Double-phosphorylated proteins and their preparation methods

Abstract

This invention provides a method for simultaneously introducing phosphorylated serine, phosphorylated tyrosine, and their non-natural amino acid analogs at specific sites in a protein, as well as a protein with dual phosphorylation modification prepared by this method and its applications.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a protein having phosphorylated serine and phosphorylated tyrosine at specific positions, and a method for preparing the same. Background Technology

[0002] Protein phosphorylation is one of the most important post-translational modifications (PTMs) of proteins. As a key mechanism in intracellular signal transduction, phosphorylation precisely regulates protein conformation, interactions, and activity. The dynamic phosphorylation-dephosphorylation cycle of proteins is a major pathway in cellular signal transduction. A major bottleneck in protein phosphorylation research is how to prepare proteins with specific phosphorylation sites. Currently, there are no efficient methods for studying phosphorylated tyrosine, mainly because there is a lack of methods to introduce phosphorylated amino acids at specific protein sites. Although amino acids can be phosphorylated using natural or modified serine / threonine and tyrosine kinases, this method lacks site selectivity, and this modification is easily removed by cellular phosphatases. Researchers often use semi-synthetic methods to modify specific sites on peptides with phosphorylated amino acids, but this method is limited to synthesizing short peptide fragments and cannot synthesize full-length proteins with native conformations. Furthermore, the extremely low yield of correctly conformated products obtained using renaturation methods also limits the widespread adoption of this approach.

[0003] Phosphorylated serine and phosphorylated tyrosine are the most common post-translational phosphorylation modifications of proteins, regulating protein structure, function, and interactions, thereby affecting cellular physiological processes. Serine phosphorylation often allosterically modulates proteins to activate their activity, while tyrosine phosphorylation, in addition to allosteric modification and activation, more importantly provides a binding site for binding proteins, promoting their interaction with other proteins to form multi-protein complexes.

[0004] Currently, there are no reported methods that can simultaneously encode phosphorylated serine and phosphorylated tyrosine or similar compounds at two or more sites in the same protein, and the development of such methods is urgently needed in this field.

[0005] Brief Description of the Invention

[0006] The present invention aims to utilize synthetic biology techniques to encode and synthesize proteins containing two post-translational modified amino acids, phosphorylated serine and phosphorylated tyrosine (and their analogues), within living cells.

[0007] This invention provides a method for simultaneously introducing phosphorylated serine and phosphorylated tyrosine at different positions in the same protein, based on gene codon expansion technology.

[0008] In a first aspect, the present invention provides a method for simultaneously introducing phosphorylated serine and phosphorylated tyrosine at different positions in the same protein.

[0009] The method of the present invention includes:

[0010] (i) Provide:

[0011] (a) M maripaludis aminoacyl that recognizes TAG codons encoding phosphorylated serine and phosphorylated tyrosine

[0012] tRNA synthetase / tRNA orthogonal pair;

[0013] (b) Methanocaldococcus janaschii (Mj.) aminoacyl-tRNA synthetase / tRNA orthogonal pair that can recognize AGGA codons encoding phosphorylated tyrosine;

[0014] (c) Nucleic acids that encode proteins containing phosphorylated serine and phosphorylated tyrosine codons at specific positions;

[0015] (d) Ortho-Ribo / Ortho-RBS improves the decoding efficiency of phosphorylated tyrosine;

[0016] (e) Components that enhance the efficiency of phosphorylated serine aminoacylation;

[0017] (ii) Construct recombinant vector 1, in which Ortho-RBS is located upstream of the protein nucleic acid sequence to drive the expression of the protein gene;

[0018] (iii) Constructing a recombinant vector 2, wherein the recombinant vector 2 contains a nucleic acid sequence capable of recognizing tRNA synthetase / tRNA orthogonal pairs that encode phosphorylated serine with TAG codon and phosphorylated tyrosine with AGGA codon, and an element that enhances the efficiency of phosphorylated serine aminoacylation.

