CN104004723B - 3,5-bis-fluoro tyrosine translation system and application thereof - Google Patents

Abstract

The present invention relates to an aminoacyl-tRNA synthetase mutant, which is an orthogonal aminoacyl-tRNA synthetase, which contains an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: 4 A group consisting of conservative variants of the amino acid sequence shown, the conservative variants have the same enzymatic activity as the amino acid sequence shown in SEQ ID NO:4. The present invention also provides a 3,5-difluorotyrosine translation system, which comprises: (i) 3,5-difluorotyrosine; (ii) the orthogonal aminoacyl-tRNA synthetase of the present invention (iii) an orthogonal tRNA, wherein said orthogonal aminoacyl-tRNA synthetase preferentially aminoacylates said orthogonal tRNA with said 3,5-difluorotyrosine; and (iv) an encoding target protein A nucleic acid, wherein said nucleic acid contains at least one selector codon specifically recognized by said orthogonal tRNA.

Description

3,5-Difluorotyrosine translation system and its application

technical field

The invention belongs to the field of biochemistry. Specifically, the present invention provides an aminoacyl-tRNA synthetase mutant, which is an orthogonal aminoacyl-tRNA synthetase, which contains an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID A group consisting of conservative variants of the amino acid sequence shown in NO:4, the conservative variants having the same enzymatic activity as the amino acid sequence shown in SEQID NO:4. The present invention also relates to a 3,5-difluorotyrosine (abbreviated as F2Y) translation system. More specifically, the present invention relates to site-specific insertion of 3,5-difluorotyrosine into 3,5-difluorotyrosine of a target protein using a pair of orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase A translation system, and a method for site-specifically inserting 3,5-difluorotyrosine into a target protein using the translation system. The invention also relates to mutant proteins containing 3,5-difluorotyrosine produced by this translation system and this method, for example, prokaryotic protein tyrosine kinases with 3,5-difluorotyrosine inserted mutants, and the use of mutant proteins inserted with 3,5-difluorotyrosine.

Background technique

Protein phosphorylation is necessary for the initiation of many biological phenomena, including cell growth, proliferation, ubiquitin-mediated protein degradation and other processes. Especially tyrosine phosphorylation, as a main way of cell signal transduction and enzyme activity regulation, usually triggers the interaction between proteins, and then mediates the growth factors, hormones and cytokines on the receptors on the cell membrane. Signal regulation. However, tyrosine phosphorylation accounts for a very low percentage of all phosphorylation modifications in the cell. About 10% of cellular proteins are covalently modified by phosphorylation, but only one tyrosine group is modified in every 100 protein phosphorylation modifications. The level of tyrosine phosphorylation is estimated to be 2000-fold lower than that of serine and threonine phosphorylation in most cells. It is precisely because the level of tyrosine phosphorylation in cells is quite low that cells can respond sensitively to the stimulation of internal and external signals, so the study of tyrosine phosphorylation on the regulation of cell signals and the research of many important biological phenomena It is of great significance, and the identification of proteins that undergo tyrosine phosphorylation and the identification of phosphorylation sites play a very important role in revealing the regulation of cellular processes and the site of action of drugs.

¹⁹ F-NMR technology has its special significance in the analysis of optical isomers and life sciences since its appearance in the 1950s due to its advantages such as large chemical shift range, low peak overlap and high sensitivity. In vivo NMR technology, compared with other methods, ¹⁹ F-NMR can perform real-time quantitative monitoring without damaging the sample. In the past 30 years, the introduction of fluorinated indicators for in vivo ¹⁹ F-NMR research, the research of fluorine-containing bioactive substances and fluorine-containing materials have received great attention, and ¹⁹ F-NMR technology and its applications have been developed. Judging from the situation in the past 10 years, the research on fluorine-containing bioactive substances and the research and application of fluorine-containing materials with excellent properties have received great attention. This is a major focus of recent ¹⁹ F-NMR research and development. features.

According to previous reports, 3,5-difluorotyrosine-containing peptides as substrates of protein tyrosine kinases exhibited similar efficiencies as the corresponding tyrosine-containing peptides as substrates, And it will not significantly affect the catalytic activity of protein tyrosine phosphatases. Therefore, the purpose of this study is to replace tyrosine with the unnatural amino acid 3,5-difluorotyrosine through the expansion of the genetic code, so that it can be used as a fluorine indicator, so that tyrosine can be detected and quantified by ¹⁹ F-NMR technology. Amino acid phosphorylation levels. Meanwhile, this study has now developed a general method for the site-specific in vivo site-specific insertion of various unnatural amino acids into proteins in both prokaryotes and eukaryotes. These methods rely on orthogonal protein translation components that recognize appropriate selector codons to enable insertion of desired unnatural amino acids 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) loads 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 (ie, it must be orthogonal). Utilizing this orthogonal tRNA-RS pairing may genetically encode a large number of structurally diverse unnatural amino acids.

The use of orthogonal translation systems suitable for making proteins containing one or more unnatural amino acids, eg, general methods for generating orthogonal translation systems, is generally known in the art. For example, see International Publication No. WO 2002/086075, whose invention title is "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS"; WO 2002/085923, whose invention title is "IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS" ; WO 2004/094593, whose invention title is "EXPANDINGTHE EUKARYOTIC GENETIC CODE". For additional discussions of orthogonal translation systems for site-specific insertion of unnatural amino acids and methods for their production and use see also 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 in Chemical Biology 9(6): 548-554 (2005); Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35:225-249 (2006).

