CN110117580B - Seleno-tyrosine translation system and application thereof - Google Patents
Seleno-tyrosine translation system and application thereof Download PDFInfo
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- CN110117580B CN110117580B CN201810116409.8A CN201810116409A CN110117580B CN 110117580 B CN110117580 B CN 110117580B CN 201810116409 A CN201810116409 A CN 201810116409A CN 110117580 B CN110117580 B CN 110117580B
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Abstract
The present invention relates to an aminoacyl-tRNA synthetase mutant, which is an orthogonal aminoacyl-tRNA synthetase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 or a conservative variant or homologue thereof having the same enzymatic activity. The present invention also provides a seleno-tyrosine translation system comprising: (i) seleno-tyrosine; (ii) the orthogonal aminoacyl-tRNA synthetase; (iii) an orthogonal tRNA, wherein said orthogonal aminoacyl-tRNA synthetase preferentially aminoacylates said orthogonal tRNA with seleno tyrosine; and (iv) a nucleic acid encoding a protein of interest, wherein the nucleic acid comprises at least one selector codon that is specifically recognized by the orthogonal tRNA. The invention also provides a method for designing and modifying protease by utilizing the seleno-tyrosine translation system, and the protease produced by the method.
Description
Technical Field
The invention belongs to the field of biochemistry. Specifically, the invention provides an aminoacyl-tRNA synthetase mutant, which is an orthogonal aminoacyl-tRNA synthetase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and the amino acid sequence shown in SEQ ID NO: 4, wherein said conservative variant has an amino acid sequence identical to SEQ ID NO: 4, and the same enzyme activity. The invention also relates to a seleno-tyrosine (SeHF for short) translation system and application thereof.
In particular, the invention relates to a seleno-tyrosine translation system for site-specific insertion of seleno-tyrosine into a target protein by using pairing of orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase, and a method for site-specific insertion of seleno-tyrosine into a target protein by using the translation system. The invention also relates to a method for designing and modifying target protease by using the set of translation system and the method, and application of mutant protease containing seleno-tyrosine produced by the method, such as agrobacterium radiobacter phosphotriesterase (abbreviated as arPTE) mutant inserted with seleno-tyrosine, and agrobacterium radiobacter phosphotriesterase mutant inserted with seleno-tyrosine.
Background
Tyrosine is ubiquitous in many enzyme catalysts and its roles include: 1. direct nucleophilic attack of substrates (e.g., DNA topoisomerase, etc.); 2. generalized bases in deprotonation processes (e.g., glycoside hydrolases, etc.); 3. generalized acid protonation of the leaving group (e.g., tyrosine phenol lyase, etc.); 4. hydrogen bond donor/acceptor or electrical repulsion of the active site (e.g., ketosteroid isomerases, etc.); 5. cation-pi interactions (e.g., terpene cyclases, etc.); 6. participate in redox and electron transfer reactions (such as ribonucleotide reductase, FtmOx1, etc.); 7. direct metal coordination (e.g., catalase, galactose oxidase, etc.). To explore the mechanism of tyrosine residues in enzyme catalysis and to better engineer enzymes, we mutated tyrosine at the active site of the enzyme to an unnatural amino acid (abbreviated UAA) with a metal or ortho substitution. However, the introduction of foreign atoms at the enzyme active site may significantly impair the enzyme activity due to steric hindrance, and therefore, it is desirable to adopt a strategy for minimizing the impairment of the enzyme activity, i.e., to adopt single atom substitution for amino acids at the enzyme active site.
Selenium is the same as the main group of oxygen, with selenium having a van der Waals radius of 1.9 angstroms and oxygen having a radius of 1.52 angstroms. We chose to synthesize and genetically insert 2-amino-3- (4-hydroselenophenyl) propionic acid (seleno-tyrosine or SeHF for short), which has a single oxygen-selenium atom substitution in one side chain compared to tyrosine, and thus can provide a valuable mechanistic tool to study the role of tyrosine in enzyme catalysts. Simultaneously, with phenol (pK)a10) pK of the phenyl enol side chaina(pKa5.9), the nucleophilicity of the phenyl selenate anion is enhanced, which also provides a good opportunity for better design of catalytic enzymes. This is achieved byIn addition, selenium can be used to determine protein structure by multi-wavelength anomalous scatter phasing. Inspired by natural or designed enzymes containing selenocysteine, selenocysteine can also be used to design artificial enzymes with peroxidase and dehalogenase activities.
Phosphotriesterase (abbreviated as PTE) is a bimetallic protein with multiple substrates, one of which, paraoxon, is one of the most commonly used insecticides. Using metal-activated water as nucleophile, PTE can be generated in a simple one-step SNThe 2-displacement reaction cleaves the P-O bond of methyl paraoxon (abbreviated DNP) to form dimethyl phosphate (DMP) and P-nitrophenol (PNP), without involving any covalent enzyme-substrate intermediate, making it an ideal enzyme design model. In addition, better understanding and improvement of PTE catalytic activity is also of some importance for protecting the environment.
The structure and mechanism of bacterial PTEs have been widely reported, with turnover rates determined by several components: mie's complex (ES) binding coefficient (k)1) And dissociation coefficient (k)2) P-O bond rupture coefficient (k)3) And product Release coefficient (k)5). It is reported that P-O bond is broken by k3Step is not rate limiting, and product release k5It is the slowest and rate limiting step in the catalytic cycle. The substrate binding pocket, and also the product release pocket, of PTEs is composed of many hydrophobic residues, including Tyr309, Phe132 and Trp131, the aromatic rings of the paraoxon substrate are surrounded by the side chains of these amino acids above, and the conversion of PTEs is believed to have approached their natural evolutionary limit. A plurality of PTEs mutants are separated by directed evolution in a laboratory or reasonable design so as to improve the catalytic efficiency on various substrates. However, the pH optima of wild-type and mutant PTEs are typically greater than 8.5, which may limit their use in environmental remediation, pesticide detoxification, and clinical applications.