[0019] (iv) Recombinant vector 1 from step (ii) and recombinant vector 2 from step (iii) were co-transformed into E. coli cells, and K-Pmp amino acid induction was added to obtain proteins with phosphorylated serine and phosphorylated tyrosine at specific positions, respectively.

[0020] Furthermore, the Mj tRNA synthetase that can recognize phosphorylated tyrosine is CMFRSq; in a specific embodiment, CMFRSq has a nucleic acid sequence as shown in SEQ ID NO:1 or an amino acid sequence as shown in SEQ ID NO:2.

[0021] Furthermore, the tRNA that recognizes phosphotyrosine is Mj.Tyr tRNA. UCCUIn one specific implementation scheme, Mj.Tyr tRNA UCCU It has a nucleic acid sequence as shown in SEQ ID NO: 3.

[0022] Furthermore, the tRNA synthetase that can recognize phosphorylated serine is SepRS; in a specific embodiment, SepRS has a nucleic acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence as shown in SEQ ID NO: 13.

[0023] Furthermore, the tRNA that recognizes phosphorylated serine is Sep tRNA; in a specific implementation, Sep tRNA CUA It has a nucleic acid sequence as shown in SEQ ID NO: 14.

[0024] Furthermore, the Ortho-RBS that enhances the decoding efficiency of phosphorylated amino acids has the nucleic acid sequence shown in SEQ ID NO: 7;

[0025] Furthermore, Ortho-Ribo, which enhances the decoding efficiency of phosphorylated amino acids, has the nucleic acid sequence shown in SEQ ID NO: 6.

[0026] Furthermore, the element that enhances the efficiency of phosphorylated serine aminoacylation is the elongation factor EF-Sep; in a specific embodiment, EF-Sep has a nucleic acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence as shown in SEQ ID NO: 16.

[0027] Furthermore, recombinant vector 1 includes Ortho-RB and the coding sequence of a protein containing phosphorylated serine and phosphorylated tyrosine codons at specific positions.

[0028] Furthermore, recombinant vector 2 includes the TacI promoter, CMFRSq, proK promoter, and Mj.Tyr tRNA. UCCU In one specific implementation, the recombinant factors include the TacI promoter, CMFRSq, proK promoter, and Mj.TyrtRNA. UCCU SepRS, Sep tRNA CUA And EF-sep.

[0029] Further, the protein containing phosphorylated serine and phosphorylated tyrosine codons at specific positions is myoglobin; in one specific embodiment, the K99 codon of myoglobin is mutated to AGGA; in one specific embodiment, the S4 codon of myoglobin is mutated to TAG; in one specific embodiment, the myoglobin with the K99 codon mutated to AGGA has the nucleic acid sequence shown in SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 9; in one specific embodiment, the myoglobin with the K99 codon mutated to AGGA and the S4 codon mutated to TAG has the nucleic acid sequence shown in SEQ ID NO: 11 or the amino acid sequence shown in SEQ ID NO: 19.

[0030] In a second aspect, the present invention provides a protein prepared by the aforementioned method.

[0031] A third aspect of the invention provides a nucleotide sequence encoding a protein other than those described above.

[0032] A fourth aspect of the present invention provides a tRNA synthetase / tRNA bioorthogonal system capable of recognizing quadrupedal codons.

[0033] In one specific implementation, the tRNA synthetase capable of recognizing a quadruple codon is CMFRSq; further, CMFRSq has a nucleic acid sequence as shown in SEQ ID NO:1 or an amino acid sequence as shown in SEQ ID NO:2.

[0034] In one specific implementation, Mj.Tyr tRNA that recognizes a quadruple codon UCCU It has a nucleic acid sequence as shown in SEQ ID NO: 3.

[0035] A fifth aspect of the present invention provides a tRNA synthetase / tRNA bioorthogonal system capable of recognizing phosphorylated serine codons.

