CN104059891A - 8-hydroxyquinoline alanine translation system and application thereof - Google Patents
8-hydroxyquinoline alanine translation system and application thereof Download PDFInfo
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Abstract
The invention relates to an aminoacyl-tRNA synthetase mutant which has an amino acid sequence selected from a group comprising an amino acid sequence as shown in SEQ ID NO:4 and conservative variants of the amino acid sequence as shown in SEQ ID NO:4, wherein the conservative variants have the same enzymatic activity as the amino acid sequence as shown in SEQ ID NO:4. The The invention also provides a translation system which comprises: (i) 8-hydroxyquinoline alanine or its structure analogs; (ii) an orthogonal aminoacyl-tRNA synthetase of the invention; (iii) orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase realizes prior aminoacylation of the orthogonal tRNA by the 8-hydroxyquinoline alanine or its structure analogs; and (iv) nucleic acid coding target protein, wherein the nucleic acid contains at least one selection codon specifically recognized by the orthogonal tRNA. Finally, the invention provides a tyrosine phenol lyase mutant which can catalyze 8-hydroxyquinoline to generate 8-hydroxyquinoline alanine, and has an amino acid sequence as shown in SEQ ID NO:5.
Description
Technical field
The invention belongs to biochemical field.Particularly; the invention provides a kind of aminoacyl-tRNA synthetase mutant; it is a kind of orthogonal aminoacyl-tRNA synthetase; its aminoacid sequence containing selects the group of the examples of conservative variations composition of the aminoacid sequence shown in the aminoacid sequence shown in free SEQ ID NO:4 and SEQ ID NO:4, and described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.The invention still further relates to a kind of 2-amino-3-(oxine-5-yl) propionic acid (be called for short oxine L-Ala, be abbreviated as HqAla) translation system.More specifically; the present invention relates to utilize the pairing of orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase that oxine L-Ala or its analog fixed point specificity are inserted to the translation system of target protein, and utilize the described translation system specificity of fixing a point in target protein to insert the method for oxine L-Ala or its analog.The invention still further relates to a kind of tyrosine phenol lyase mutant, its aminoacid sequence containing is by shown in SEQ ID NO:5, and described tyrosine phenol lyase mutant catalysis oxine generates oxine L-Ala.Finally, the present invention also provides a kind of mutein that comprises alpha-non-natural amino acid that can chelated metal ions by genetic coding to expand the method for protein function.
Background technology
The alpha-non-natural amino acid of genetic coding chelated metal ions is a kind of Study on Protein sensor design, the powerful of metalloenzyme engineering and pyrenoids mr, but, be limited to the synthetic complicacy of alpha-non-natural amino acid, the method can not be applied widely by biologist.The present invention passes through orthogenesis tyrosine phenol lyase, the biosynthesizing of a kind of novel alpha-non-natural amino acid-oxine L-Ala of catalysis efficiently, and this alpha-non-natural amino acid can form stable compound with multiple transition metal ion.Tyrosine phenol lyase (Tyrosine phenol lyase, TPL), has another name called beta-Tyrosinase, taking pyridoxal phosphate (pyridoxal-phosphate, PLP) as coenzyme.1987, the subunit that the discovery TPL molecules such as Kazakov are about 50kDa by 4 identical molecular weight formed, and each subunit tetramer and 4 molecule pyridoxal phosphates (PLP) combine.Can there is the generation of β-elimination reaction phenol, pyruvic acid and ammonia by catalysis TYR in TPL.Because this reaction is reversible, oxine is replaced after phenol, can under TPL catalysis, generate oxine L-Ala by oxine, Sodium.alpha.-ketopropionate and ammonium chloride.
Genetic coding oxine L-Ala also needs a kind of method of the special insertion alpha-non-natural amino acid of fixing a point in target protein, and this research has now been developed in protokaryon and eukaryote and will in various alpha-non-natural amino acid bodies, have been fixed a point to insert the universal method of protein locus specificity.These methods depend on orthogonal protein translation component, and described component identifies suitable selection codon (selector codon) thereby polypeptide translate duration inserts required alpha-non-natural amino acid to limit position in vivo.These methods are utilized the orthogonal tRNA (O-tRNA) of identification selection codon, and the corresponding orthogonal aminoacyl-tRNA synthetase of specificity (O-RS) loads this O-tRNA with alpha-non-natural amino acid.These components not with host organisms in any endogenous tRNA, aminoacyl-tRNA synthetase (RS), amino acid or codon cross reaction (, it must be orthogonal).Utilize the alpha-non-natural amino acid of a large amount of configurations of this orthogonal tRNA-RS pairing possibility genetic coding.
This area generally knows that utilization is suitable for the orthogonal translation system of preparation containing the protein of one or more alpha-non-natural amino acids, for example, produce the universal method of orthogonal translation system.For example, referring to international publication number WO 2002/086075, its denomination of invention is " METHODS AND COMPOSITIONFOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNASYNTHETASE PAIRS "; WO 2002/085923, its denomination of invention is " IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS "; WO 2004/094593, its denomination of invention is " EXPANDING THE EUKARYOTIC GENETIC CODE ".Fixed point specificity inserts the orthogonal translation system of alpha-non-natural amino acid and other discussion of their generation and using method also can be referring to Wang and Schultz, Chem.Commun. (Camb) 1:1-11 (2002); Wang and Schultz, Angewandte Chemie Int.Ed.44 (1): 34-66 (2005); Xie and Schultz, Methods 36 (3): 227-238 (2005); Xie and Schultz, Curr.Opinion inChemical Biology9 (6): 548-554 (2005); Wang etc., Annu.Rev.Biophys.Biomol.Struct.35:225-249 (2006).Although the existing a set of perfect system in current this area can be fixed a point, specificity is inserted multiple alpha-non-natural amino acid, but due to oxine L-Ala self structure, still can successfully in albumen, insert oxine L-Ala or its analog without report at present.Meanwhile, other alpha-non-natural amino acids that in report, success is inserted are at present all larger with oxine L-Ala textural difference, cause those skilled in the art can not expect using identical system desintegration oxine L-Ala or its analog.In addition; the screening operation amount of orthogonal aminoacyl-tRNA synthetase is very large; need to take turns positive-negative selection by 3, altogether 6 take turns screening, just from mutation library (comprising even up to ten million clones up to a million), screen and obtain the orthogonal aminoacyl-tRNA synthetase described in the present invention.Owing to often there will be false positive clone in screening process, we need to go to verify by further test the ability of their insertion alpha-non-natural amino acids successively, have virtually increased again many workloads simultaneously.Therefore, content of the present invention has great importance, also be the special insertion oxine L-Ala of successfully fixing a point in albumen first, the mutein producing not only has the ability of chelated metal ions, and can serve as specificity metal ion sensor.