We speculate that the negatively charged SeHF may promote product release and that the pK of the SeHF side chainaThe value (pH5.9) can obviously improve the catalytic activity of the PTE in a neutral pH environment. The present study therefore aims to significantly improve the enzymatic activity of PTE by replacing tyrosine by the unnatural amino acid, SeHF, by an extension of the genetic code. All in oneNow, the present study has developed a general method for site-specific in vivo site-specific insertion of various unnatural amino acids into proteins in prokaryotes and eukaryotes. These methods rely on orthogonal protein translation components that recognize appropriate selector codons (selector codon) to enable insertion of the desired unnatural amino acid at defined positions during in vivo translation of the polypeptide. These methods utilize an orthogonal tRNA (O-tRNA) that recognizes a selector codon, and a corresponding specific orthogonal aminoacyl-tRNA synthetase (O-RS) charges the O-tRNA with an unnatural amino acid. These components do not cross-react with any endogenous tRNA, aminoacyl-tRNA synthetase (RS), amino acid, or codon in the host organism (i.e., it must be orthogonal). Using such orthogonal tRNA-RS pairs, it is possible to genetically encode a large number of structurally diverse unnatural amino acids.
It is generally known in the art to utilize orthogonal translation systems suitable for preparing proteins containing one or more unnatural amino acid, e.g., to generate a general method for orthogonal translation systems. See, FOR example, International publication No. WO 2002/086075 entitled "METHOD AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOCYL-tRNA SYNTHETASE PAIRS"; WO 2002/085923 entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS"; WO 2004/094593 entitled "EXPANDING THE EUKARYOTIC GENETIC CODE". Additional discussion of orthogonal translation systems for site-specific insertion of unnatural amino acids and methods for their generation and use can also be found in Wang and Schultz, chem. 1-11 (2002); wang and Schultz, angelwan Chemie int.ed.44 (1): 34-66 (2005); xie and Schultz, Methods 36 (3): 227-; xie and Schultz, curr. opinion in Chemical Biology 9 (6): 548-554 (2005); wang et al, annu, rev, biophysis, biomol, struct.35: 225-249(2006).
Disclosure of Invention
I. Technical scheme
The inventor obtains an aminoacyl-tRNA synthetase mutant for the first time through screening, wherein the aminoacyl-tRNA synthetase mutant is an orthogonal aminoacyl-tRNA synthetase and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, and the amino acid sequence shown in SEQ ID NO: 4 and conservative variants of the amino acid sequence shown in SEQ ID NO: 4, wherein said conservative variants and homologues have a homology to the amino acid sequence set forth in SEQ ID NO: 4, wherein the sequence homology may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 99% or more. Such aminoacyl-tRNA synthetase mutants can preferentially aminoacylate the orthogonal tRNA with seleno tyrosine (SeHF for short), thereby inserting SeHF into the translated amino acid sequence. This was the first discovery by the present inventors and, accordingly, it was named orthogonal selenoyltyrosyl aminoacyl-tRNA synthetase (abbreviated as SeHFRS) in the present invention.
In a preferred embodiment, the amino acid sequence of the orthogonal selenoyltyrosyl aminoacyl-tRNA synthetase is as set forth in SEQ ID NO: 4, respectively.
Based on the above findings, the present invention provides a seleno-tyrosine translation system for site-specific insertion of seleno-tyrosine into a target protein using a pair of orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase, and a method for site-specific insertion of seleno-tyrosine into a target protein using the translation system. The invention also relates to seleno-tyrosine-containing mutant proteins produced by such translation systems and such methods, and uses thereof.
Accordingly, it is an object of the present invention to provide an orthogonal selenoyltyrosine aminoacyl-tRNA synthetase, a selenoyltyrosine translation system for site-specific insertion of selenoyltyrosine into a protein using a pair of orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase, and a method for site-specific insertion of selenoyltyrosine into a target protein using the translation system.
The invention also provides a method for designing and modifying target protease by using the seleno-tyrosine translation system, and mutant protease containing at least one seleno-tyrosine produced by using the method. In a preferred aspect of the invention, the present inventors utilize this approach to site-specifically insert seleno-tyrosine into a protease of interest, including, but not limited to, phosphotriesterase, e.g., Agrobacterium radiobacter phosphotriesterase, to enhance enzymatic activity. However, it will be appreciated by those skilled in the art that the method of the present invention may also be used in a variety of proteases other than phosphotriesterase, and is not limited to such proteases.
It will be appreciated by those skilled in the art that, in the present invention, in addition to SEQ ID NO: 4, the term "orthogonal aminoacyl-tRNA synthetase of the invention" or "orthogonal seleno-tyrosine aminoacyl-tRNA synthetase" also includes, in addition to the amino acid sequence shown in SEQ ID NO: 4, provided that the conservative variant has an amino acid sequence that is identical to SEQ ID NO: 4, or an enzyme activity having the same amino acid sequence as shown in 4; and further comprising converting SEQ ID NO: 4 by substitution, deletion or addition of one or more amino acids, and has a sequence identical to that of SEQ ID NO: 4, the enzyme activity of the same amino acid sequence as shown in SEQ ID NO: 4, or a derivative thereof; and further comprising a sequence identical to SEQ ID NO: 4, wherein the sequence homology may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 99% or more, and the homologue has a sequence homology with the amino acid sequence shown in SEQ ID NO: 4, and the same enzyme activity.
Also, nucleotide sequences encoding the orthogonal selenoyltyrosyl aminoacyl-tRNA synthetases (SeHFRS) of the invention are also included within the scope of the invention. Preferably, the coding nucleotide sequence is SEQ ID NO: 3, respectively.
In particular, the present invention provides a seleno tyrosine translation system that recognizes a Selector codon (Selector codon), such as an amber stop codon (TAG), in vivo (e.g., in a host cell) to site-specifically insert a non-natural amino acid, seleno tyrosine, into a polypeptide chain. The seleno-tyrosine translation system comprises an orthogonal-tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS) pair that do not interact with the host cell translation machinery. That is, the host cell's endogenous aminoacyl-tRNA synthetases do not recognize the O-tRNA. Similarly, the O-RSs provided herein do not recognize endogenous tRNA's at significant or, in some cases, detectable levels. The translation system can be used for producing a large amount of protein with specific site-specific insertion of seleno-tyrosine in the translation process.