[0036] In one specific implementation, the tRNA synthetase that can recognize phosphorylated serine codons is SepRS; further, SepRS has a nucleic acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence as shown in SEQ ID NO: 13;

[0037] In one specific implementation, the tRNA that can recognize phosphorylated serine codons is Sep tRNA, said Sep tRNA CUA It has a nucleic acid sequence as shown in SEQ ID NO: 14.

[0038] A sixth aspect of the invention provides an orthogonal system combination of aminoacyl-tRNA synthetase / tRNA capable of simultaneously introducing phosphorylated serine and phosphorylated tyrosine at different sites in a protein, the orthogonal system combination comprising the aforementioned CMFRSq / Mj.Tyr tRNA that recognizes the phosphorylated tyrosine AGGA codon. UCCU Orthogonal systems, and the aforementioned SepRS / Sep tRNA that recognizes phosphorylated serine codons. CUA Orthogonal system.

[0039] A seventh aspect of the present invention provides the aforementioned Ortho-Ribo / Ortho-RBS which can improve the decoding efficiency of phosphorylated tyrosine.

[0040] An eighth aspect of the present invention provides the aforementioned element that can improve the efficiency of phosphorylated serine aminoacylation. Attached Figure Description

[0041] The accompanying drawings constituting this application are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention. In the drawings:

[0042] Figure 1 The chemical structures of different amino acids such as tyrosine, phosphorylated tyrosine (pY) and their analogues CMF and Pmp, as well as the chemical structures of serine and phosphorylated serine (Sep).

[0043] Figure 2 SDS-PAGE gel image of purified protein. Lane M is the protein marker ladder; lane 1 contains pAux-T5-oRBS-Myo-K99AGGA plasmid and pUltra-CMFRSq-Mj Tyr tRNA. UCCU The plasmids were co-transformed into E. coli TOP10 strains and induced to express and purify the products in medium containing 1 mM IPTG and 2 mM CMF; lane 2 contained pAux-T5-oRBS-Myo-K99AGGA plasmid and pUltra-CMFRSq-Mj Tyr tRNAU CCU The purified product was induced to be expressed by co-transformation of E. coli TOP10 strain with plasmid in medium containing 1 mM IPTG.

[0044] Figure 3 Myo-K99Pmp protein spectroscopy results.

[0045] Figure 4SDS-PAGE electrophoresis images of Myoglobin wild-type protein (wt), single-site serine phosphorylated incorporation protein (S4pS), single-site tyrosine phosphorylated analog incorporation protein (K99Pmp), and dual-site double phosphorylated protein (S4pS+K99Pmp).

[0046] Figure 5 Myo-wt protein proteometry results.

[0047] Figure 6 Myo-S4Sep protein proteometry results.

[0048] Figure 7 Myo-S4Sep-K99Pmp mass spectrometry results.

[0049] Detailed Description of the Invention

[0050] This invention is described in detail herein by reference to the definitions and embodiments described below. All patents and publications mentioned herein, including all sequences disclosed in such patents and publications, are expressly incorporated herein by reference.

[0051] In this invention, the "bioorthogonal translation system" refers to a system composed of an engineered orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pair. aaRS specifically aminoacylates homologous tRNA with specific non-natural amino acids, dedicated to decoding specific codons (usually the stop codon UAG), and subsequently recognizes the tRNA via cellular ribosomes. CUA This allows for the specific incorporation of the UAA-cord site into the synthetic peptide during translation. In this invention, aaRS / tRNA includes CMFRSq / Mj.Tyr tRNA capable of recognizing tetracodons. UCCU Biological orthogonal systems, and SepRS / Sep tRNA that can recognize phosphorylated serine codons. CUA Biological orthogonal system.

[0052] In this invention, a "double phosphorylated protein" refers to a protein with different phosphorylated amino acids at two sites, such as a protein with phosphorylated tyrosine at both sites, or a protein with phosphorylated serine at both sites, or a protein with phosphorylated tyrosine at one site and phosphorylated serine at the other site. Example

[0053] It should be noted that, unless otherwise specified, the embodiments in this application are merely illustrative and are not intended to limit the invention in any way.