Summary of the invention
1, technical problem
The invention provides a kind of aminoacyl-tRNA synthetase mutant; it is a kind of orthogonal aminoacyl-tRNA synthetase; its aminoacid sequence containing selects the group of the examples of conservative variations composition of the aminoacid sequence shown in amino acid shown in free SEQ ID NO:4 and SEQ ID NO:4, and described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.The orthogonal tRNA that this aminoacyl-tRNA synthetase mutant can match with it with oxine L-Ala (being abbreviated as HqAla) or the preferential aminoacylation of its analog, thus in the aminoacid sequence of translation, insert HqAla or its analog.This is that the inventor finds first, correspondingly, and in the present invention by orthogonal its called after oxine L-Ala aminoacyl-tRNA synthetase (HqAlaRS).
In whole specification sheets of the present invention, term " analog of oxine L-Ala " refers to the salt or ester, p-hydroxybenzene L-Ala or the salt of p-hydroxybenzene L-Ala or the compound of ester that are selected from oxine L-Ala.The present invention also provides a kind of tyrosine phenol lyase (Tyrosinephenol lyase, be abbreviated as TPL) mutant, described tyrosine phenol lyase mutant efficiently catalysis oxine generates oxine L-Ala, and its aminoacid sequence is:
(1) aminoacid sequence shown in SEQ ID NO:5, or
(2) aminoacid sequence shown in SEQ ID NO:5 is passed through to one or more amino acid whose replacements, disappearance or interpolation and there is the catalysis oxine identical with the aminoacid sequence shown in SEQ ID NO:5 the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 that generates the enzymic activity of oxine L-Ala.
In addition, those skilled in the art should understand that, in the present invention, term " can catalysis oxine generates the tyrosine phenol lyase mutant of oxine L-Ala " and not only comprises the aminoacid sequence shown in SEQID NO:5, also comprise function fragment or the functional deriv of the aminoacid sequence shown in SEQ ID NO:5, , described function fragment retains the enzymic activity of catalysis oxine generation oxine L-Ala, described functional deriv refers to the aminoacid sequence shown in SEQ ID NO:5 through one or more amino acid whose replacements, lack or add and have the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 of the enzymic activity of the catalysis oxine generation oxine L-Ala identical with the aminoacid sequence shown in SEQ ID NO:5.
This tyrosine phenol lyase mutant that can catalysis oxine generates oxine L-Ala is that the inventor finds first.
On the basis of above-mentioned discovery; the invention provides a kind of pairing that utilizes orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase oxine L-Ala or its analog fixed point specificity are inserted to the translation system of target protein, and utilize the described translation system specificity of fixing a point in target protein to insert the method for oxine L-Ala or its analog.The invention still further relates to the mutein that contains oxine L-Ala or its analog and the application thereof that produce by this translation system and this method.
Therefore; the object of the present invention is to provide the pairing that utilizes orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase that oxine L-Ala or its analog fixed point specificity are inserted to the translation system of protein, and provide and utilize this translation system specificity of fixing a point in target protein to insert the method for oxine L-Ala or its analog.
The present invention also provides the mutein that contains at least one oxine L-Ala or its analog that utilizes translation system of the present invention to produce.In preferred aspects of the invention, inventor's profit is inserted oxine L-Ala or its analog fixed point specificity in target protein in this way, and described target protein includes, but are not limited to, fluorescin (Fluorescent Proteins, is abbreviated as FP).But, it should be appreciated by those skilled in the art that method of the present invention also can, for specificity insertion oxine L-Ala or its analog of fixing a point in the multiple protein outside fluorescin, be not limited to this albumen.
Finally, the present invention also provides a kind of mutein that comprises alpha-non-natural amino acid that can chelated metal ions by genetic coding to expand the method for protein function, described alpha-non-natural amino acid energy chelating many kinds of metal ions is preferably introduced oxine L-Ala or its analog in the aminoacid sequence of albumen in the present invention.
2, technical scheme
The inventor is through screening; obtain a kind of orthogonal aminoacyl-tRNA synthetase; it is a kind of orthogonal aminoacyl-tRNA synthetase; its aminoacid sequence containing selects the group of the examples of conservative variations composition of the aminoacid sequence shown in aminoacid sequence shown in free SEQ ID NO:4 and SEQ ID NO:4; described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4, in the present invention by orthogonal its called after oxine L-Ala aminoacyl-tRNA synthetase (HqAlaRS).And; the inventor utilizes described orthogonal aminoacyl-tRNA synthetase; researched and developed a kind of translation system of introducing oxine L-Ala or its analog in the aminoacid sequence of albumen, this translation system is in this article referred to as oxine L-Ala translation system (sometimes also referred to as " translation system of the present invention ").
Those skilled in the art should understand that, in the present invention, except the aminoacid sequence shown in SEQ ID NO:4, term " orthogonal aminoacyl-tRNA synthetase of the present invention " or " orthogonal oxine L-Ala aminoacyl-tRNA synthetase " also comprise the examples of conservative variations of aminoacid sequence shown in SEQ ID NO:4, as long as described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4; And comprise aminoacid sequence shown in SEQ ID NO:4 through one or more amino acid whose replacements, disappearance or interpolation and there is the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:4 of the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.
Specifically, the invention provides in vivo (for example, in host cell) identification selection codon (selector codon) as amber terminator codon (TAG) thus alpha-non-natural amino acid oxine L-Ala or its analog fixed point specificity is inserted into the oxine L-Ala translation system in polypeptide chain.Described oxine L-Ala translation system comprise not with host cell translating mechanism interactional orthogonal-tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synthetase (O-RS) pairing., host cell endogenous aminoacyl-tRNA synthetase can not identified O-tRNA.Similarly, O-RS provided by the invention not with in conspicuous level or some situation not can identify endogenous tRNA detection level.Utilize described translation system to be created in to fix a point in translation process specificity to insert a large amount of protein of oxine L-Ala or its analog.
In certain aspects, the invention provides oxine L-Ala translation system.Described translation system comprises:
(a) oxine L-Ala or its analog,
(b) orthogonal aminoacyl-tRNA synthetic enzyme of the present invention (O-RS), and
(c) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, O-tRNA described in oxine L-Ala or the preferential aminoacylation of its analog for wherein said orthogonal aminoacyl-tRNA synthetic enzyme.
Wherein, term " analog of oxine L-Ala " refers to the salt or ester, p-hydroxybenzene L-Ala or the salt of p-hydroxybenzene L-Ala or the compound of ester that are selected from oxine L-Ala.
Preferably, oxine L-Ala translation system of the present invention also comprises the nucleic acid of the target protein of encoding, wherein said nucleic acid contains at least one the selection codon by orthogonal tRNA (O-tRNA) specific recognition, is preferably amber codon.More preferably, oxine L-Ala translation system of the present invention also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
Orthogonal aminoacyl-tRNA synthetase (O-RS) used in described system is the aminoacyl tRNA synthetase mutant that the inventor finds first; its aminoacid sequence containing selects the group of the examples of conservative variations composition of the aminoacid sequence shown in aminoacid sequence shown in free SEQ IDNO:4 and SEQ ID NO:4, and described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.