In a preferred aspect of the invention, the invention provides a seleno tyrosine translation system. The translation system includes:
(a) the selenium substituted tyrosine is replaced by the selenium substituted tyrosine,
(b) an orthogonal aminoacyl-tRNA synthetase (O-RS) of the invention, and
(c) an orthogonal tRNA (O-tRNA) comprising SEQ ID NO:1, wherein the orthogonal aminoacyl-tRNA synthetase preferentially aminoacylates the O-tRNA with seleno-tyrosine;
(d) a nucleic acid encoding a protein of interest, wherein the nucleic acid comprises at least one selector codon (optionally an amber codon) specifically recognized by the orthogonal tRNA.
Preferably, the seleno tyrosine translation system of the present invention further comprises a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase.
The "orthogonal aminoacyl-tRNA synthetase (O-RS) of the invention" used in the system is an aminoacyl-tRNA synthetase mutant that has been discovered for the first time by the present inventors, and has an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 4, wherein said conservative variant has an amino acid sequence identical to SEQ ID NO: 4, and the same enzyme activity. In other words, in the present invention, the term "orthogonal aminoacyl-tRNA synthetase of the invention" can be used interchangeably with "orthogonal selenoyltyrosine aminoacyl-tRNA synthetase of the invention".
The various components of the translation system may be derived from various species sources, for example, the components of the translation system are derived from Methanococcus jannaschii (Methanococcus jannaschii). For example, an orthogonal tRNA (O-tRNA) is an archaeal anticodon mutated to a tyrosine tRNA that is complementary to the amber codon. In some embodiments, the O-tRNA is an amber suppressor tRNA. In some embodiments, the O-tRNA comprises SEQ ID NO:1, preferably, the sequence of the O-tRNA is as set forth in SEQ ID NO:1 is shown. In one embodiment, the orthogonal aminoacyl-tRNA synthetase for use in the system can comprise SEQ ID NO: 4 and conservative variants of the sequence. In a preferred embodiment, the amino acid sequence of the orthogonal aminoacyl-tRNA synthetase used in the system is SEQ ID NO: 4, respectively.
In some aspects, a seleno tyrosine translation system of the invention further comprises a nucleic acid encoding a protein of interest, wherein the nucleic acid has at least one selector codon that is specifically recognized by an orthogonal tRNA (O-tRNA). In a preferred aspect, the orthogonal tRNA is an amber suppressor tRNA, and the selector codon is an amber codon.
In some aspects, the invention provides host cells comprising a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase of the invention, and a corresponding orthogonal tRNA sequence. The host cell used is not particularly limited so long as the orthogonal aminoacyl-tRNA synthetase and the orthogonal tRNA retain their orthogonality in their host cell environment. For example, the host cell may be a eubacterial cell, preferably E.coli.
The invention also provides methods for producing mutant proteins having site-specific insertion of seleno-tyrosine at least one selected position. The method utilizes the seleno-tyrosine translation system. The method generally comprises the steps of:
(a) providing a seleno-tyrosine translation system comprising the steps of:
(i) seleno-tyrosine;
(ii) an orthogonal aminoacyl-tRNA synthetase (O-RS) of the invention;
(iii) an orthogonal tRNA (O-tRNA) comprising SEQ ID NO:1, wherein the O-RS preferentially aminoacylates the O-tRNA with seleno-tyrosine; and
(iv) a nucleic acid encoding a protein of interest, wherein the nucleic acid comprises at least one selector codon (optionally an amber codon) specifically recognized by the O-tRNA;
(b) cloning and transforming the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding the target protein into an appropriate host cell, adding seleno-tyrosine to the culture medium, wherein during translation of the target protein, the seleno-tyrosine aminoacylated orthogonal tRNA recognizes a selector codon on the mRNA encoding the target protein and the seleno-tyrosine, thereby site-specifically inserting the seleno-tyrosine into an amino acid position corresponding to the selector codon, thereby producing a mutein comprising seleno-tyrosine at the selected position.
It will be appreciated by those skilled in the art that the construction of suitable recombinant vectors and the screening of host cells may be achieved by conventional molecular cloning and screening techniques.
It will be appreciated by those skilled in the art that cloning and transforming the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding the target protein into a suitable host cell in step (b) can be performed in a variety of ways, e.g., by operably linking the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding the target protein, respectively, to a suitable vector, and then transforming into a suitable host cell in any order or in combination; alternatively, the orthogonal tRNA sequence and the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase can be operably linked to a suitable vector (with or without a suitable linker between the two sequences), the nucleic acid sequence encoding the protein of interest can be operably linked to a different suitable vector, and the two recombinant vectors constructed can be co-transformed into a suitable host cell; alternatively, the orthogonal tRNA sequence and the nucleic acid sequence encoding the protein of interest can be operably linked to a suitable vector (with or without a suitable linker between the two sequences), the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase can be operably linked to a different suitable vector, and the two recombinant vectors constructed can be co-transformed into a suitable host cell. Alternatively, the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding the protein of interest can be operably linked together in any suitable order, cloned into a vector, and finally transformed into a suitable host cell. The above cloning protocols are all possible and can be readily selected by one skilled in the art as required by the experiment.
In addition, it will be understood by those skilled in the art that vectors carrying different antibiotic markers are often selected for the construction of nucleic acid sequence fragments that need to be co-transformed into the same host cell in order to avoid the "kicking" effect of the host cell on the foreign recombinant vector. Selection of an appropriate vector, construction of a recombinant vector, transformation or transfection of a host cell, and the like are routine in the art, and for example, see molecular cloning handbook, published by the Cold spring harbor laboratory, USA.