Contents of the invention

1. Technical issues

The present invention provides an aminoacyl-tRNA synthetase mutant, which is an orthogonal aminoacyl-tRNA synthetase, which contains an amino acid sequence selected from amino acids shown in SEQ ID NO: 4 and shown in SEQ ID NO: 4 A group consisting of conservative variants of the amino acid sequence, said conservative variants have the same enzymatic activity as the amino acid sequence shown in SEQ ID NO:4. This aminoacyl-tRNA synthetase mutant can preferentially aminoacylate the paired orthogonal tRNA with 3,5-difluorotyrosine (abbreviated as F2Y), thereby inserting F2Y in the translated amino acid sequence. This was discovered by the inventors for the first time, and at the same time, the inventors also analyzed its high-resolution crystal structure, and accordingly, it is named as orthogonal 3,5-difluorotyrosine aminoacyl in the present invention - tRNA synthetase (F2YRS).

On the basis of the above findings, the present invention provides a 3,5-difluorotyrosine that uses the pairing of an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase to specifically insert 3,5-difluorotyrosine into a target protein. A fluorotyrosine translation system, and a method for site-specific insertion of 3,5-difluorotyrosine in a target protein using the translation system. The present invention also relates to the mutant protein containing 3,5-difluorotyrosine produced by this translation system and this method and its application.

Therefore, the object of the present invention is to provide a 3,5-difluorotyrosine that utilizes the pairing of an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase to insert 3,5-difluorotyrosine into a protein site-specifically. A translation system and a method for site-specific insertion of 3,5-difluorotyrosine in a target protein by using the translation system are provided.

The present invention also provides muteins containing at least one 3,5-difluorotyrosine produced by using the 3,5-difluorotyrosine translation system of the present invention. In a preferred aspect of the invention, the inventors have utilized this method to site-specifically insert 3,5-difluorotyrosine into proteins of interest, including, but not limited to, prokaryotic protein tyrosine kinases Etk. The prokaryotic protein tyrosine kinase mutant protein containing 3,5-difluorotyrosine obtained by the method of the present invention can be used as a fluorine indicator to detect and quantify tyrosine phosphorylation by ¹⁹ F-NMR technology level. However, those skilled in the art should understand that the method of the present invention can also be used for site-specific insertion of 3,5-difluorotyrosine in various proteins other than protein tyrosine kinases, and is not limited to this protein. 2. Technical solution

After screening, the present inventors obtained an orthogonal aminoacyl-tRNA synthetase mutant, which is an orthogonal aminoacyl-tRNA synthetase, which contains an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 4 and A group consisting of conservative variants of the amino acid sequence shown in SEQ ID NO: 4, which has the same enzymatic activity as the amino acid sequence shown in SEQ ID NO: 4, which is named in the present invention Orthogonal 3,5-difluorotyrosine aminoacyl-tRNA synthetase (F2YRS). At the same time, the inventors also analyzed its high-resolution crystal structure. And, the present inventors developed a 3,5-difluorotyrosine translation system using the orthogonal aminoacyl-tRNA synthetase.

Those skilled in the art should understand that in the present invention, in addition to the amino acid sequence shown in SEQ ID NO: 4, the term "orthogonal aminoacyl-tRNA synthetase of the present invention" or "orthogonal 3,5-difluoro Substituting tyrosine aminoacyl-tRNA synthetase" also includes conservative variants of the amino acid sequence shown in SEQ ID NO: 4, as long as the conservative variant has the same enzymatic activity as the amino acid sequence shown in SEQ ID NO: 4 and also include the amino acid sequence shown in SEQ ID NO: 4 through one or more amino acid substitutions, deletions or additions and having the same enzymatic activity as the amino acid sequence shown in SEQ ID NO: 4 by SEQ ID The amino acid sequence derived from the amino acid sequence shown in NO:4.

Furthermore, the nucleotide sequence encoding the orthogonal 3,5-difluorotyrosine aminoacyl-tRNA synthetase (F2YRS) of the present invention is also included in the scope of the present invention. Preferably, the coding nucleotide sequence is shown in SEQ ID NO:3.

Specifically, the present invention provides in vivo (for example, in a host cell) recognition of a selector codon (selector codon) such as an amber stop codon (TAG) so as to site-specifically insert the unnatural amino acid 3,5-difluorotyrosine 3,5-difluorotyrosine translation system into the polypeptide chain. The 3,5-difluorotyrosine translation system comprises an orthogonal-tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS) pair that does not interact with the host cell translation machinery. That is, the host cell's endogenous aminoacyl-tRNA synthetase does not recognize the O-tRNA. Similarly, the O-RS provided herein do not recognize endogenous tRNAs at a significant level or in some cases not at a detectable level. The use of the translation system enables the production of large quantities of proteins with site-specific insertion of 3,5-difluorotyrosine during translation.

In some aspects, the invention provides 3,5-difluorotyrosine translation systems. The translation system includes:

(a) an unnatural amino acid, namely 3,5-difluorotyrosine,

(b) an orthogonal aminoacyl-tRNA synthetase (O-RS) of the invention, and

(c) Orthogonal tRNA (O-tRNA), which comprises the polynucleotide sequence shown in SEQ ID NO: 1, wherein the orthogonal aminoacyl-tRNA synthetase uses the unnatural amino acid (ie 3,5- difluorotyrosine), preferentially aminoacylates the O-tRNA.

Preferably, the 3,5-difluorotyrosine translation system of the present invention further comprises a nucleic acid encoding a target protein, wherein the nucleic acid contains at least one selector codon specifically recognized by an orthogonal tRNA (O-tRNA), Amber codons are preferred. More preferably, the 3,5-difluorotyrosine translation system of the present invention further comprises a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase.