The invention still further relates to the nucleotide sequence of the described orthogonal aminoacyl-tRNA synthetic enzyme of coding (O-RS).One preferred aspect in, described nucleotides sequence is classified SEQ ID NO:3 as.
In preferred aspects of the invention, the invention provides a kind of oxine L-Ala translation system, described system comprises:
(i) oxine L-Ala or its analog;
(ii) orthogonal aminoacyl-tRNA synthetase of the present invention;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in described oxine L-Ala or the preferential aminoacylation of its analog for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) nucleic acid of coding target protein, at least one selection codon that wherein said nucleic acid contains described orthogonal tRNA specific recognition.
Wherein, term " analog of oxine L-Ala " refers to the salt or ester, p-hydroxybenzene L-Ala or the salt of p-hydroxybenzene L-Ala or the compound of ester that are selected from oxine L-Ala.
Preferably, described oxine L-Ala translation system also comprises the nucleotide sequence of the orthogonal aminoacyl-tRNA synthetase of the present invention of encoding.In a preferred embodiment, the nucleotides sequence of described coding orthogonal aminoacyl-tRNA synthetase of the present invention is classified as shown in SEQ ID NO:3.
Various components in this translation system can be derived from various source of species, and for example, the each component in this translation system is derived from Methanococcus jannaschii (Methanococcus jannaschii).For example, the anticodon that orthogonal tRNA (O-tRNA) originates for ancient bacterium sports the tyrosine tRNA with amber codon complementation.In some embodiments, O-tRNA is amber inhibition type tRNA.In some embodiments, O-tRNA comprises the polynucleotide sequence shown in SEQ ID NO:1, and preferably, the sequence of O-tRNA is as shown in SEQ ID NO:1.In one embodiment, can comprise the aminoacid sequence shown in SEQ ID NO:4 and the conservative variant of this sequence for the orthogonal aminoacyl-tRNA synthetase of this system.In preferred embodiments, be shown in SEQ ID NO:4 for the aminoacid sequence of the orthogonal aminoacyl-tRNA synthetase of this system.
In certain aspects, oxine L-Ala translation system of the present invention also comprises the nucleic acid of the target protein of encoding, and wherein said nucleic acid has at least one the selection codon by orthogonal tRNA (O-tRNA) specific recognition.In aspect preferably, described orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
In certain aspects, the invention provides the host cell of the nucleotide sequence that comprises the orthogonal aminoacyl-tRNA synthetase of the present invention of encoding and corresponding orthogonal tRNA sequence.Host cell used is not done concrete restriction, as long as orthogonal aminoacyl-tRNA synthetase and orthogonal tRNA retain their orthogonality in their host cell environment.For example, described host cell can be eubacterium cell, preferably intestinal bacteria.
The present invention also provides the method for the mutein that is created at least one selected location fixed point specificity insertion oxine L-Ala.Described method is utilized above-mentioned oxine L-Ala translation system.Described method generally includes following step:
(a) provide the step of the oxine L-Ala translation system that contains following component:
(i) oxine L-Ala or its analog;
(ii) orthogonal aminoacyl-tRNA synthetase of the present invention (O-RS);
(iii) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, O-tRNA described in oxine L-Ala or the preferential aminoacylation of its analog for wherein said O-RS; With
(iv) nucleic acid of coding target protein, at least one selection codon (being optionally amber codon) that wherein said nucleic acid contains O-tRNA specific recognition;
(b) described orthogonal tRNA sequence and the coding nucleotide sequence of described orthogonal aminoacyl-tRNA synthetase and the nucleotide sequence of the described target protein of encoding are cloned and are transformed in suitable host cell, in substratum, add oxine L-Ala or its analog, in the translation process of described target protein, selection codon described in the orthogonal RNA recognition coding of oxine L-Ala or its analog aminoacylation on the mRNA of target protein and oxine L-Ala or its analog, thereby mediation oxine L-Ala or its analog fixed point specificity are inserted amino acid position corresponding to described selection codon, thereby be created in the mutein that oxine L-Ala or its analog are contained in selected location.
Wherein, term " analog of oxine L-Ala " refers to the salt or ester, p-hydroxybenzene L-Ala or the salt of p-hydroxybenzene L-Ala or the compound of ester that are selected from oxine L-Ala.
It should be appreciated by those skilled in the art that the structure of suitable recombinant vectors and the screening of host cell can realize by conventional molecule clone technology and triage techniques.
Those skilled in the art should understand that, in step (b), described orthogonal tRNA sequence and the coding nucleotide sequence of described orthogonal aminoacyl-tRNA synthetase and the nucleotide sequence of the described target protein of encoding are cloned and are transformed in suitable host cell and can carry out in several ways, for example, by described orthogonal tRNA sequence, the encode nucleotide sequence of described orthogonal aminoacyl-tRNA synthetase and the nucleotide sequence of the described target protein of encoding is connected to respectively in suitable carrier operably, jointly be transformed in suitable host cell with any order or three again, or, also the nucleotide sequence of described orthogonal tRNA sequence and the described orthogonal aminoacyl-tRNA synthetase of coding can be connected to operably in a suitable carrier and (between two kinds of sequences, have or connect without suitable joint), the nucleotide sequence of the described target protein of coding is connected in another kind of different suitable carrier operably, then two kinds of recombinant vectorss that build is transformed in suitable host cell jointly, or, also the nucleotide sequence of described orthogonal tRNA sequence and the described target protein of coding can be connected to operably in a suitable carrier and (between two kinds of sequences, have or connect without suitable joint), the nucleotide sequence of the described orthogonal aminoacyl-tRNA synthetase of coding is connected in another kind of different suitable carrier operably, then two kinds of recombinant vectorss that build is transformed in suitable host cell jointly.Or; also the nucleotide sequence of the nucleotide sequence of orthogonal tRNA sequence and the described orthogonal aminoacyl-tRNA synthetase of coding and coding target protein can be linked together operably with any suitable order; then be cloned on a carrier, be finally transformed in suitable host cell.Above-mentioned clone's scheme is all feasible, and those skilled in the art can experimentally need to easily carry out appropriate selection.
In addition, those skilled in the art be also to be understood that " play and remove " effect to external source recombinant vectors for fear of host cell, often select to use with the carrier of different antibiotic markers to build and need to jointly be transformed into the nucleic acid sequence fragments in same host cell.For the conversion of the structure of the selection of suitable carrier, recombinant vectors, host cell or transfection etc., be all the ordinary skill in the art, for example, the molecular cloning handbook that can publish referring to Cold Spring Harbor Laboratory.