In some embodiments of the methods, the step of providing a translation system comprises screening for aminoacyl-tRNA synthetase mutants that preferentially aminoacylate the O-tRNA with the unnatural amino acid (i.e., seleno tyrosine) (i.e., orthogonal aminoacyl-tRNA synthetases used in the invention) by mutating the amino acid binding pocket of a wild-type aminoacyl-tRNA synthetase by site-directed mutagenesis. The screening step involves positive and negative selection of the O-RS from the pool of aminoacyl-tRNA synthetase molecules obtained following site-directed mutagenesis (see example 2 below). In some embodiments, the step of providing a translation system further comprises providing a sequence of an O-tRNA that is an archaea-derived anticodon mutated to a tyrosine tRNA that is complementary to the amber codon, e.g., the O-tRNA is an amber suppressor tRNA, or the O-tRNA comprises the amino acid sequence of SEQ ID NO: 1. In these methods, the step of providing a translation system further comprises providing a nucleic acid encoding a protein of interest comprising an amber selector codon for use in said translation system.
In one embodiment, the method of producing a seleno tyrosine-containing mutein can also be practiced within a host cell. In these cases, a host cell is provided that comprises a seleno tyrosine translation system of the invention (i.e., comprises a nucleotide sequence encoding an O-RS of the invention, an O-tRNA sequence, and a nucleic acid encoding a protein of interest that comprises at least one selector codon), and culturing the host cell under suitable culture conditions (e.g., addition of seleno tyrosine to the culture medium, etc.) results in site-specific insertion of seleno tyrosine into the protein of interest. In some embodiments, the providing step comprises providing a eubacterial host cell (e.g., e.
The invention also provides a method for producing a seleno-tyrosine-containing protease mutant by using the method for producing a mutant protein in which seleno-tyrosine is site-specifically inserted at least one selected position, wherein the nucleic acid sequence used for encoding the protease mutant comprises a selector codon specifically recognized by the orthogonal tRNA at the selected position, and seleno-tyrosine is site-specifically inserted into an amino acid position corresponding to the selector codon during translation of the protease, thereby producing the protease mutant containing seleno-tyrosine at the selected position.
Preferably, the present invention also provides a method of producing an agrobacterium radiobacter phosphotriesterase (arpe) mutant containing selenocytidylcholine, the method being performed using the above-described selenocytidylcholine translation system, the method generally comprising the steps of:
(a) providing a seleno-tyrosine translation system comprising the steps of:
(i) seleno-tyrosine;
(ii) an orthogonal aminoacyl-tRNA synthetase (O-RS);
(iii) an orthogonal tRNA (O-tRNA) comprising SEQ ID NO:1, wherein the O-RS preferentially aminoacylates the O-tRNA with the seleno-tyrosine; and
(iv) nucleic acids encoding the agrobacterium radiobacter phosphotriesterase (arpe), such as, but not limited to, SEQ ID NO: 7 (note: to express a purified active arPTE protein in E.coli, the 1 st to 24 th amino acids of arPTE are deleted, and the V at position 364 is mutated to S, so that a nucleic acid sequence encoding amino acids 1 to 24 is not included in SEQ ID NO: 7), wherein said nucleic acid contains at least one selector codon (optionally an amber codon) specifically recognized by said O-tRNA;
(b) cloning and transforming the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding Agrobacterium radiobacter phosphotriesterase into an appropriate host cell, adding selenoyltyrosine to the culture medium, wherein during translation of the Agrobacterium radiobacter phosphotriesterase (arPTE), the selenoyltyrosine aminoacylated orthogonal tRNA recognizes a selector codon on the mRNA encoding phosphotriesterase and selenoyltyrosine, thereby inserting a selenoyltyrosine site at a specific position (i.e., an amino acid position corresponding to the selector codon) of the phosphotriesterase.
The invention also provides a novel method for designing and modifying protease, which comprises the step of substituting tyrosine on the active site of the target protease into seleno-tyrosine by using the seleno-tyrosine translation system, so that a target protease mutant containing seleno-tyrosine is obtained, and the enzymatic activity is enhanced.
Preferably, the present inventors designed to engineer agrobacterium radiobacter phosphotriesterase (arPTE) using the seleno-tyrosine translation system, and generated an arPTE mutant containing seleno-tyrosine. Specifically, seleno tyrosine was introduced at position 309 of wild-type arPTE (note: amino acid 309 of wild-type arPTE corresponds to amino acid 285 of arPTE of the present invention because arPTE expressed by the present invention removes amino acids 1 to 24 of wild-type), and the amino acid sequence of the arPTE mutant is SEQ ID NO: 8, in SEQ ID NO: in 8, the 285 th amino acid is seleno-tyrosine (indicated by "Y" in SEQ ID NO: 8 shown in fig. 5), and the activity of the arPTE mutant is significantly improved as compared with wild-type arPTE.
In summary, the present invention provides the following technical solutions:
1. an orthogonal aminoacyl-tRNA synthetase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, and the amino acid sequence shown in SEQ ID NO: 4 and conservative variants of the amino acid sequence shown in SEQ ID NO: 4, wherein the conservative variant or homologue has a homology with the amino acid sequence set forth in SEQ ID NO: 4, and the same enzyme activity.
2. The orthogonal aminoacyl-tRNA synthetase of claim 1, comprising an amino acid sequence that differs from SEQ ID NO: 4, wherein the homologue has a sequence homology of more than 95% to the amino acid sequence set forth in SEQ ID NO: 4, and the same enzyme activity.
3. The orthogonal aminoacyl-tRNA synthetase of claim 2, comprising an amino acid sequence that is identical to SEQ ID NO: 4, wherein the homologue has a sequence homology of more than 99% with the amino acid sequence shown in SEQ ID NO: 4, and the same enzyme activity.
4. A seleno tyrosine translation system, the system comprising:
(i) seleno-tyrosine;
(ii) an orthogonal aminoacyl-tRNA synthetase of any of items 1-3;
(iii) an orthogonal tRNA comprising SEQ ID NO: 1; wherein the orthogonal aminoacyl-tRNA synthetase preferentially aminoacylates the orthogonal tRNA with the seleno tyrosine; and
(iv) a nucleic acid encoding a protein of interest, wherein the nucleic acid comprises at least one selector codon that is specifically recognized by the orthogonal tRNA.
5. The translation system of claim 4, wherein said orthogonal tRNA is an amber suppressor tRNA, said selector codon is an amber codon, and said translation system further comprises a nucleotide sequence encoding said orthogonal aminoacyl-tRNA synthetase.