The orthogonal aminoacyl-tRNA synthetase (O-RS) used in the system is the aminoacyl tRNA synthetase mutant discovered for the first time by the inventors, and the amino acid sequence it contains is selected from the amino acids shown in SEQ ID NO: 4 sequence and a conservative variant of the amino acid sequence shown in SEQ ID NO: 4, and the conservative variant has the same enzymatic activity as the amino acid sequence shown in SEQ ID NO: 4.

In a preferred aspect of the present invention, the present invention provides a 3,5-difluorotyrosine translation system, said system comprising:

(i) 3,5-difluorotyrosine;

(ii) an orthogonal aminoacyl-tRNA synthetase of the invention;

(iii) an orthogonal tRNA comprising the polynucleotide sequence shown in SEQ ID NO: 1; wherein said orthogonal aminoacyl-tRNA synthetase is preferentially aminoacylated with said 3,5-difluorotyrosine said orthogonal tRNA; and

(iv) a nucleic acid encoding a protein of interest, wherein said nucleic acid contains at least one selector codon specifically recognized by said orthogonal tRNA.

Preferably, the 3,5-difluorotyrosine translation system further comprises a nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase of the present invention.

The various components of the translation system can be derived from various species sources, for example, the components of the translation system are derived from Methanococcus jannaschii. For example, an orthogonal tRNA (O-tRNA) is an anticodon of archaeal origin mutated into a tyrosine tRNA complementary to the amber codon. In some embodiments, the O-tRNA is an amber suppressor tRNA. In some embodiments, the O-tRNA comprises the polynucleotide sequence shown in SEQ ID NO: 1, preferably, the sequence of the O-tRNA is shown in SEQ ID NO: 1. In one embodiment, the orthogonal aminoacyl-tRNA synthetase used in the system may comprise the amino acid sequence shown in 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 shown in SEQ ID NO:4.

In some aspects, the 3,5-difluorotyrosine 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 specifically recognized by an orthogonal tRNA (O-tRNA) son. 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 a host cell 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 as 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 present invention also provides methods for producing muteins with site-specific insertion of 3,5-difluorotyrosine at at least one selected position. The method utilizes the 3,5-difluorotyrosine translation system described above. The method generally includes the steps of:

(a) the step of providing a 3,5-difluorotyrosine translation system comprising:

(i) an unnatural amino acid, namely 3,5-difluorotyrosine;

(ii) Orthogonal aminoacyl-tRNA synthetase (O-RS) of the present invention;

(iii) Orthogonal tRNA (O-tRNA), which comprises the polynucleotide sequence shown in SEQ ID NO: 1, wherein the O-RS is preferentially aminoacylated with 3,5-difluorotyrosine O-tRNA; and

(iv) a nucleic acid encoding a protein of interest, wherein the nucleic acid contains 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, in a culture medium Adding 3,5-difluorotyrosine, during translation of the target protein, 3,5-difluorotyrosine aminoacylated orthogonal RNA recognizes selection on the mRNA encoding the target protein codon and 3,5-difluorotyrosine, thereby mediating the site-specific insertion of 3,5-difluorotyrosine into the amino acid position corresponding to the selector codon, thereby generating the selected position containing 3, Mutant proteins of 5-difluorotyrosine.

Those skilled in the art should understand that construction of appropriate recombinant vectors and screening of host cells can be achieved by conventional molecular cloning and screening techniques.

Those skilled in the art should understand that in step (b), the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase and the nucleic acid sequence encoding the target protein are cloned and transformed into Into a suitable host cell can be carried out in a variety of ways, for example, the orthogonal tRNA sequence, the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase and the nucleic acid sequence encoding the target protein can be respectively operably linked to an appropriate vector, and then transformed into an appropriate host cell in any order or jointly by the three; or, the orthogonal tRNA sequence and the orthogonal aminoacyl-tRNA synthetase encoding The nucleotide sequence is operably linked into an appropriate vector (with or without a suitable linker between the two sequences), and the nucleic acid sequence encoding the target protein is operably linked to another different vector. An appropriate vector, and then co-transform the two constructed recombinant vectors into an appropriate host cell; or, the orthogonal tRNA sequence and the nucleic acid sequence encoding the target protein can also be operably linked to a In a suitable vector (with or without suitable linker ligation between the two sequences), the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase is operably linked to another different suitable vector , and then co-transform the constructed two recombinant vectors into appropriate host cells. Alternatively, the orthogonal tRNA sequence and the nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase and the nucleic acid sequence encoding the target protein can also be operably linked together in any appropriate order, and then cloned into a vector and finally transformed into an appropriate host cell. The above cloning schemes are all feasible, and those skilled in the art can easily make appropriate selections according to the needs of experiments.

In addition, those skilled in the art should also understand that, in order to avoid the "kick-out" effect of host cells on foreign recombinant vectors, vectors with different antibiotic markers are often selected to construct nucleic acid sequence fragments that need to be co-transformed into the same host cell. Selection of appropriate vectors, construction of recombinant vectors, transformation or transfection of host cells, etc. are all routine techniques in the art, for example, refer to the Molecular Cloning Handbook published by Cold Spring Harbor Laboratory, USA.