In some embodiments of described method; providing the step of translation system to comprise by site-directed mutagenesis undergos mutation the amino acid binding pocket of wild-type aminoacyl-tRNA synthetase; select the aminoacyl-tRNA synthetase mutant (, the present invention's orthogonal aminoacyl-tRNA synthetase used) with O-tRNA described in the preferential aminoacylation of described alpha-non-natural amino acid (being oxine L-Ala or its analog).Described selection step is carried out just selection and the negative selection (referring to following embodiment 2) of described O-RS from the aminoacyl-tRNA synthetase library of molecules obtaining after comprising site-directed mutagenesis.In some embodiments, provide the step of translation system also to comprise the sequence that O-tRNA is provided, O-tRNA is that the anticodon in ancient bacterium source sports and the tyrosine tRNA of amber codon complementation, for example, described O-tRNA is amber inhibition type tRNA, or O-tRNA comprises the polynucleotide sequence shown in SEQ ID NO:1.In these methods, provide the step of translation system also to comprise the nucleic acid that the coding target protein that contains described translation system amber selection codon used is provided.
Also can in host cell, implement to produce the method for the mutein that contains oxine L-Ala or its analog.In these situations, (the host cell providing comprises oxine L-Ala translation system of the present invention, nucleotide sequence, the O-tRNA sequence that comprises the O-RS of the present invention that encodes and contain at least one and select the nucleic acid of coding target protein of codon), for example, insert oxine L-Ala or its analog and (, add oxine L-Ala or its analog etc. in substratum) and cultivate this host cell specificity that can cause fixing a point under suitable culture condition in described target protein.In some embodiments, provide step to comprise eubacterium host cell (for example, intestinal bacteria) is provided.
The present invention also provides the method for producing the fluorescin mutant that contains oxine L-Ala or its analog, it utilizes the method for the above-mentioned mutein that is created at least one selected location fixed point specificity insertion oxine L-Ala or its analog, wherein the nucleotide sequence of coding fluorescence protein mutant used comprises the selection codon of described orthogonal tRNA specific recognition in position, at the translate duration of fluorescin, oxine L-Ala or its analog fixed point are inserted into amino acid position corresponding to described selection codon, thereby produce the fluorescin mutant that contains oxine L-Ala or its analog.
Preferably, the present invention also provides the method for producing the modified enhanced green fluorescent protein that contains oxine L-Ala or its analog, and described method utilizes above-mentioned oxine L-Ala translation system to carry out, and described method generally includes following step:
(a) provide the step of the oxine L-Ala translation system that contains following component:
(i) oxine L-Ala or its analog;
(ii) orthogonal aminoacyl-tRNA synthetase (O-RS);
(iii) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, O-tRNA described in described oxine L-Ala or the preferential aminoacylation of its analog for wherein said O-RS; With
(iv) the encode nucleic acid of described green fluorescent protein, such as, but not limited to, SEQ IDNO:6, wherein said nucleic acid contain described O-tRNA specific recognition at least one select codon (being optionally amber codon);
(b) described orthogonal tRNA sequence and the coding nucleotide sequence of described orthogonal aminoacyl-tRNA synthetase and the nucleotide sequence of the described target protein of encoding are cloned and are transformed in suitable host cell, in substratum, add oxine L-Ala or its analog, in the translation process of described target protein (being green fluorescent protein), selection codon and oxine L-Ala on the mRNA of the orthogonal RNA recognition coding green fluorescent protein of oxine L-Ala or its analog aminoacylation, thereby the specific position of mediation oxine L-Ala or the described target protein of its analog fixed point insertion (, amino acid position corresponding to described selection codon).
Wherein, term " analog of oxine L-Ala " refers to the salt or ester, p-hydroxybenzene L-Ala or the salt of p-hydroxybenzene L-Ala or the compound of ester that are selected from oxine L-Ala.
The present invention also provides the fluorescin mutant that contains oxine L-Ala or its analog that utilizes oxine L-Ala translation system of the present invention to produce, and the aminoacid sequence of described fluorescin mutant is SEQ ID NO:7.
In addition, the inventor has further studied the application of the protein mutant of having introduced oxine L-Ala or its analog in aminoacid sequence, result of study is found, super easy folding modified enhanced green fluorescent protein sfGFP-151-HqAla has very strong cupric ion binding ability, be 0.1fM to the avidity of cupric ion, can remove cupric ion for chelating; And sfGFP-66-HqAla also adds respectively the mutain cpsfGFP-66-HqAla forming after the joint GGTGGS of one section of six peptide to can be used as specificity zine ion sensor at N-terminal and C-terminal.
The invention still further relates to a kind of mutein that comprises oxine L-Ala or its analog by genetic coding and expand the method for protein function, described method comprises: utilize oxine L-Ala translation system of the present invention or described at least one selected location fixed point specificity that is created in to insert the method for the mutein of oxine L-Ala and produce the mutein that comprises oxine L-Ala or its analog, the mutein producing has the ability of chelated metal ions, and can serve as specificity metal ion sensor.
Finally, the inventor, through screening, has also obtained a kind of tyrosine phenol lyase (being abbreviated as TPL) mutant that can catalysis oxine generates oxine L-Ala, and its aminoacid sequence is:
(1) aminoacid sequence shown in SEQ ID NO:5, or
(2) aminoacid sequence shown in SEQ ID NO:5 is passed through to one or more amino acid whose replacements, disappearance or interpolation and there is the catalysis oxine identical with the aminoacid sequence shown in SEQ ID NO:5 the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 that generates the enzymic activity of oxine L-Ala.
Preferably, the aminoacid sequence of described tyrosine phenol lyase mutant is SEQ ID NO:5.Molecular model shows, compare with wild-type tyrosine phenol lyase (aminoacid sequence is SEQ ID NO:11), after sporting respectively Serine and halfcystine, 288 of this mutant and 448 amino acids expand significantly enzyme pocket, enzyme and substrate can be acted on better, thereby catalysis oxine generate oxine L-Ala efficiently.Enzymic catalytic reaction liquid productive rate after HPLC purifying can reach 40%, finally obtains the oxine L-Ala of high yield.
In addition, those skilled in the art should understand that, in the present invention, term " can catalysis oxine generates the tyrosine phenol lyase mutant of oxine L-Ala " and not only comprises the aminoacid sequence shown in SEQID NO:5, also comprise function fragment or the functional deriv of the aminoacid sequence shown in SEQ ID NO:5, , described function fragment retains the enzymic activity of catalysis oxine generation oxine L-Ala, described functional deriv refers to the aminoacid sequence shown in SEQ ID NO:5 through one or more amino acid whose replacements, lack or add and have the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 of the enzymic activity of the catalysis oxine generation oxine L-Ala identical with the aminoacid sequence shown in SEQ ID NO:5.
The invention still further relates to the nucleotide sequence of the described tyrosine phenol lyase mutant of coding.
The invention still further relates to a kind of method of preparing oxine L-Ala, described method comprises with above-mentioned tyrosine phenol lyase mutant catalysis oxine, generates described oxine L-Ala.
In sum, the invention provides following:
1. an orthogonal aminoacyl-tRNA synthetase; its aminoacid sequence containing selects the group of the examples of conservative variations composition of the aminoacid sequence shown in aminoacid sequence shown in free SEQID NO:4 and SEQ ID NO:4, and described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.