[0054] Example 1. Preparation of phosphorylated tyrosine analogs for site-specific modification of myoglobin protein based on gene codon expansion, tetranucleotide codons and orthogonal ribosome technology

[0055] To achieve the simultaneous encoding of two different types of non-natural amino acids in a protein, it is necessary to address the issues of codon allocation and orthogonality between codons and non-natural amino acids. Here, we combine the use of the quadruplet codon to encode phosphorylated tyrosine and its analogues, while using the amber codon UAG to encode phosphorylated serine.

[0056] Methanocaldococcus janaschii(Mj)TyrRS / Tyr tRNA CUA The orthogonal pair was modified to recognize the quadruple codon AGGA and encode phosphorylated tyrosine (pY) and its analogues. The Mj TyrRS protein was modified to contain the following site mutations (Y32S, L65A, F108K, Q109H, D158G, L162K, Y230K, C231K, P232K, F261P, H283W, D286G), and named CMFRSq.

[0057] Modified Mj Tyr tRN CUA Mj Tyr tRNA UCCU This enables it to encode the AGGA quadcote.

[0058] Combine CMFRSq with Mj Tyr tRNA UCCU The expression of CMFRSq was driven by the TacI promoter and Mj Tyr tRNA was driven by the proK promoter. UCCU Transcription, plasmid named pUltra-CMFRSq-Mj Tyr tRNA UCCU .

[0059] The introduction of orthogonal ribosomal elements Ortho-Ribo and Ortho-RBS sequence orthogonal pairs improves the decoding efficiency of quadcodons and ensures orthogonal codons with natural ribosomes, thus preventing the translation of quadcodons from affecting the operation of the natural translation system. Ortho-Ribo is modified from the E. coli 16S ribosomal RNA sequence. The Ortho-RBS sequence is shown in Table 1.

[0060] Here, myoglobin protein was used as the model protein. Its coding sequence was modified, with the K99 codon changed to an AGGA quadruple codon, and a 6xHis tag sequence fused to the C-terminus, named myo-K99AGGA. The Ortho-RBS sequence and myoglobin-K99AGGA were cloned into the pAUX plasmid. Myoglobin-K99AGGA expression was driven by the T5 promoter and Ortho-RBS, while Ortho-Ribo transcription was driven by the natural 16S rRNA promoter. The plasmid was named pAux-T5-oRBS-Myo-K99AGGA.

[0061] To achieve the expression, preparation, and purification of Myo-K99Pmp protein, which is site-directedly modified with the phosphorylated tyrosine analog Pmp encoded by a quadruple codon, the plasmid pAux-T5-oRBS-Myo-K99AGGA and the tRNA pUltra-CMFRSq-Mj Tyr were prepared. UCCU The plasmid was co-transformed into E. coli TOP10 competent cells, cultured in LB agar dishes containing ampicillin (AMP) and spectinomycin (Spec) antibiotics, and clones were picked and transferred to LB liquid medium containing AMP+Spec and cultured overnight in a shaker at 37°C.

[0062] Dilute the overnight cultured bacterial solution at a ratio of 1:100 and transfer it into a 50 mL shake flask containing TB medium containing Amp, Spec and K-Pmp (5 mM). Incubate at 37°C with shaking for 4-6 hours until the OD600 of the bacterial solution reaches 0.6-0.8.

[0063] Add IPTG (final concentration 1mM) to the above bacterial culture, place it in a constant temperature shaker at 30℃ and incubate for 16-24 hours to induce expression.

[0064] Collect the bacterial culture and centrifuge at 8000-12000 rpm to collect the bacterial cells. Resuspend the bacterial cells in DPBS buffer and sonicate them in an ice bath at 4°C to release intracellular proteins. Centrifuge the bacterial lysate at 10000-15000 rpm and collect the supernatant for Ni-NTA resin affinity chromatography.