6. A host cell comprising a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase of any of claims 1-3, and a corresponding orthogonal tRNA sequence.
7. The host cell according to item 4, wherein the host cell is a eubacterial cell, preferably an E.coli cell.
8. A method of producing a mutant protein having a site-specific insertion of seleno tyrosine at least one selected position, the method comprising the steps of:
(a) providing the seleno tyrosine translation system of item 4, the system comprising:
(i) seleno-tyrosine;
(ii) an orthogonal aminoacyl-tRNA synthetase of any of items 1-3;
(iii) an orthogonal tRNA comprising SEQ ID NO: 1; wherein the orthogonal aminoacyl-tRNA synthetase preferentially aminoacylates the orthogonal tRNA with the seleno tyrosine; and
(iv) a nucleic acid encoding the target protein, wherein the nucleic acid comprises at least one selector codon at a selected position that is specifically recognized by the orthogonal tRNA; and
(b) cloning and transforming the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding the target protein into an appropriate host cell, adding seleno-tyrosine to the culture medium, wherein during translation of the target protein, the seleno-tyrosine aminoacylated orthogonal tRNA recognizes a selector codon on the mRNA encoding the target protein and seleno-tyrosine, thereby site-specifically inserting seleno-tyrosine into an amino acid position corresponding to the selector codon, thereby producing the target protein comprising seleno-tyrosine at the selected position.
9. The method of item 8, wherein the orthogonal tRNA is an amber suppressor tRNA and the selector codon is an amber codon.
10. A method for producing a seleno tyrosine-containing mutant of a target protease using the method of item 8, wherein the nucleic acid sequence encoding the mutant of the target protease comprises a selector codon specifically recognized by the orthogonal tRNA at a selected position, and seleno tyrosine is inserted at a site corresponding to the amino acid position of the selector codon during translation of the target protease, thereby producing a mutant of the target protease containing seleno tyrosine at the selected position.
11. A method for designing and modifying protease by the method of item 10, wherein the method comprises the step of substituting tyrosine on the active site of target protease with seleno-tyrosine by the method of item 10, so as to obtain a target protease mutant containing seleno-tyrosine, thereby obviously improving the enzymatic activity.
12. The selenoyltyrosine-containing protease mutant of interest obtained by the method of claim 11, including, but not limited to, Agrobacterium radiobacter phosphotriesterase, having an amino acid sequence as set forth in SEQ ID NO: shown in fig. 8.
II. Advantageous effects
This study resulted in the screening of an orthogonal aminoacyl-tRNA synthetase, and, based thereon, a seleno-tyrosine translation system was developed. By this system, specific site-directed insertion of seleno-tyrosine (SeHF) into a protease of interest, such as, but not limited to, arPTE, results in an arPTE mutant containing SeHF at position 309 (corresponding to amino acid 285 in SEQ ID NO: 8), which has significantly improved enzymatic activity and K is significantly higher than wild-type arPTEcatIncrease by 12 times, k at pH7.0cat/KmThe increase was 3.2 times. Through MD simulation, this study revealed an mechanism of enhancement of the enzymatic activity of the arpe mutant, i.e., a mechanism of hydrogen bond conversion that opens the product release channel.
Since SeHF and tyrosine differ by only a single atom, replacement of tyrosine by SeHF at the site of enzymatic activity results in only minimal structural changes and steric hindrance. Importantly, the method comprises the following steps: 1. given the high nucleophilicity of SeHF, enzymes containing tyrosine nucleophiles may exhibit higher activity through incorporation of SeHF. 2. Tyrosine pK as a generalized acid/baseaMust be regulated by surrounding residues to facilitate proton extraction/addition at near neutral pH conditions while possessing low pKaSeHF of (5.9) can act as a generalized acid/base at neutral pH without help from surrounding residues, and this unique property can significantly simplify a reasonable computational enzyme design. 3. Since SeHF carries a negative charge at neutral pH, it can exhibit strong cation- π interactions for stabilizing the switching state of the enzyme.
The results of this study indicate that the incorporation of SeHF can be used as a powerful tool to improve the efficiency of enzyme catalysis and as a probe to study tyrosine function in the enzyme active site. In addition, if the introduction of selenium ligand can significantly adjust the oxidation-reduction potential and catalytic performance of metalloenzymes, SeHF is also very useful in the design of metalloenzymes.
The above method can also be used to design and explore proteases with tyrosine residues in the active sites, such as: tyrosine phenol lyase, steroid isomerase, terpene cyclase, ribonucleotide reductase, FtmOx1, galactose oxidase, and the like.
Drawings
The above features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a scheme of the chemical synthesis scheme for seleno-tyrosine (SeHF);
FIG. 2: (A) is an SDS-PAGE electrophoretogram of a seleno-tyrosine-containing phosphotriesterase mutant of Agrobacterium radiobacter (SeHF-arPTE); (B) is a comparison graph of the activity curves of the wild type and 309SeHF mutant PTE enzymes;
FIG. 3 is a mass spectrum of trypsin digested PTE309 SeHF;
FIG. 4: (A) is the pH-Rate Curve (logk) of the hydrolysis of methylprotophos at room temperature by the Wild Type (WT) and the 309SeHF mutant PTEcatTo pH); (B) is the pH-Rate Curve (logk) of the wild type and 309SeHF mutant PTE hydrolysis of methylprotophos at room temperaturecat/KmTo pH); (C) is a graph of the reaction rate of PTE hydrolysis of methyl paraoxon by wild type and 309SeHF mutants under room temperature and neutral conditions (pH7.0, pH7.5);
FIG. 5 is a sequence of an orthogonal tRNA, a wild-type tyrosyl tRNA synthetase, an orthogonal aminoacyl-tRNA synthetase of the invention, and an Agrobacterium radiobacter phosphotriester enzyme mutant.
DESCRIPTION OF THE SEQUENCES
Detailed Description
The invention is further illustrated by the following examples. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.
It will be understood by those skilled in the art that unless otherwise indicated, all of the chemical reagents used in the following examples are commercially available reagents of analytical grade.