2. a translation system, described system comprises:
(i) oxine L-Ala or its analog;
(ii) the orthogonal aminoacyl-tRNA synthetase described in the 1st;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in described oxine L-Ala or the preferential aminoacylation of its analog for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) nucleic acid of coding target protein, at least one selection codon that wherein said nucleic acid contains described orthogonal tRNA specific recognition,
The analog of wherein said oxine L-Ala is selected from the salt of oxine L-Ala or salt or the ester of ester, p-hydroxybenzene L-Ala or p-hydroxybenzene L-Ala.
3. the translation system as described in the 2nd, is characterized in that, described orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
4. the translation system as described in the 2nd, wherein said translation system also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
5. a host cell, it comprises the encode nucleotide sequence of the orthogonal aminoacyl-tRNA synthetase described in the 1st and corresponding orthogonal tRNA sequence, and wherein said host cell is eubacterium cell, preferably Bacillus coli cells.
6. be created at least one selected location fixed point specificity and insert a method for the mutein of oxine L-Ala or its analog, described method comprises the steps:
(a) provide the translation system described in the 2nd, this system comprises:
(i) oxine L-Ala or its analog;
(ii) the orthogonal aminoacyl-tRNA synthetase described in the 1st;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in described oxine L-Ala or the preferential aminoacylation of its analog for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) the encode nucleic acid of described target protein, wherein said nucleic acid comprise described orthogonal tRNA specific recognition in selected position at least one select codon; With
(b) described orthogonal tRNA sequence and the coding nucleotide sequence of described orthogonal aminoacyl-tRNA synthetase and the nucleotide sequence of the described target protein of encoding are cloned and are transformed in suitable host cell, in substratum, add oxine L-Ala or its analog, at the translate duration of described target protein, selection codon and oxine L-Ala described in the orthogonal tRNA recognition coding of oxine L-Ala or its analog aminoacylation on the mRNA of target protein, thereby mediation oxine L-Ala or its analog fixed point specificity are inserted amino acid position corresponding to described selection codon, thereby be created in the described target protein of selected location containing oxine L-Ala or its analog,
The analog of wherein said oxine L-Ala is selected from the salt of oxine L-Ala or salt or the ester of ester, p-hydroxybenzene L-Ala or p-hydroxybenzene L-Ala.
7. a mutein that comprises oxine L-Ala or its analog by genetic coding is expanded the method for protein function, described method comprises: utilize the method described in the 6th to produce the mutein that comprises oxine L-Ala or its analog, the mutein producing has the ability of chelated metal ions, and can serve as specificity metal ion sensor.
8. a tyrosine phenol lyase mutant, described tyrosine phenol lyase mutant catalysis oxine generates oxine L-Ala, and its aminoacid sequence is:
(1) aminoacid sequence shown in SEQ ID NO:5, or
(2) aminoacid sequence shown in SEQ ID NO:5 is passed through to one or more amino acid whose replacements, disappearance or interpolation and there is the catalysis oxine identical with the aminoacid sequence shown in SEQ ID NO:5 the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 that generates the enzymic activity of oxine L-Ala.
9. the nucleotide sequence of tyrosine phenol lyase mutant of the 8th of coding.
10. prepare a method for oxine L-Ala, described method comprises the tyrosine phenol lyase mutant catalysis oxine with the 8th, generates described oxine L-Ala.
3, beneficial effect
The present invention is intended to expand protein function by the alpha-non-natural amino acid of genetic coding chelated metal ions, and wherein the alpha-non-natural amino acid of chelated metal ions is preferably oxine L-Ala, and target protein is preferably fluorescin.By at super easy folding green fluorescent protein sfGFP, super easy folding yellow fluorescence protein sfYFP, in light conversion monomer orange fluorescent protein PsmOrange and scarlet fluorescin eqFP650, fix a point to insert specifically after oxine L-Ala, all there is movement in various degree in the excitation/emission wavelength of these several mutains, the equal red shift 30nm of emission wavelength left and right, especially eqFP650 mutant, excite with emission wavelength and be respectively 622 and 680nm, this is the maximum emission wavelength in the similar green fluorescent protein mid and far infrared district that reports up to now.These protein mutants all can be served as the marker of in-vivo imaging research, increase the sensitivity of surveying, and carry out deep tissues imaging or as fluorescent energy resonance transfer sensor.Simultaneously, sfGFP-151-HqAla mutant in the present invention (, be oxine L-Ala (HqAla) by the amino acid mutation of the 151st of wild-type green fluorescent protein sfGFP) than GFP-151-pyTyr (, be pyrazoles tyrosine (pyTyr) by the amino acid mutation of the 151st of wild-type green fluorescent protein GFP, be 201210285659.7 referring to application number, denomination of invention is the patent application of " 3-pyrazolyl tyrosine translation system and application thereof ") have a stronger cupric ion binding ability, its avidity to cupric ion is 9,000,000 times of GFP-151-pyTyr, prove that thereby oxine L-Ala transmits the electronics transmission in probe research albumen than the 3-pyrazoles tyrosine Photoinduced Electron that does preferably.By further genetic evolution, mutant cpsfGFP-66-HqAla can also be served as zine ion sensor, and zine ion is being brought into play vital effect in cell, responsive special zine ion inductor block makes us can more in depth study various Regulation Mechanism, as enzymic catalytic reaction, cellular metabolism, genetic expression and neurotransmission etc.
In addition, because wild-type tyrosine phenol lyase cannot catalysis generate oxine L-Ala, we are by evolution tyrosine phenol lyase (TPL), make its directly catalysis oxine generation oxine L-Ala, obtain a kind of tyrosine phenol lyase mutant, its aminoacid sequence is SEQ ID NO:5, use this tyrosine phenol lyase mutant only to need single step reaction just can reach 40% productive rate, finally can obtain relatively easily the mutein of a large amount of fixed points insertion oxine L-Ala.Compared with synthesizing with traditional chemical method, this kind of microbial enzyme method biosynthesizing alpha-non-natural amino acid has the advantages such as technique is simple, stable yield, simultaneously, avoid heavy metal catalysis involved in chemosynthesis process, carcinogenic solvent and strong acid and strong base, and do not need just can obtain a large amount of target products through the column purification step excessively of follow-up complexity.
