic acid contains at least one selector's codon, and wherein said selector's codon is described O-tRNA identification.

8. translation system as claimed in claim 7; also comprise the 2nd O-RS and the 2nd O-tRNA; wherein said the 2nd O-RS described the 2nd O-tRNA of the preferential aminoacylation of second alpha-non-natural amino acid that is different from described first alpha-non-natural amino acid, and wherein said the 2nd O-tRNA identification is different from selector's codon of selector's codon of described O-tRNA identification.

9. translation system as claimed in claim 1 is characterized in that, described system comprises the host cell that contains described first alpha-non-natural amino acid, a described O-RS and a described O-tRNA.

10. translation system as claimed in claim 9 is characterized in that, described host cell is the eubacterium cell.

11. translation system as claimed in claim 10 is characterized in that, described eubacterium cell is a Bacillus coli cells.

12. translation system as claimed in claim 9 is characterized in that, described host cell contains the polynucleotide of the described O-RS that encodes.

13. translation system as claimed in claim 12 is characterized in that, described polynucleotide comprise nucleotide sequence shown in the SEQ ID NO:5.

14. translation system as claimed in claim 9 is characterized in that, described host cell comprises the polynucleotide of the described O-tRNA that encodes.

15. one kind is created in the method for protein that contains alpha-non-natural amino acid on the selected location in translation system, described method comprises:

(a) provide a kind of translation system, it comprises:

(i) first alpha-non-natural amino acid, i.e. L-(umbelliferone-4-yl) ethyl glycine alpha-non-natural amino acid;

(ii) first quadrature aminoacyl-tRNA synthetic enzyme (O-RS);

The (iii) first quadrature tRNA (O-tRNA), a wherein said O-RS is with the described O-tRNA of the preferential aminoacylation of described alpha-non-natural amino acid; With

The (iv) nucleic acid of code for said proteins, wherein said nucleic acid comprise at least one selector's codon of described O-tRNA identification; With

(b) in described proteinic translation process, described selector's codon is reacted and described alpha-non-natural amino acid is mixed described proteinic described selected location, thereby be created in the described protein that selected location contains described alpha-non-natural amino acid.

16. method as claimed in claim 15 is characterized in that, the described polynucleotide that provide translation system to comprise to provide the described O-RS of coding.

17. method as claimed in claim 15 is characterized in that, the described O-RS that provides translation system to comprise to provide derived from Methanococcus jannaschii aminoacyl-tRNA synthetic enzyme.

18. method as claimed in claim 15 is characterized in that, the described O-RS that provides translation system to comprise to provide derived from wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase.

19. method as claimed in claim 15 is characterized in that, describedly provides translation system to comprise to provide O-RS and its conservative variant that contains aminoacid sequence shown in the SEQ ID NO:4.

20. method as claimed in claim 15; it is characterized in that; the described translation system that provides comprises amino acid binding pocket by site-directed mutagenesis sudden change wild-type aminoacyl-tRNA synthetic enzyme, and select can be with the gained O-RS of the described O-tRNA of the preferential aminoacylation of described alpha-non-natural amino acid.

21. method as claimed in claim 20 is characterized in that, selection is just being selected and born to described selection step to described O-RS after being included in site-directed mutagenesis from the set that contains a plurality of gained aminoacyls-tRNA synthetic enzyme molecule.

22. method as claimed in claim 15 is characterized in that, the described polynucleotide that provide translation system to comprise to provide the described O-tRNA of coding.

23. method as claimed in claim 15 is characterized in that, described provide translation system to comprise O-tRNA, this O-tRNA to be provided be amber inhibition type tRNA.

24. method as claimed in claim 15 is characterized in that, described provide translation system to comprise to provide contain polynucleotide sequence shown in the SEQ ID NO:1 or by the O-tRNA of its coding.

25. method as claimed in claim 15 is characterized in that, describedly provides translation system to comprise the nucleic acid that contains amber selector codon is provided.

26. method as claimed in claim 15; its feature also is; described protein contains second alpha-non-natural amino acid that is different from described first alpha-non-natural amino acid; and wherein said translation system also comprises the 2nd O-RS and the 2nd O-tRNA; wherein said the 2nd O-RS described the 2nd O-tRNA of the preferential aminoacylation of second alpha-non-natural amino acid that is different from described first alpha-non-natural amino acid, and the 2nd O-tRNA wherein discerns selector's codon of the selector's codon that is different from described O-tRNA identification in the nucleic acid.

27. method as claimed in claim 15, it is characterized in that, describedly provide translation system to comprise host cell is provided, wherein said host cell contains described first alpha-non-natural amino acid, a described O-RS, a described O-tRNA and described nucleic acid, and the wherein said step of mixing comprises the described host cell of cultivation.

28. method as claimed in claim 27 is characterized in that, describedly provides host cell to comprise the eubacterium host cell is provided.

29. method as claimed in claim 28 is characterized in that, describedly provides the eubacterium host cell to comprise e. coli host cell is provided.

30. method as claimed in claim 27 is characterized in that, describedly provides host cell to comprise the host cell of the polynucleotide that contain the described O-RS that encodes is provided.

31. method as claimed in claim 30 is characterized in that, the described step that the host cell of the polynucleotide that contain the described O-RS that encodes is provided comprises provides the host cell that contains the polynucleotide of nucleotide sequence shown in the SEQ ID NO:5.

32. method as claimed in claim 15 is characterized in that, describedly provides translation system to comprise cell extract is provided.

33. composition that comprises polypeptide; described polypeptide contains aminoacid sequence shown in the SEQ ID NO:4; or its conservative variant; wherein said conservative variant polypeptide is with the quadrature tRNA (O-tRNA) of alpha-non-natural amino acid aminoacylation association, and efficient is at least 50% of the translation system efficient of observing the aminoacyl-tRNA synthetic enzyme that contains described O-tRNA, described alpha-non-natural amino acid and contain aminoacid sequence shown in the SEQ ID NO:4.

34. the polynucleotide of the described polypeptide of claim 33 of encoding.

35. polynucleotide as claimed in claim 34 is characterized in that, described polynucleotide comprise nucleotide sequence shown in the SEQ ID NO:5.

36. composition as claimed in claim 33 is characterized in that, described composition is the cell that contains described polypeptide.

37. a composition that contains polynucleotide, described polynucleotide comprise nucleotide sequence shown in the SEQ ID NO:5.

38. carrier that contains the described polynucleotide of claim 37.

39 1 kinds of expression vectors that contain the described polynucleotide of claim 37.

40. a cell that comprises carrier, described carrier contain the described polynucleotide of claim 37.

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