Example 1: chemical Synthesis of Seleno tyrosine (SeHF) (FIG. 1)
The method comprises the following steps: synthesis of diethyl 2-acetylamino-2- (4-nitrobenzyl) malonate (Compound 2)
A mixture of 1- (bromomethyl) -4-nitrobenzene (1.0g, 1 equivalent), diethyl 2-acetamidomalonate (1.2g, 1.2 equivalents) and tBuOK (0.63g, 1.2 equivalents) was placed in a round bottom flask containing 20mL EtOH and monitored by TLC for reflux overnight. After completion of the reaction, the mixture was cooled to room temperature, filtered and washed with EtOH. The residue was applied to flash chromatography on silica gel using ethyl acetate/hexane (1: 1 mix) as eluent to give the title compound as a white solid (1.23g, 75.5% yield).
1H NMR(500MHz，CDCl3)δ8.12(d，2H)，7.18(d，2H)，6.56(s，1H)，4.28(q，4H)，3.77(s，2H)，2.04(s，3H)，1.3(t，6H).13C NMR(CDCl3)δ169.40，167.05，147.27，143.15，130.72，123.46，66.86，63.02，37.59，23.02，14.01。
Step two: synthesis of diethyl 2-acetylamino-2- (4-aminobenzyl) malonate (Compound 3)
15mL of a mixture of Compound 2(1.0g, 1 eq) and Pd/C (0.6g, 0.1 eq) in MeOH in hydrogen (4atm) were stirred under TLC monitoring, after completion of the reaction, the mixture was filtered, concentrated under reduced pressure, and flash chromatographed on silica gel using ethyl acetate/hexane (1: 1 mix) as eluent to give the title compound as a white solid (1.0g, 100% yield).
1H NMR(500MHz，CDCl3)δ6.78(d，2H)，6.57(d，2H)，4.25(q，4H)，3.52(s，2H)，2.01(s，3H)，1.28(t，6H).13C NMR(CDCl3)δ168.94，167.67，145.50，130.67，124.78，115.01，67.39，62.50，37.07，23.04，14.02。
Step three: synthesis of diethyl 2-acetylamino-2- (4-selenocyanobenzyl) malonate (Compound 5)
Compound 3(1.0g) and 6N HCl (5mL) were placed in a round bottom flask, cooled to 0 deg.C and NaNO was added dropwise2Solution (0.25g, 1.25 equiv in 5mL H2O in) (note: this step may generate toxic nitrogen-oxygen compounds and present an explosion hazard, necessitating the necessary protective procedures). After 5 minutes, use Na2CO3The pH was adjusted to 6. The mixture was maintained at 0 ℃ and KSeCN (0.5g, 1.1 equiv in 5mL H) was added dropwise2In O). The mixture was then warmed to room temperature and the reaction was continued for 1 hour, and 50mL of water was poured into the reaction mixture. The aqueous layer was extracted 3 times with dichloromethane and the organic layer was purified over anhydrous Na2SO4Drying, filtration, concentration and chromatography on silica gel using ethyl acetate/hexane (1: 2 mix) as eluent gave the title compound (0.55g, 40% yield). 1H NMR (500MHz, CDCl 3). delta.7.54 (d, 2H), 7.05(d, 2H), 6.55(s, 1H), 4.33-4.22(m, 4H), 3.69-3.66(m, 2H), 2.04(s, 3H), 1.3(m, 6H).
Step four: synthesis of 2, 2' - ((Alkylenebis (4, 1-phenylene)) bis (methylene)) bis (2-acetamidomalonate) (Compound 6)
Compound 5(0.5g, 1 eq) was dissolved in 5mL EtOH in a three-neck round-bottom flask under nitrogen, and KOH solution (0.34g, 5 eq) was injected via syringe, followed by stirring at room temperature for 1 hour. 10mL of water was then added, and the precipitated solid product was filtered, washed and dried to give the title compound (0.42g, 90% yield) (note that this step generates toxic KCN compound and the necessary protection and work-up procedures had to be taken).
Step five: synthesis of 3, 3' - (Alkylenedi (4, 1-phenylene)) bis (2-aminopropionic acid) (Compound 7, Seleno-tyrosine)
To a round bottom flask was added compound 6(0.42 g). Concentrated HCl (10mL) was added and the mixture was stirred backFlow 10 hours, monitored by TLC. After the reaction was completed, the solvent was concentrated under reduced pressure by half. The mixture was left to stand overnight in a refrigerator at 4 ℃. The precipitated product was filtered and dried to give the title compound (0.19g, 70% yield). 1H NMR (500MHz, D2O) Δ 7.55(D, 2H), 7.26(D, 2H), 4.10(t, 1H), 3.09(m, 2H). Mass spectrum: m/z 489, 487, 485, [ M + H ]]+。
The chemical reagents required for the above synthesis reaction are purchased from Bailingwei science and technology Co., Ltd, Sigma Aldrich trade Co., Ltd or Beijing chemical plant, all of which are analytically pure or higher, unless otherwise specified. Recording Using a Bruker AMX-600 Instrument1H NMR spectra, chemical shifts are reported in ppm using TMS as an internal standard (TMS, 0.00). Multiplicity is reported as follows: s is singlet, d is doublet, t is triplet, q is quartet, and m is multiplet. Recording at 75.4MHz13C NMR spectrum, and chemical shifts are reported in ppm using deuterated solvents as internal standard (CDCl 3, 77.0).