Brief description of the drawings
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 is the reaction formula that TPL mutant (SEQ ID NO:5) catalyzes and synthesizes oxine L-Ala;
Fig. 2 is thin layer chromatography screening TPL mutant;
Fig. 3 is the HPLC purifying collection of illustrative plates of HqAla;
Fig. 4 is the mass spectrum of HqAla;
Fig. 5 is the sequence of orthogonal tRNA, wild-type tyrosyl-t RNA synthetase, orthogonal aminoacyl-tRNA synthetase of the present invention, tyrosine phenol lyase (TPL) mutant, fluorescin mutant;
Fig. 6 is the SDS-PAGE electrophorogram of sfGFP-66-HqAla;
Fig. 7 is the mass spectrum of sfGFP-66-HqAla;
Fig. 8 is the high resolving power crystalline structure figure of sfGFP-66-HqAla;
Fig. 9: Fig. 9 A is sfGFP-66-HqAla, sfYFP-66-HqAla, the Absorption and emission spectra figure of PsmOrange-72-HqAla and eqFP650-67-HqAla; Fig. 9 B is wild-type sfGFP, sfYFP, PsmOrange, eqFP650 and mutant sfGFP-66-HqAla, sfYFP-66-HqAla, the chromophoric group chemical structure schematic diagram of PsmOrange-72-HqAla and eqFP650-67-HqAla;
Figure 10: Figure 10 A is that 1 μ M cpsfGFP-66-HqAla adds the fluorescence intensity after different metal ion; Figure 10 B expresses the Bacillus coli cells of cpsfGFP-66-HqAla before zine ion, (the picture left above is confocal fluorescent figure adding respectively, top right plot is visible ray figure) and add the fluorescence imaging figure of (lower-left figure is confocal fluorescent figure, and bottom-right graph is visible ray figure) after zine ion; Figure 10 C is the structure iron of sfGFP-66-HqAla; Figure 10 D is the structure iron of cpsfGFP-66-HqAla.
Embodiment
Carry out by the following examples further to illustrate the present invention.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
It should be appreciated by those skilled in the art that unless stated otherwise, in following embodiment, chemical reagent used is the reagent of the analytical pure rank that can buy by commercial sources.
The biocatalysis synthetic (Fig. 1-Fig. 4) of embodiment 1:8-hydroxyquinoline L-Ala (HqAla)
The present invention adopts the method for biological enzyme, utilize from Citrobacter freundii (ATCC8090, purchased from American type culture collection (ATCC)) in the synthetic oxine L-Ala of tyrosine phenol lyase (TPL) the catalysis oxine that clones, catalyzed reaction formula is shown in Fig. 1.
But experimental result shows, wild-type tyrosine phenol lyase (TPL, aminoacid sequence is SEQID NO:11) cannot catalysis generate oxine L-Ala.Therefore, the inventor has analyzed the crystalline structure figure of TPL, picks out 448 phenylalanines, 36 phenylalanines and 288 methionine(Met)s, introduces NNK sudden change (N=A+T+C+G; K=T+G), be built into pEt-TPL mutation library and carry out the orthogenesis of TPL.Pick out 1024 mono-clonals from mutation library, with after 96 orifice plate overnight incubation, add lysozyme lysis cell, then add oxine, ammonium chloride and Sodium.alpha.-ketopropionate, hatch 4h for 37 DEG C, detect amino acid whose formation by triketohydrindene hydrate tlc.Result shows (Fig. 2), and one of them is cloned successfully catalysis and has formed oxine L-Ala.Sequencing result shows, the aminoacid sequence of this tyrosine phenol lyase mutant is SEQ ID NO:5, compared with wild-type TPL (aminoacid sequence is SEQ ID NO:11), 288 of this mutant have been mutated into Serine by methionine(Met), and 448 are suddenlyd change for halfcystine by phenylalanine.Use this tyrosine phenol lyase mutant only to need single step reaction just can reach 40% productive rate, finally can obtain relatively easily the mutein of a large amount of fixed points insertion oxine L-Ala.
The TPL mutant that we obtain screening carries out amplification culture, then receives bacterium, centrifugal, and ultrasonication adopts ni-sepharose purification, obtains protease mutant.Get 30mM ammonium acetate, 20mM8-hydroxyquinoline (purchased from Beijing lark prestige company), 60mM Sodium.alpha.-ketopropionate, 5mM mercaptoethanol, 50 μ M pyridoxal phosphates and 10mg protease mutant, be settled to 1L, pH8.0, then room temperature lucifuge stirs 7 days.Collect water, obtain white powder (Fig. 3), productive rate 40% by HPLC separation and purification.Product, through mass spectrometric detection, finds that its molecular weight is 235Da (Fig. 4), coincide with theoretical molecular 235Da.
The required chemical reagent of above building-up reactions if no special instructions, all purchased from Sigma company, is the above rank of analytical pure.
In addition, those skilled in the art should understand that, in the present invention, term " can catalysis oxine generates the tyrosine phenol lyase mutant of oxine L-Ala " and not only comprises the aminoacid sequence shown in SEQID NO:5, also comprise function fragment or the functional deriv of the aminoacid sequence shown in SEQ ID NO:5, , described function fragment retains the enzymic activity of catalysis oxine generation oxine L-Ala, described functional deriv is through one or more amino acid whose replacements by the aminoacid sequence shown in SEQ ID NO:5, lack or add and have the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 of the enzymic activity of the catalysis oxine generation oxine L-Ala identical with the aminoacid sequence shown in SEQ ID NO:5.
Embodiment 2: evolution HqAla specificity aminoacyl-tRNA synthetase
For locus specificity in gene inserts HqAla; need in E.coli host cell used, introduce aminoacyl-tRNA synthetase/tRNA orthogonal right, this is orthogonal to deriving from Methanococcus jannaschii (Methanococcus jannaschii) amber inhibition tyrosyl tRNA (MjtRNA
cUA tyr)/tyrosyl-t RNA synthetase (its aminoacid sequence is SEQ ID NO:2 for MjTyrRS, wild-type) is right.MjTyrRS mutation library is structured in card and receives in mycin resistance pBK plasmid (purchased from the Peter G.Schultz of scripps institute of U.S. laboratory), on this plasmid between the promotor and terminator of E.coli glutamine synthetase.The synthetic enzyme mutation library using is pBk-lib-jw1 storehouse, and the construction process of this mutation library is: on MjTyrRS gene, select 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) introducing NNK sudden change (N=A+T+C+G; K=T+G), other 6 sites (Ile63, Ala67, His70, Tyr114, Ile159, Val164) or random mutation be Gly or remain unchanged (referring to Xie, J.; Liu, W.S.; Schultz, P.G.Angew.Chem., Int.Ed.2007,46,9239-9242; Wang, JY.; Zhang W.; Song WJ; Et al.J.Am.Chem.Soc.2010,132,14812-14818).