[0065] The supernatant obtained from the centrifugation was added to a gravity column packed with Ni-NTA resin. After loading the sample twice, the column packing was washed with DPBS buffer (pH 7.5) containing 50 mM imidazole. The target protein was then eluted with DPBS buffer (pH 7.5) containing 300 mM imidazole. The protein sample was then concentrated by ultrafiltration using an ultrafiltration concentrator (3 kDa molecular weight cutoff) and replaced with DPBS (pH 7.5) buffer.

[0066] The concentrated protein samples were subjected to SDS-PAGE electrophoresis and mass spectrometry identification. SDS-PAGE results showed that with the addition of 5 mM K-Pmp amino acids, the AGGA codon was successfully decoded to obtain the full-length myoglobin protein after induction, while the non-specifically encoded protein was not expressed without the addition of exogenous K-Pmp. Figure 2 Mass spectrometry analysis revealed that the purified Myo-K99Pmp protein had a molecular weight of 18468.3 Da. Figure 3 The result is consistent with the theoretical molecular weight of 18468.1 Da, indicating that the Myo-K99Pmp protein encoded by the quadruple codon AGGA was successfully expressed.

[0067] Table 1. Gene and amino acid sequence list

[0068] CMFRSq Nucleic acid >SEQ ID NO: 1 CMFRSq amino acids >SEQ ID NO: 2 <![CDATA[Mj Tyr tRNA UCCU ]]> Nucleic acid >SEQ ID NO: 3 TacI promoter Nucleic acid >SEQ ID NO: 4 proK promoter Nucleic acid >SEQ ID NO: 5 E. coli 16S ribosomal RNA Nucleic acid >SEQ ID NO: 6 Ortho-RBS SEQ Nucleic acid >SEQ ID NO: 7 myo-K99AGGA Nucleic acid >SEQ ID NO: 8 T5 starter Nucleic acid >SEQ ID NO: 9 Myo-K99Pmp amino acids >SEQ ID NO: 10 myo-S4TAG-K99AGGA Nucleic acid >SEQ ID NO: 11 SepRS Nucleic acid >SEQ ID NO: 12 SepRS amino acids >SEQ ID NO: 13 <![CDATA[Sep tRNA CUA ]]> Nucleic acid >SEQ ID NO: 14 EF-Sep Nucleic acid >SEQ ID NO: 15 EF-Sep amino acids >SEQ ID NO: 16 GlnS promoter Nucleic acid >SEQ ID NO: 17 LPP promoter Nucleic acid >SEQ ID NO: 18 Myo-S4Sep-K99Pmp amino acids >SEQ ID NO: 19 Myo-wt Nucleic acid >SEQ ID NO: 20 Myo-wt amino acids >SEQ ID NO: 21 Myo-S4TAG Nucleic acid >SEQ ID NO: 22 Myo-S4sep amino acids >SEQ ID NO: 23

[0069] Example 2: Expression and purification of proteins simultaneously encoding phosphorylated serine and phosphorylated tyrosine analogues for site-directed modification.

[0070] To verify the synchronous site-specific encoding of phosphorylated serine and phosphorylated tyrosine analogs in proteins, myoglobin protein was selected as the model protein. Its K99 amino acid encoded the phosphorylated tyrosine analog Pmp, and its S4 amino acid encoded the phosphorylated serine Sep. The myoglobin coding sequence was modified by changing the K99 codon to an AGGA quadruple codon and the S4 codon to a TAG codon. A 6xHis tag sequence was fused to the C-terminus, naming the result myo-S4TAG-K99AGGA. The Ortho-RBS sequence and myo-S4TAG-K99AGGA were cloned into the pAUX plasmid. Myo-S4TAG-K99AGGA expression was driven by the T5 promoter and Ortho-RBS, while Ortho-Ribo transcription was driven by the natural 16S rRNA promoter. The plasmid was named pAux-T5-oRBS-myo-S4TAG-K99AGGA.