Example 2: evolved SeHF-specific aminoacyl-tRNA synthetases
Coli host cells used need to be introduced with an aminoacyl-tRNA synthetase/tRNA orthogonal pair derived from Methanococcus jannaschii amber suppressor tyrosyltRNA (MjtRNA)TyrCUA)/tyrosyl tRNA synthetase (MjYRS, wild type, having the amino acid sequence of SEQ ID NO: 2) and (4) carrying out pairing. The MjYRS mutant library was constructed in the kanamycin resistant pBK plasmid (purchased from scrips institute Peter g schultz laboratories, usa) between the promoter and terminator of e.coli glutamine synthetase on this plasmid. The synthetase mutation library is pBk-lib-iw1 library, and the construction method of the mutation library comprises the following steps: NNK mutations (N ═ A + T + C + G; K ═ T + G) were introduced into 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) selected from the MjYRS gene, and the other 6 sites (Ile63, Ala67, His70, Tyr114, Ile159, Val164) were or randomly mutated to Gly or remained unchanged (see Xie, J.; Liu, W.S.; Schultz, P.G.Angew. chem., Int.Ed.2007, 46, 9239-containing 9242; Wang, JY.; Zhang W.; Song WJ; et al.J.Am.Chem.Soc.2010, 132, 12-containing14818)。
aminoacyl-tRNA synthetases that specifically recognize SeHF were evolved by positive and negative screens. Positive selection plasmids contain MjtRNATyr CUAA TAG mutated chloramphenicol acetyl transferase gene, an amber mutated T7RNA polymerase which initiates expression of green fluorescent protein, a tetracycline resistance gene. Negative selection plasmids contain MjtRNATyr CUAAmber mutant bacillus rnase gene under the arabinose operon, and ampicillin resistance gene. And (3) carrying out positive and negative screening: coli DH10B cells containing the positive selection plasmid were used as positive selection host cells. Cells were electroporated into the pbk-lib-jw1 library, SOC Medium (2% (W/V) tryptone, 0.5% (W/V) Yeast powder, 0.05% (W/V) NaCl, 2.5mM KCl, 10mM MgCl220mM glucose) was incubated at 37 ℃ for 1 hour. Then the medium was replaced with minimal medium (formulation of GMML minimal medium: M9 salt/glycerol: 764g Na2HPO4.7H2O or 30g Na2HPO4，15g KH2PO4，2.5g NaCl，5g NH4Cl, 50ml glycerol, autoclaved, pH7.0; 1M MgSO4: sterilizing under high pressure; 50mM CaCl2: sterilizing under high pressure; 25mM FeCl2: filtering and sterilizing; 0.3M leucine: dissolving in 0.3M NaOH, filtering, and sterilizing; 1L liquid GMML medium: 200ml M9 salt/Glycerol, 2ml MgSO4，2ml CaCl2，2ml FeCl21ml leucine) was washed twice, and a solid minimal medium (500 ml of 3% agar powder, 1mM SeHF, 50mg/L kanamycin, 60mg/L chloramphenicol, 15mg/L tetracycline was added to the liquid GMML medium) was plated and cultured at 37 ℃ for 60 hours. Collecting cells, extracting plasmid DNA, performing electrophoretic separation, and recovering gel. The positively selected pBK-lib-jw1 was then transformed into DH10B competent cells containing the negative selection plasmid. Recovery in SOC medium was 1 hour. Plates were then plated containing 0.2% arabinose (purchased from sigma) on LB solid medium (10 g tryptone, 5g yeast powder, 10g NaCl per liter of medium). Culturing at 37 deg.C for 8-12 hr. Repeat 3 rounds in total.
The last round of positive selection picked 384 clones, which were spotted separately on GMML solid medium containing 1mM SeHF, chloramphenicol 60, 80, 120, 160. mu.g/mL, and GMML solid medium not containing SeHF but containing chloramphenicol 0, 20, 40, 60. mu.g/mL. Clones that grew on 1mM SeHF 160. mu.g/mL chloramphenicol but did not grow on 0mM SeHF at concentrations greater than 20. mu.g/mL chloramphenicol were selected for further validation. Finally 1 clone is picked out, seleno-tyrosine is poked in with highest efficiency, and sequencing shows that the amino acid sequence of aminoacyl-tRNA synthetase mutant (SeHFRS) contained in the clone is SEQ ID NO: 4, wherein the mutation sites are as follows: tyr32Leu, Leu65Ser, Asp87Asn, Gly97Arg, Tyr114Ser, Asp158Gly and Leu162 Thr.
It will be appreciated by those skilled in the art that, in the present invention, in addition to SEQ ID NO: 4, the term "orthogonal aminoacyl-tRNA synthetase" or "orthogonal seleno-tyrosine aminoacyl-tRNA synthetase" also includes the amino acid sequences set forth in SEQ ID NOs: 4, provided that the conservative variant has an amino acid sequence that is identical to SEQ ID NO: 4, or an enzyme activity having the same amino acid sequence as shown in 4; and further comprising converting SEQ ID NO: 4 by substitution, deletion or addition of one or more amino acids, and has a sequence identical to that of SEQ ID NO: 4, the enzyme activity of the same amino acid sequence as shown in SEQ ID NO: 4, or a derivative thereof; and further comprising a sequence identical to SEQ ID NO: 4, wherein the sequence homology may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 99% or more, and the homologue has a sequence homology with the amino acid sequence shown in SEQ ID NO: 4, and the same enzyme activity.
Example 3: expression of SeHF-arPTE and identification by mass spectrometry
To determine the efficiency and fidelity of SeHF incorporation into proteins, we replaced Tyr309 with an amber stop codon in ARPTE containing a His6 tag at the C-terminus. In SeHFRS, MjtRNATyr CUAArPTE was expressed by E.coli in the presence of 0.5mM SeHF, while SeHF was not added as a negative control.
The method comprises the following specific steps: orthogonal tRNA (SEQ ID NO: 1) and the selected SeHFRS encoding geneThe nucleotide sequence (SEQ ID NO: 3) was constructed on a pEVOL vector (purchased from the U.S. script institute, Peter G.Schultz laboratory); the nucleotide sequence encoding arpe (SEQ ID NO: 5) was constructed on a pET-22b vector (purchased from novagen), and a Y309TAG mutation was introduced into the arpe-pET 22b by PCR using TransStartFastPfu (purchased from allyaau) DNA polymerase and primer design. Both plasmids were co-transformed into BL21(DE3) cells (purchased from holo-gold). Selecting single clone, culturing at 37 deg.C to OD600About 0.6 deg.C, the temperature was lowered to 30 deg.C and added to TB medium (1.2% tryptone, 2.4% yeast extract, 72mM K)2HPO4，17mM KH2PO4, 0.4% glycerol) was added 0.2mM IPTG, 0.2% arabinose, 0.2mM CoCl2And 0.5mM SeHF, control without SeHF. After 16 hours, the cells were harvested and the proteins were purified by Ni-NTA (from Nanjing Kinseri) followed by Superdex 20010/300 gel filtration column (from GE Healthcare) and finally subjected to SDS-PAGE analysis and LC/MS analysis.