In some embodiments of the method, the step of providing a translation system comprises mutating the amino acid binding pocket of wild-type aminoacyl-tRNA synthetase by site-directed mutagenesis, selected for use with said unnatural amino acid (i.e., 3,5-di fluorotyrosine) preferentially aminoacylates the O-tRNA aminoacyl-tRNA synthetase mutant (ie, the orthogonal aminoacyl-tRNA synthetase used in the present invention). The selection step includes positive selection and negative selection of the O-RS from the obtained library of aminoacyl-tRNA synthetase molecules after site-directed mutagenesis (see Example 2 below). In some embodiments, the step of providing a translation system further includes providing the sequence of an O-tRNA, the O-tRNA is an archaeal-derived anticodon mutated into a tyrosine tRNA complementary to an amber codon, for example, the O-tRNA It is an amber suppressor tRNA, or the O-tRNA comprises the polynucleotide sequence shown in 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.

The method of producing a 3,5-difluorotyrosine-containing mutein can also be performed in a host cell. In these cases, host cells are provided comprising a 3,5-difluorotyrosine translation system of the invention (i.e., comprising a nucleotide sequence encoding an O-RS of the invention, an O-tRNA sequence and containing at least one Nucleic acid encoding the target protein that selects codons), and culturing the host cell under appropriate culture conditions (for example, adding 3,5-difluorotyrosine to the medium, etc.) can result in Site-specific insertion of 3,5-difluorotyrosine. In some embodiments, the providing step comprises providing a eubacterial host cell (eg, E. coli).

The present invention also provides a method for producing a 3,5-difluorotyrosine-containing tyrosine kinase mutant, which uses the above-mentioned production method for site-specific insertion of 3,5-difluorotyrosine at at least one selected position. A method for mutating a protein, wherein the nucleic acid sequence encoding a prokaryotic protein tyrosine kinase mutant comprises at a selected position a selector codon specifically recognized by said orthogonal tRNA, during translation of the tyrosine kinase, 3 , 5-difluorotyrosine is site-specifically inserted into the amino acid position corresponding to the selector codon, thereby generating a tyrosine kinase mutant containing 3,5-difluorotyrosine at the selected position.

Preferably, the present invention also provides a method for producing a prokaryotic protein tyrosine kinase mutant containing 3,5-difluorotyrosine, which method is carried out using the above-mentioned 3,5-difluorotyrosine translation system, The method generally includes the steps of:

(a) the step of providing a 3,5-difluorotyrosine translation system comprising:

(i) 3,5-difluorotyrosine;

(ii) Orthogonal aminoacyl-tRNA synthetase (O-RS);

(iii) Orthogonal tRNA (O-tRNA), which comprises the polynucleotide sequence shown in SEQ ID NO: 1, wherein the O-RS is preferentially aminoacylated with the 3,5-difluorotyrosine the O-tRNA; and

(iv) nucleic acid encoding said prokaryotic protein tyrosine kinase, such as, but not limited to, SEQ ID NO: 7, wherein said nucleic acid contains at least one selector codon specifically recognized by said O-tRNA (optionally is the amber codon);

(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, in a culture medium Addition of 3,5-difluorotyrosine, an orthogonal RNA recognition code for aminoacylation of 3,5-difluorotyrosine during translation of the target protein (i.e. prokaryotic protein tyrosine kinase) A selector codon and 3,5-difluorotyrosine on the mRNA of tyrosine kinase, thereby mediating the site-specific insertion of 3,5-difluorotyrosine into the specific position of the target protein (i.e., the Select the amino acid position corresponding to the codon).

The present invention also provides a prokaryotic protein tyrosine kinase mutant containing 3,5-difluorotyrosine as a fluorinated indicator produced by the 3,5-difluorotyrosine translation system of the present invention. 3,5-difluorotyrosine is introduced into position 574 of the wild-type prokaryotic protein tyrosine kinase, and the amino acid sequence of the tyrosine kinase mutant is SEQ ID NO:8. The kinase mutant, like the wild-type tyrosine kinase, can undergo autophosphorylation of active center tyrosine under suitable conditions, thereby activating its own protein kinase activity.

The present invention also provides a novel method for identifying a tyrosine phosphorylation site of a target protein, the method comprising utilizing the 3,5-difluorotyrosine translation system of the present invention to putatively Phosphorylated tyrosine is replaced by 3,5-difluorotyrosine, thereby obtaining a target protein mutant containing 3,5-difluorotyrosine, which is used as a fluorine indicator , using ¹⁹ F-NMR technology to detect the phosphorylation of 3,5-difluorotyrosine, so as to identify whether the tyrosine site is a tyrosine phosphorylation site.

For example, prokaryotic protein tyrosine kinases are known to have a low degree of autophosphorylation ability, and there is currently no suitable technology to detect and quantify their autophosphorylation level. However, using the 3,5-difluorotyrosine translation system of the present invention to replace the 574-position tyrosine residue in the active center of the prokaryotic protein tyrosine kinase with 3,5-difluorotyrosine, to obtain a protein containing 3 , a prokaryotic protein tyrosine kinase mutant of 5-difluorotyrosine, and then detect and quantify the self-tyrosine phosphorylation level of the prokaryotic protein tyrosine kinase mutant by ¹⁹ F-NMR technology. The tyrosine phosphorylation level of the prokaryotic protein tyrosine kinase mutant can represent the autophosphorylation level of the wild type prokaryotic protein tyrosine kinase to a certain extent. In addition, this detection result also proves that the 574th tyrosine in the active center of the prokaryotic protein tyrosine kinase is its own tyrosine phosphorylation site.

Accordingly, the present invention provides the following:

1. An orthogonal aminoacyl-tRNA synthetase, which contains an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 4 and the conservative variant of the amino acid sequence shown in SEQ ID NO: 4, the The conservative variant has the same enzymatic activity as the amino acid sequence shown in SEQ ID NO:4.