By the evolve aminoacyl-tRNA synthetase of specific recognition HqAla of positive-negative selection.Just screen plasmid and comprising MjtRNA
cUA tyr, the chloramphenicol acetyl transferasegene of TAG sudden change, the t7 rna polymerase of the amber mutation of startup expressing green fluorescent protein, tetracycline resistance gene.Negative screening plasmid comprises MjtRNA
cUA tyr, the amber mutation barnase gene under arabinose operon, and ampicillin resistance gene.Carry out 3 and take turns positive-negative selection: include the E.coli DH10B cell that just screens plasmid as just screening host cell.Cell electricity turns pbk-lib-jw1 storehouse, SOC substratum (2% (W/V) Tryptones, 0.5% (W/V) yeast powder, 0.05% (W/V) NaCl, 2.5mM KCl, 10mM MgCl
2, 20mM glucose) cultivate 1 hour at 37 DEG C.Use afterwards the minimal medium (formula of GMML minimal medium: M9 salt/glycerine: 764g Na instead
2hPO
4.7H
2o or 30g Na
2hPO
4, 15g KH
2pO
4, 2.5g NaCl, 5g NH
4cl, 50ml glycerine, autoclaving, pH7.0; 1M MgSO
4: autoclaving; 50mM CaCl
2: autoclaving; 25mM FeCl
2: filtration sterilization; 0.3M leucine: be dissolved in 0.3M NaOH filtration sterilization; 1L liquid GMML substratum: 200ml M9 salt/glycerine, 2ml MgSO
4, 2ml CaCl
2, 2ml FeCl
2, 1ml leucine) and wash twice, bed board solid minimal medium (adding 500ml3% agar powder in liquid GMML substratum, 1mM HqAla, 50mg/L kantlex, 60mg/L paraxin, 15mg/L tsiklomitsin), cultivates 60 hours for 37 DEG C.Collect cell, extract plasmid DNA, electrophoretic separation, glue reclaims.Then, will be transformed into through the pBK-lib-jw1 just screening in the DH10B competent cell that comprises negative screening plasmid.In SOC substratum, recover 1 hour.The LB solid medium (every liter of substratum is containing 10g Tryptones, 5g yeast powder, 10g NaCl) that coated plate comprises 0.2% pectinose (purchased from sigma company) afterwards.Cultivate 8-12 hour for 37 DEG C.Repeating altogether 3 takes turns.
Last is taken turns just to screen and chooses 384 clones, puts respectively plate and is containing 1mM HqAla, paraxin 60,80,100, on the GMML solid medium of 120mg/L, and does not comprise HqAla but comprises paraxin 0,20,40, the GMML solid medium of 60mg/L.Select on the substratum at 1mM HqAla100mg/L paraxin and grow, and at 0mM F2Y, concentration is greater than the clone who does not grow in 20 μ g/mL paraxin substratum and further verifies.Finally choose 1 clone; insert oxine L-Ala most effective, order-checking shows, the aminoacid sequence of the aminoacyl-tRNA synthetase mutant (HqAlaRS) that clone comprises is shown in SEQ ID NO:4; wherein mutational site is Y32H; 153V, L65H, H70G; F108R; Q109V, D158N, L162D and V164G.
Those skilled in the art should understand that, in the present invention, except the aminoacid sequence shown in SEQ ID NO:4, term " orthogonal aminoacyl-tRNA synthetase " or " orthogonal oxine L-Ala aminoacyl-tRNA synthetase " also comprise the examples of conservative variations of aminoacid sequence shown in SEQ ID NO:4, as long as described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4; And comprise aminoacid sequence shown in SEQ ID NO:4 through one or more amino acid whose replacements, disappearance or interpolation and there is the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:4 of the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.
Embodiment 3: express HqAla-green fluorescent protein and Mass Spectrometric Identification
The nucleotide sequence (SEQ ID NO:3) of orthogonal tRNA (SEQ ID NO:1) and the coding HqAlaRS that screens is building up on pEVOL carrier (purchased from the U.S. PeterG.Schultz of scripps institute laboratory), it is upper that the super easy nucleotide sequence (66TAG) (SEQ ID NO:6) that folds green fluorescent protein of encoding is building up to pET22b carrier (purchased from Novagen company), and then cotransformation is in e. coli bl21 sense state cell (purchased from Quan Shi King Company).Picking is single to be cloned in to the 5ml LB substratum that contains 50 μ g/mL ammonia benzyl mycins and 30 μ g/mL paraxin, cultivates 12 hours for 37 DEG C.Then by above 1ml nutrient solution containing enlarged culturing in above antibiotic 100ml LB substratum, to OD
600approximate at 1.1 o'clock, in LB substratum, add 1mMHqAla, 1mM IPTG and 0.2% pectinose (purchased from sigma company) culturing cell, contrast does not add HqAla.After 12 hours, receive bacterium, Ni-NTA purifying protein, and with SDS-PAGE electrophoretic analysis (Fig. 6).
We find, only in the substratum that has HqAla, just can be purified into the super folding green fluorescent protein of total length, and the HqAlaRS that this explanation screens can specific identification HqAla.In LB substratum, the productive rate of the super folding green fluorescent protein of HqAla-is 20mg/L, and the productive rate of wild-type protein is 100mg/L.Only be inserted into 66 amber mutation sites of super folding green fluorescent protein in order to detect HqAla, we have carried out ESI-TOF mass spectrometric detection to sfGFP-66-HqAla, detected result molecular weight is 27342Da (Fig. 7), coincide with the molecular weight 27342Da calculating.
Excite and the emission wavelength of mutein sfGFP-66-HqAla is respectively 537 and 544nm, compare the red shift that wild-type sfGFP has produced about 30nm, we have resolved the crystalline structure (Fig. 8) of sfGFP-66-HqAla, find that HqAla has participated in chromophoric formation in sfGFP albumen, and this copline of imidazolone cyclic group in quinoline ring and the chromophoric group of HqAla, this Red Shift Phenomena that is mutein provides architecture basics.
Embodiment 4: express HqAla-fluorescin mutant and carry out excitation/emission spectral investigation
We have built super easy folding yellow fluorescence protein sfYFP (SEQ IDNO:8) by gene engineering method, light conversion monomer orange fluorescent protein PsmOrange (SEQ ID NO:9) and scarlet fluorescin eqFP650 (SEQ ID NO:10) mutant, then insert HqAla by the same procedure in embodiment 3 in the specific fixed point specificity of fluorescin mutant, express and produce mutain sfYFP-66-HqAla, PsmOrange-72-HqAla and eqFP650-67-HqAla, then measured respectively the excitation/emission spectrum of these mutains, result as shown in Figure 9, fixed point is inserted the equal red shift 30nm of the emission wavelength left and right that causes these several mutains after oxine L-Ala specifically, especially eqFP650 mutant, excite with emission wavelength and be respectively 622 and 680nm, this is the maximum emission wavelength in the class green fluorescent protein mid and far infrared district that reports up to now.
Embodiment 5: express HqAla-green fluorescent protein and carry out cupric ion avidity mensuration
We use the method identical with embodiment 3 to express and have produced mutain sfGFP-151-HqAla and measured its cupric ion binding ability.At 60mM Tris-HCl, in pH7.0 damping fluid, add 1 μ M sfGFP-151-HqAla and 1 μ M Cu (II), contrast does not add Cu (II), then carries out fluorescent strength determining.Result demonstration adds 1 μ M Cu (II) can cause 65% fluorescent quenching, show that oxine L-Ala and 3-pyrazoles tyrosine (are 201210285659.7 referring to application number, the patent application that denomination of invention is " 3-pyrazolyl tyrosine translation system and application thereof ") in conjunction with all can be used as electron acceptor(EA) after cupric ion, cause fluorescent quenching thereby produce Photoinduced Electron transmission with the GFP chromophoric group as electron donor.But sfGFP-151-HqAla has stronger cupric ion binding ability, and it is 0.1fM to the avidity of cupric ion, be 9,000,000 times of GFP-151-pyTyr.