SDS-PAGE showed that the full-length arPTE309SeHF mutant (hereinafter abbreviated as PTE309SeHF) could be purified only in the presence of SeHF (FIG. 2A), indicating that the selected SeHFRS could specifically recognize SeHF.
LC/MS profiling showed that trypsin digested PTE309SeHF showed 93.5% coverage of full length protein, where peptides containing 309SeHF could be well identified (fig. 3), suggesting that we successfully obtained full length arPTE protein containing SeHF 309.
Sequencing shows that the amino acid sequence of the wild-type arPTE is shown as SEQ ID NO: 6 (corresponding to amino acid sequences 25-365 of the full-length wild type), the amino acid sequence of the arPTE309SeHF mutant is shown as SEQ ID NO: 8, wherein amino acid 285 (i.e., position 309 corresponding to the full length wild type) is replaced by seleno tyrosine (figure 5).
Example 4: PTE enzyme activity assay
To determine whether SeHF could be used to enhance enzyme activity, we assayed the catalytic activity of wild-type (WT for short) and 309SeHF mutant PTE. Since SeHF and tyrosine are structurally similar, the inserted 309SeHF should not significantly affect the PTE structure.
At room temperature, when the concentration of the substrate methylparatophosphonium (purchased from Sigma, CAS #: 950-35-6) was gradually increased from 12.5. mu.M to 1mM, we monitored the product 4-nitrophenol at 347nm (. epsilon.) (ε)347＝5176M-1cm-1) UV-Vis spectra of (A) to determine PTE activity. The data were then fitted to the Mie equation to calculate the kinetic constant kcatAnd KmPlotting the logk of WT and 309SeHF mutant PTE, respectivelycatVersus pH curve (pH-rate curve, fig. 4A). The WT and 309SeHF mutant PTE have the same pK at pH 4.7e/pKesThe value is obtained. However, at pH 6.0, only the PTE309SeHF mutant showed the second pKes. According to the curve (fig. 4B), the pH-rate curve of the WT PTE reached the plateau at pH 5.0, while the pH-rate curve of the 309SeHF mutant reached the plateau at pH 6.5, reflecting deprotonation of the SeHF side chain.
To further validate the relationship between deprotonation of SeHF and increased enzyme activity, we compared the reaction rates of WT and 309SeHF mutant PTE at near neutral pH (pH7.0-pH 7.5). As shown in fig. 4C, the 309SeHF mutant exhibited a significantly accelerated enzyme reaction rate. The 309SeHF mutant has a 12-fold higher k than WT PTE at pH7.0catValues indicating that the 309SeHF mutant has a faster rate of product release compared to WT PTE, while demonstrating that 309SeHF participates in a key rate-limiting step of product release. KmIncreasing the representation k1Reduced, or less efficient, binding of the michaelis complex (ES) formed, further demonstrating that 309SeHF is involved in the formation of the michaelis complex.
Overall, the 309SeHF mutation resulted in a K for PTE at pH7cat/KmThe increase was 3.2 times. Although the negative charge and increased van der waals radius of SeHF lead to a decrease in substrate binding affinity, SeHF can significantly accelerate the product release step, ultimately resulting in a greatly increased enzymatic activity of PTE under neutral pH conditions.
It should be understood that while the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein, and any combination of the various embodiments may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
Claims (10)
1. An orthogonal aminoacyl-tRNA synthetase, whose amino acid sequence is SEQ ID NO 4.
2. A seleno tyrosine translation system, the system comprising:
(i) seleno-tyrosine;
(ii) the orthogonal aminoacyl-tRNA synthetase of claim 1;
(iii) an orthogonal tRNA comprising the polynucleotide sequence shown in SEQ ID NO. 1; wherein the orthogonal aminoacyl-tRNA synthetase preferentially aminoacylates the orthogonal tRNA with the seleno tyrosine; and
(iv) a nucleic acid encoding a protein of interest, wherein the nucleic acid comprises at least one selector codon that is specifically recognized by the orthogonal tRNA.
3. The translation system of claim 2, wherein said orthogonal tRNA is an amber suppressor tRNA, said selector codon is an amber codon, and said translation system further comprises a nucleotide sequence encoding said orthogonal aminoacyl-tRNA synthetase.
4. A host cell comprising a nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase of claim 1, and a corresponding orthogonal tRNA sequence.
5. The host cell of claim 4, wherein the host cell is a eubacterial cell.
6. The host cell of claim 4, wherein the host cell is an E.
7. A method of producing a mutant protein having a site-specific insertion of seleno tyrosine at least one selected position, the method comprising the steps of:
(a) providing a seleno tyrosine translation system of claim 2,
and
(b) cloning and transforming the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase, and the nucleic acid sequence encoding the target protein into an appropriate host cell, adding seleno-tyrosine to the culture medium, wherein during translation of the target protein, the seleno-tyrosine aminoacylated orthogonal tRNA recognizes a selector codon on the mRNA encoding the target protein and seleno-tyrosine, thereby site-specifically inserting seleno-tyrosine into an amino acid position corresponding to the selector codon, thereby producing the target protein comprising seleno-tyrosine at the selected position.
8. The method of claim 7, wherein the orthogonal tRNA is an amber suppressor tRNA and the selector codon is an amber codon.
9. A method of producing a seleno tyrosine-containing mutant of a target protease using the method of claim 7, wherein the nucleic acid sequence encoding the mutant of the target protease used comprises a selector codon specifically recognized by the orthogonal tRNA at a selected position, and seleno tyrosine is inserted at a site-specific position into an amino acid position corresponding to the selector codon during translation of the target protease, thereby producing a mutant of the target protease containing seleno tyrosine at the selected position.
10. A method for designing and modifying protease by the method of claim 9, which comprises substituting tyrosine in the active site of target protease with seleno-tyrosine by the method of claim 9, thereby obtaining a target protease mutant containing seleno-tyrosine, and thus significantly improving the enzymatic activity.
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