2. A 3,5-difluorotyrosine translation system, said system comprising:

(i) 3,5-difluorotyrosine;

(ii) the orthogonal aminoacyl-tRNA synthetase described in item 1;

(iii) an orthogonal tRNA comprising the polynucleotide sequence shown in SEQ ID NO: 1; wherein said orthogonal aminoacyl-tRNA synthetase is preferentially aminoacylated with said 3,5-difluorotyrosine said orthogonal tRNA; and

(iv) a nucleic acid encoding a protein of interest, wherein said nucleic acid contains at least one selector codon specifically recognized by said orthogonal tRNA.

3. The translation system according to item 2, wherein the orthogonal tRNA is an amber suppressor tRNA, the selector codon is an amber codon, and the translation system further comprises Nucleotide sequence of acyl-tRNA synthetase.

4. A host cell comprising a nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase described in item 1 and a corresponding orthogonal tRNA sequence.

5. The host cell according to item 4, wherein the host cell is a eubacterial cell, preferably an E. coli cell.

6. A method of producing a mutein with site-specific insertion of 3,5-difluorotyrosine at at least one selected position, said method comprising the steps of:

(a) providing the 3,5-difluorotyrosine translation system described in item 2, which system comprises:

(i) 3,5-difluorotyrosine;

(ii) the orthogonal aminoacyl-tRNA synthetase described in item 1;

(iii) an orthogonal tRNA comprising the polynucleotide sequence shown in SEQ ID NO: 1; wherein said orthogonal aminoacyl-tRNA synthetase is preferentially aminoacylated with said 3,5-difluorotyrosine said orthogonal tRNA; and

(iv) a nucleic acid encoding said protein of interest, wherein said nucleic acid comprises at a selected position at least one selector codon specifically recognized by said 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, in a culture medium Addition of 3,5-difluorotyrosine, 3,5-difluorotyrosine aminoacylated orthogonal tRNA recognizes the selector codon on the mRNA encoding the target protein during translation of the target protein codon and 3,5-difluorotyrosine, thereby mediating the site-specific insertion of 3,5-difluorotyrosine into the amino acid position corresponding to the selector codon, thereby generating a codon containing 3,5 at the selected position - said target protein of difluorotyrosine.

7. The method of item 6, wherein said orthogonal tRNA is an amber suppressor tRNA and said selector codon is an amber codon.

8. A method for producing a prokaryotic protein tyrosine kinase mutant containing 3,5-difluorotyrosine, which utilizes the method described in item 6, wherein the nucleic acid encoding the prokaryotic protein tyrosine kinase mutant used The sequence contains a selector codon specifically recognized by the orthogonal tRNA at a selected position, and during translation of the kinase, 3,5-difluorotyrosine is site-specifically inserted into the amino acid position corresponding to the selector codon, thereby Generation of prokaryotic protein tyrosine kinase mutants containing 3,5-difluorotyrosine at selected positions.

9. The prokaryotic protein tyrosine kinase mutant containing 3,5-difluorotyrosine obtained by the method described in item 8, the amino acid sequence of which is SEQ ID NO:8.

10. A method for identifying a tyrosine phosphorylation site of a target protein, said method comprising substituting 3,5-difluoro tyrosine presumed to be phosphorylated in said target protein using the method of item 6 Substituting tyrosine, thereby obtaining a target protein mutant containing 3,5-difluorotyrosine, using the target protein mutant as a fluorine indicator, and detecting 3,5-difluorotyrosine by ¹⁹ F-NMR technology Phosphorylation of fluorotyrosine, thereby identifying whether the tyrosine site is a tyrosine phosphorylation site.

11. A nucleotide sequence encoding the orthogonal aminoacyl-tRNA synthetase of item 1.

13. The nucleotide sequence according to item 11, which is SEQ ID NO:3.

3. Beneficial effects

In this study, an orthogonal aminoacyl-tRNA synthetase was obtained through screening, and a 3,5-difluorotyrosine translation system was developed on this basis. Through this system, 3,5-difluorotyrosine can be specifically inserted into the target protein—for example, prokaryotic protein tyrosine kinase Etk, to generate a tyrosine containing 3,5-difluorotyrosine at position 574 The tyrosine kinase mutant was used as a fluorinated indicator, so that the tyrosine phosphorylation level could be detected and quantified by ¹⁹ F-NMR technology.

Our new method for detecting tyrosine phosphorylation levels has many advantages over traditional methods. First, the level of tyrosine phosphorylation at a specific site of the target protein can be directly detected and quantified. Second, samples can be frozen at any time prior to ¹⁹ FNMR spectroscopic analysis, which minimizes sample preparation time for more precise quantification of phosphorylation levels in protein samples. Third, due to the high sensitivity of ^19F , the chemical shift anisotropy relative to the environment, and the lack of direct ^19F - ^19F coupling, single-site integration of ^19F combined with NMR analysis can be more effectively used to elucidate the dynamic properties of proteins. Finally, NMR studies may help to reveal the motility properties of the active and inactive states of eukaryotic and prokaryotic protein tyrosine kinases, as well as the dynamic exchange between these states. These unique advantages allowed us to obtain direct evidence to demonstrate and quantify the phosphorylation level of the active site of the prokaryotic protein tyrosine kinase Etk, which was not possible before. Moreover, the method for monitoring the activation and activity of tyrosine kinase has high sensitivity and strong selectivity, and can provide an effective way for screening new inhibitors of tyrosine kinase/substrate protein interaction.