Embodiment 6: express HqAla-modified enhanced green fluorescent protein as zine ion sensor
In order to verify after sfGFP fixed point is inserted HqAla whether can be used as zine ion sensor, we add 2 μ M sfGFP-66-HqAla and 100 μ M zinc chloride at 60mM Tris-HCl in pH7.0 damping fluid, but do not detect that any fluorescent signal changes.So, we further transform sfGFP-66-HqAla, add respectively the joint GGTGGS of one section of six peptide at the N-terminal of albumen and C-terminal, form mutain cpsfGFP-66-HqAla (Figure 10 C, Figure 10 D), and then add zinc chloride, find that fluorescent signal has strengthened 7.2 times.Then, we have detected the ability of cpsfGFP-66-HqAla in conjunction with other metal ions, toward the Cu (II) that adds respectively 100 μ M in mutain, Fe (II), the many kinds of metal ions such as Co (II) and Ni (II), result as shown in Figure 10 A, except zine ion, other metal ions all, less than the fluorescence intensity that significantly increases mutain, prove that cpsfGFP-66-HqAla can be used as specificity zine ion sensor.
In viable cell, whether also can be used as zine ion sensor in order to detect cpsfGFP-66-HqAla, we by cpsfGFP-66-HqAla and pEOV-HqAlaRS plasmid cotransformation in Bacillus coli cells, in the substratum that contains 50 μ g/mL ammonia benzyl mycins and 30 μ g/mL paraxin, 37 DEG C of cultivations are to OD
600approximate at 1.1 o'clock, in LB substratum, add 0.5mM HqAla, 1mM IPTG and 0.2% pectinose, after continuing to cultivate 12 hours, receive bacterium, add 100 μ M Zn (II), simultaneously with the bacterium liquid that do not add Zn (II) in contrast, at fluorescence microscopy Microscopic observation, find not add the cell fluorescence intensity of Zn (II) very weak, and add after Zn (II), fluorescence intensity significantly strengthens (Figure 10 B), proves that cpsfGFP-66-HqAla also can be effectively in conjunction with zine ion in viable cell.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the defined the spirit and scope of the present invention of claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (10)
1. an orthogonal aminoacyl-tRNA synthetase; its aminoacid sequence containing selects the group of the examples of conservative variations composition of the aminoacid sequence shown in aminoacid sequence shown in free SEQID NO:4 and SEQ ID NO:4, and described examples of conservative variations has the enzymic activity identical with the aminoacid sequence shown in SEQ ID NO:4.
2. a translation system, described system comprises:
(i) oxine L-Ala or its analog;
(ii) orthogonal aminoacyl-tRNA synthetase claimed in claim 1;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in described oxine L-Ala or the preferential aminoacylation of its analog for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) nucleic acid of coding target protein, at least one selection codon that wherein said nucleic acid contains described orthogonal tRNA specific recognition,
The analog of wherein said oxine L-Ala is selected from the salt of oxine L-Ala or salt or the ester of ester, p-hydroxybenzene L-Ala or p-hydroxybenzene L-Ala.
3. translation system as claimed in claim 2, is characterized in that, described orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
4. translation system as claimed in claim 2, wherein said translation system also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
5. a host cell, the nucleotide sequence that it comprises the orthogonal aminoacyl-tRNA synthetase claimed in claim 1 of encoding and corresponding orthogonal tRNA sequence, wherein said host cell is eubacterium cell, preferably Bacillus coli cells.
6. be created at least one selected location fixed point specificity and insert a method for the mutein of oxine L-Ala or its analog, described method comprises the steps:
(a) provide translation system claimed in claim 2, this system comprises:
(i) oxine L-Ala or its analog;
(ii) orthogonal aminoacyl-tRNA synthetase claimed in claim 1;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in described oxine L-Ala or the preferential aminoacylation of its analog for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) the encode nucleic acid of described target protein, wherein said nucleic acid comprise described orthogonal tRNA specific recognition in selected position at least one select codon; With
(b) described orthogonal tRNA sequence and the coding nucleotide sequence of described orthogonal aminoacyl-tRNA synthetase and the nucleotide sequence of the described target protein of encoding are cloned and are transformed in suitable host cell, in substratum, add oxine L-Ala or its analog, at the translate duration of described target protein, selection codon and oxine L-Ala described in the orthogonal tRNA recognition coding of oxine L-Ala or its analog aminoacylation on the mRNA of target protein, thereby mediation oxine L-Ala or its analog fixed point specificity are inserted amino acid position corresponding to described selection codon, thereby be created in the described target protein of selected location containing oxine L-Ala or its analog,
The analog of wherein said oxine L-Ala is selected from the salt of oxine L-Ala or salt or the ester of ester, p-hydroxybenzene L-Ala or p-hydroxybenzene L-Ala.
7. a mutein that comprises oxine L-Ala or its analog by genetic coding is expanded the method for protein function, described method comprises: utilize the method described in claim 6 to produce the mutein that comprises oxine L-Ala or its analog, the mutein producing has the ability of chelated metal ions, and can serve as specificity metal ion sensor.
8. a tyrosine phenol lyase mutant, described tyrosine phenol lyase mutant catalysis oxine generates oxine L-Ala, and its aminoacid sequence is:
(1) aminoacid sequence shown in SEQ ID NO:5, or
(2) aminoacid sequence shown in SEQ ID NO:5 is passed through to one or more amino acid whose replacements, disappearance or interpolation and there is the catalysis oxine identical with the aminoacid sequence shown in SEQ ID NO:5 the derivative aminoacid sequence of the aminoacid sequence by shown in SEQ ID NO:5 that generates the enzymic activity of oxine L-Ala.
9. the nucleotide sequence of the tyrosine phenol lyase mutant of coding claim 8.
10. prepare a method for oxine L-Ala, described method comprises the tyrosine phenol lyase mutant catalysis oxine by claim 8, generates described oxine L-Ala.
Priority Applications (2)
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| CN105176939A (en) * | 2015-09-02 | 2015-12-23 | 北京安生绿源科技有限公司 | Laccase containing unnatural amino acid, as well as preparation method and application of laccase |
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| CN114134141A (en) * | 2021-11-03 | 2022-03-04 | 浙江大学 | Chimeric phenylalanine translation system introducing unnatural amino acids and construction method thereof |
| CN114134141B (en) * | 2021-11-03 | 2024-01-30 | 杭州嵌化合生医药科技有限公司 | Chimeric phenylalanine translation system with introduced unnatural amino acid and construction method thereof |
| WO2024040885A1 (en) * | 2022-08-25 | 2024-02-29 | 凯莱英医药集团（天津）股份有限公司 | Aminoacyl-trna synthase mutant and use thereof |
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| CN104059891B (en) | 2016-08-17 |
| CN106047846A (en) | 2016-10-26 |
| CN106047846B (en) | 2020-02-04 |
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