Description of drawings

From the following detailed description in conjunction with the accompanying drawings, the above-mentioned features and advantages of the present invention will be more apparent, wherein:

Fig. 1 is the crystal structure diagram of orthogonal 3,5-difluorotyrosine aminoacyl-tRNA synthetase (F2YRS) of the present invention;

Figure 2: Figure 2A is the acid urea polyacrylamide gel electrophoresis image of in vitro aminoacylation, Figure 2B is the SDS-PAGE electrophoresis image of F2Y-GFP, Figure 2C and Figure 2D are the mass spectra of F2Y-GFP picture;

Figure 3 is the ¹⁹ F-NMR spectrum of 3,5-difluorotyrosine (F2Y) (lower figure) and o-phospho-3,5-difluorotyrosine (pF2Y) (upper figure);

Figure 4: Figure 4A is the SDS-PAGE electrophoresis of wild-type prokaryotic protein tyrosine kinase (Etk, lane 1) and Etk-574-F2Y (lane 2), and Figure 4B is Etk-574-F2Y and tyrosine phosphate The Western detection chart of the enzyme PTP1B (the upper picture is the pTyr antibody, the lower picture is the His antibody), Figure 4C is the ¹⁹ F-NMR spectrum of Etk-574-F2Y phosphorylation, and Figure 4D is the Etk-574-F2Y dephosphorylated ¹⁹ F-NMR spectrum;

Fig. 5 is orthogonal tRNA, wild type tyrosyl tRNA synthetase, orthogonal aminoacyl-tRNA synthetase of the present invention, green fluorescent protein mutant, prokaryotic protein tyrosine kinase mutant and protein tyrosine phosphatase sequence.

detailed description

The present invention is further illustrated by the following examples. It should be understood, however, that the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the invention.

It should be understood by those skilled in the art that, unless otherwise specified, the chemical reagents used in the following examples are commercially available reagents of analytical grade.

Embodiment 1: The chemical synthesis of o-phospho-3,5-difluorotyrosine (pF2Y)

Take 100mM phosphorus oxychloride, 100mM 3,5-difluorotyrosine (purchased from Shanghai Jier Biochemical Company) and 500mM sodium hydroxide, dissolve to 5mL, and then stir at room temperature for 1h. The aqueous phase was collected and separated and purified by HPLC to obtain a white powder with a yield of 10%. (YMC AA12S052503WT column, 12ml/min flow rate, from10%to90%CH3CN, 0.1%TFA(w/v) in water, over the course of 30 min).MS: m/z: 298[M+H]+; 1H-NMR (600 MHz, D2O): 7.03 (d, 2H) 4.01 (dd, 1H) 3.21 (m, 2H).

Unless otherwise specified, the chemical reagents required for the above synthesis reactions were purchased from Beijing Chemical Plant and were of analytical grade or above.

Example 2: Evolution of F2Y-specific aminoacyl-tRNA synthetases

In order to site-specifically insert F2Y into the gene, it is necessary to introduce an aminoacyl-tRNA synthetase/tRNA orthogonal pair in the E.coli host cell used. Aminoacyl tRNA (MjtRNA _CUA ^Y )/tyrosyl tRNA synthetase (MjYRS, wild type, its amino acid sequence is SEQ ID NO: 2) pair. The MjYRS mutation library was constructed in the kanamycin-resistant pBK plasmid (purchased from the PeterG. Schultz laboratory of Scripps Research Institute, USA), and located between the promoter and terminator of E. coli glutamine synthetase on the plasmid. The synthetic enzyme mutant library used is the pBk-lib-jw1 library, and the construction method of the mutant library is: select 6 sites (Y32, Leu65, Phe108, Gln109, Asp158, and Leu162) on the MjYRS gene to introduce NNK mutation ( N=A+T+C+G; K=T+G), the other 6 sites (Ile63, Ala67, His70, Y114, Ile159, Vall64) were either randomly mutated to Gly or remained unchanged (see Xie, J. ; Liu, WS; Schultz, PGAngew.Chem., Int.Ed.2007, 46, 9239-9242; Wang, JY.; Zhang W.; Song WJ; et al.J.Am.Chem.Soc.2010, 132 , 14812-14818).

Aminoacyl-tRNA synthetases that specifically recognize F2Y were evolved by positive and negative selection. The positive selection plasmid contains MjtRNA _CUA ^Y , a TAG-mutated chloramphenicol acetyltransferase gene, an amber-mutated T7 RNA polymerase that initiates the expression of green fluorescent protein, and a tetracycline resistance gene. The negative selection plasmid contained _MjtRNACUAY , the amber mutant ^barnase gene under the arabinose operon, and the ampicillin resistance gene. Three rounds of positive and negative selection were carried out: E.coli DH10B cells containing positive selection plasmids were used as positive selection host cells. Cell electroporation 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 MgCl ₂ , 20 mM glucose) at 37°C for 1 hour. Then change to minimal medium (recipe of GMML minimal medium: M9 salt/glycerol: 764gNa ₂ HPO ₄ .7H ₂ O or 30g Na ₂ HPO ₄ , 15g KH ₂ PO ₄ , 2.5g NaCl, 5g NH ₄ Cl, 50ml Glycerol, autoclaved, pH 7.0; 1M _MgSO4 : autoclaved; 50mM _CaCl2 : autoclaved; 25mM FeCl2 _: filter sterilized; 0.3M Leucine: dissolved in 0.3M NaOH, filter sterilized ; 1L liquid GMML medium: 200ml M9 salt/glycerol, 2ml MgSO ₄ , 2ml CaCl ₂ , 2ml FeCl ₂ , 1ml leucine) wash twice, plate solid minimal medium (add 500ml 3% agar to the liquid GMML medium powder, 1mM F2Y, 50mg/L kanamycin, 60mg/L chloramphenicol, 15mg/L tetracycline), and cultivated at 37°C for 60 hours. Collect cells, extract plasmid DNA, separate by electrophoresis, and recover by gel. Then, the positively selected pBK-lib-jw1 was transformed into DH10B competent cells containing the negatively selected plasmid. Recover in SOC medium for 1 hour. Then plate LB solid medium containing 0.2% arabinose (purchased from sigma company) (per liter medium contains 10 g tryptone, 5 g yeast powder, 10 g NaCl). Incubate at 37°C for 8-12 hours. Repeat for 3 rounds.

In the last round of positive screening, 384 clones were selected and plated on GMML solid medium containing 1 mM F2Y, chloramphenicol 60, 80, 120, and 160 μg/mL, and those that did not contain F2Y but contained chloramphenicol 0 and 20 μg/mL. , 40, 60 μg/mL GMML solid medium. Select the clones that grow on the medium of 1 mM F2Y160 μg/mL chloramphenicol, but do not grow in the medium of 0 mM F2Y with a concentration greater than 20 μg/mL of chloramphenicol for further verification. Finally, one clone was selected with the highest efficiency of inserting 3,5-difluorotyrosine. Sequencing showed that the amino acid sequence of the aminoacyl-tRNA synthetase mutant (F2YRS) contained in the clone was shown in SEQ ID NO: 4, The mutation sites are Tyr32Arg, Leu65Tyr, His70Gly, Phe108Asn, Gln109Cys, Asp158Asn and Leu162Ser.

Those skilled in the art should understand that in the present invention, in addition to the amino acid sequence shown in SEQ ID NO: 4, the term "orthogonal aminoacyl-tRNA synthetase" or "orthogonal 3,5-difluorotyrosine Aminoacyl-tRNA synthetase" also includes conservative variants of the amino acid sequence shown in SEQ ID NO: 4, as long as the conservative variant has the same enzymatic activity as the amino acid sequence shown in SEQ ID NO: 4 and also include the amino acid sequence shown in SEQ ID NO: 4 through one or more amino acid substitutions, deletions or additions and having the same enzymatic activity as the amino acid sequence shown in SEQ ID NO: 4 by SEQ ID NO: Amino acid sequence derived from the amino acid sequence shown in 4.

At the same time, the inventors also analyzed the crystal structure of F2YRS (Fig. 1), which provided a structural basis for the specific integration of F2Y by F2YRS.

Example 3: Analysis of aminoacylation in vitro

In order to verify the high efficiency and fidelity of F2YRS integration of F2Y in the target protein, we performed an in vitro aminoacylation assay. Take 50mM sodium chloride, 20mM magnesium chloride, 4mM dithiothreitol, 2mM ATP, 10μM Tyr tRNA, 3μM F2YRS and 2mM tyrosine or F2Y, dissolve in 20mM Tris buffer, pH 8.0, and incubate at 37°C for 1h. The reaction solution was analyzed by 24h acidic urea polyacrylamide gel electrophoresis. As shown in FIG. 2A , F2YRS can only integrate F2Y, but not tyrosine.

Example 4: Expression of F2Y-green fluorescent protein and identification by mass spectrometry

The orthogonal tRNA (SEQ ID NO: 1) and the screened nucleotide sequence encoding F2YRS (SEQ ID NO: 3) were constructed on the pEVOL vector (purchased from the laboratory of Peter G. Schultz, Scripps Research Institute, USA), encoding The nucleotide sequence (151TAG) (SEQ ID NO: 5) of green fluorescent protein was constructed on the pET vector (purchased from PeterG. )middle. A single clone was picked and cultured at 37°C until the _OD600 was approximately equal to 1.2, and 1 mM IPTG, 0.02% arabinose (purchased from sigma) and 0.5 mM F2Y were added to the LB medium to culture the cells, and the control did not add F2Y. After 8 hours, the bacteria were harvested, and the protein was purified by Ni-NTA and analyzed by SDS-PAGE electrophoresis ( FIG. 2B ).

We found that the full-length green fluorescent protein could be purified only in the medium with F2Y, which indicated that the screened F2YRS could specifically recognize F2Y. The yield of F2Y-GFP in LB medium was 60 mg/L, while that of wild-type GFP was 100 mg/L. In order to detect that F2Y is only inserted into the 151-position amber mutation site of green fluorescent protein, we performed ESI-TOF mass spectrometry detection on 151-F2Y-green protein, and the molecular weight of the detection result was 27746Da (Fig. match.

Example 5: Expression of F2Y-prokaryotic protein tyrosine kinase mutant and detection of tyrosine phosphorylation

We selected the C-terminal cytoplasmic region fragment of the prokaryotic protein tyrosine kinase Etk (position 451-726, the nucleotide sequence is shown in SEQ ID NO: 7), and constructed the Etk mutant by genetic engineering method, wherein the 574-position Mutated to a TAG stop codon, then using the same method in Example 4 to insert F2Y at the 574th position of the Etk mutant, and express the mutant protein Etk-574-F2Y (the amino acid sequence is shown in SEQ ID NO: 8) , the yield was 12.5 mg/L, while that of wild-type Etk was 20 mg/L (Fig. 4A).

In order to verify whether the 574-position tyrosine in the active center of Etk can be autophosphorylated, we took the mutant protein Etk-574-F2Y and put it in a pH 8.5 buffer (containing: 20mM Tris, 20mM NaCl, 2mM ATP and 5mM MgCl2) Incubate at 25°C for 30min. As a control, we added 0.125 mg/mL protein tyrosine phosphatase PTP1B (obtained by cloning and expressing by conventional molecular biology methods, or commercially available, to the mutant protein Etk-574-F2Y, the amino acid sequence is as SEQ ID NO : 9) for dephosphorylation, and then incubated at 25° C. for 30 min in a pH 8.5 buffer (containing: 20 mM Tris, 20 mM NaCl and 1 mM EDTA). After the incubation, a small amount of the mixture was taken for Western verification, and the remaining samples were immediately freeze-dried and analyzed by ¹⁹ F-NMR. At the same time, we took F2Y and the newly synthesized pF2Y in Example 1 for ¹⁹ F-NMR analysis, as a control to verify whether F2Y was phosphorylated. The results are shown in Fig. 3 and Fig. 4C, D, the ¹⁹ F chemical shift of F2Y is -133.1 ppm, and that of pF2Y is -126.7 ppm (Fig. 3). Figure 4C and D show that signal peaks appear at -122.3ppm and -134.5ppm respectively in the mixed solution containing only Etk-574-F2Y, while the signal peak appears only at -134.5ppm in the mixed solution containing PTP1B, indicating that The 574-difluorotyrosine of Etk-574-F2Y was autophosphorylated in the buffer, which was completely consistent with the detection results of western (Fig. 4B). At the same time, through signal intensity comparison analysis, it was found that only 3.8% of difluorotyrosine was phosphorylated, which is consistent with the low phosphorylation level of the active center of prokaryotic protein tyrosine kinase in previous reports. .

It should be understood that while the invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that, without departing from the spirit and scope of the invention as defined by the appended claims, Various changes in form and details can be made therein, and any combination of various embodiments can be made.

Claims (10)

1. an orthogonal aminoacyl-tRNA synzyme, its aminoacid sequence is as shown in SEQ ID NO:4.

2. one kind 3,5-bis-fluoro tyrosine translation system, described system comprises:

(i) 3,5-bis-fluorotyrosine；

(ii) orthogonal aminoacyl-tRNA synzyme described in claim 1；

(iii) orthogonal tRNA, it is as shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-tRNA synzyme is with described 3,5- Orthogonal tRNA described in the two preferential aminoacylations of fluorotyrosine；With

(iv) nucleic acid of encoding target protein, wherein said nucleic acid contains at least one of described orthogonal tRNA specific recognition Select codon.

3. translation system as claimed in claim 2, it is characterised in that described selection codon is amber codon, and institute State translation system and also comprise the nucleotide sequence encoding described orthogonal aminoacyl-tRNA synzyme.

4. a host cell, it comprises the nucleotide sequence of coding orthogonal aminoacyl-tRNA synzyme described in claim 1 With corresponding orthogonal tRNA sequence.

5. host cell as claimed in claim 4, wherein said host cell is eubacteria cell.

6. host cell as claimed in claim 5, it is Bacillus coli cells.

7. one kind produces at least one selected location fixed point specificity insertion 3, the mutein of 5-bis-fluorotyrosine Method, described method comprises the steps:

A () provides 3 described in claim 2,5-bis-fluoro tyrosine translation system, this system comprises:

(i) 3,5-bis-fluorotyrosine；

(ii) orthogonal aminoacyl-tRNA synzyme described in claim 1；

(iii) orthogonal tRNA, it is for shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-tRNA synzyme is with described 3,5- Orthogonal tRNA described in the two preferential aminoacylations of fluorotyrosine；With

(iv) encoding the nucleic acid of described target protein, it is special that wherein said nucleic acid comprises described orthogonal tRNA in selected position Property identification at least one select codon；With

B () is by described orthogonal tRNA sequence and the nucleotide sequence and the coding that encode described orthogonal aminoacyl-tRNA synzyme The nucleotide sequence of described target protein clones and is transformed in suitable host cell, adds 3 in the medium, 5-bis-fluoro Tyrosine, during the translation of described target protein, the orthogonal tRNA recognition coding institute of 3,5-bis-fluorotyrosine aminoacylations State the selection codon and 3 on the mRNA of target protein, 5-bis-fluorotyrosine, thus mediate 3,5-bis-fluorotyrosine Fixed point specificity inserts the amino acid position that described selection codon is corresponding, produces in selected location containing 3,5-bis-fluorotyrosine Described target protein.

8. method as claimed in claim 7, wherein said selection codon is amber codon.

9. producing containing 3, the method for the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine, it utilizes claim Method described in 7, the nucleotide sequence of coding protokaryon protein tyrosine kinase mutant wherein used comprises in selected position The selection codon of described orthogonal tRNA specific recognition, during kinase whose translation, 3,5-bis-fluorotyrosine fixed points are inserted To the amino acid position that described selection codon is corresponding, thus produce and contain 3 at select location, the protokaryon of 5-bis-fluorotyrosine Protein tyrosine kinase mutant.

10. the method identifying the Tyr phosphorylation site of target protein, described method includes the side utilizing claim 7 Presumption in described target protein is carried out the tyrosine of phosphorylation and is substituted by 3 by method, and 5-bis-fluorotyrosine thus obtains containing 3, The target protein mutant of 5-bis-fluorotyrosine, utilizes this target protein mutant as tetraalkylammonium fluoride, passes through¹⁹F-NMR Technology detects 3, the phosphorylation of 5-bis-fluorotyrosine, thus identifies whether described tyrosine site is tyrosine phosphorylation position Point.