[0071] CMFRSq and Mj Tyr tRNA UCCU , SepRS (SEQ ID NO: 12), Sep tRNA CUA (SEQ ID NO: 14) The EF-Sep element was cloned into the pUltra vector plasmid, and CMFRSq expression was driven by the TacI promoter, SepRS expression by the GInS promoter, EF-Sep expression by the TacI promoter, and Mj Tyr tRNA expression by the proK promoter. UCCU Transcription, LPP promoter drives Sep tRNACUA Transcription, plasmid named pUltra-CMFRSq-Mj Tyr tRNA UCCU -SepRS-Sep tRNA CUA -EFsep.

[0072] The expression system incorporates the elongation factor EF-Sep element to enhance the aminoacylation efficiency of Sep-tRNA, thereby improving the coding efficiency of phosphorylated serine.

[0073] To achieve the expression, preparation, and purification of the phosphorylated tyrosine analog Pmp and the phosphorylated serine Sep-modified Myo-S4TAG-K99Pmp protein, the pAux-T5-oRBS-myo-S4TAG-K99AGGA plasmid and pUltra-CMFRSq-Mj TyrtRNA were prepared. UCCU -SepRS-Sep tRNA CUA -EFsep plasmid was co-transformed into E. coli TOP10-δserB competent cells, cultured in LB agar dishes containing ampicillin (AMP) and spectinomycin (Spec) antibiotics, and clones were picked and transferred to LB liquid medium containing AMP+Spec and cultured overnight in a shaker at 37°C.

[0074] Dilute the overnight cultured bacterial solution at a ratio of 1:100 and transfer it into a 50 mL shake flask containing TB medium containing Amp, Spec and K-Pmp (5 mM). Incubate at 37°C with shaking for 4-6 hours until the OD600 of the bacterial solution reaches 0.6-0.8.

[0075] For the expression preparation of wild-type Myoglobin protein, the plasmid pAux-T5-oRBS-myo-wt containing the wild-type Myoglobin gene sequence (SEQ ID NO: 20) was transformed into E. coli TOP10-δserB competent cells. The cells were cultured in LB agar dishes containing ampicillin (AMP), and clones were picked and transferred to LB liquid medium containing AMP. The cells were then cultured overnight at 37°C in a shaker. The overnight culture was diluted 1:100 and transferred to 50 mL of TB medium containing AMP in a shake flask. The cells were then cultured at 37°C in a shaker for 4-6 hours until the OD600 reached 0.6-0.8.

[0076] For the expression preparation of Myoglobin protein with single-site phosphorylated serine incorporation, the plasmid pAux-T5-oRBS-Myo-S4TAG containing the Myo-S4TAG gene sequence (SEQ ID NO: 22) and the plasmid pUltra-CMFRSq-Mj TyrtRNA were prepared. UCCU-SepRS-Sep tRNA CUA The -EFsep plasmid was co-transformed into E. coli TOP10-δserB competent cells, cultured in LB agar dishes containing ampicillin (AMP) and spectinomycin (Spec) antibiotics, and clones were picked and transferred to LB liquid medium containing AMP+Spec. The culture was incubated overnight at 37°C with a shaker. The overnight culture was then diluted 1:100 and transferred to 50 mL TB medium containing Amp and Spec in a shaker flask, and incubated at 37°C with shaking for 4-6 hours until the OD600 reached 0.6-0.8.

[0077] Add IPTG (final concentration 1mM) to the above bacterial culture, place it in a constant temperature shaker at 30℃ and incubate for 16-24 hours to induce expression.

[0078] Collect the bacterial culture and centrifuge at 8000-12000 rpm to collect the bacterial cells. Resuspend the bacterial cells in DPBS buffer and sonicate them in an ice bath at 4°C to release intracellular proteins. Centrifuge the bacterial lysate at 10000-15000 rpm and collect the supernatant for Ni-NTA resin affinity chromatography.

[0079] The supernatant obtained from the centrifugation was added to a gravity column packed with Ni-NTA resin. After loading the sample twice, the column packing was washed with DPBS buffer (pH 7.5) containing 50 mM imidazole. The target protein was then eluted with DPBS buffer (pH 7.5) containing 300 mM imidazole. The protein sample was then concentrated by ultrafiltration using an ultrafiltration concentrator (3 kDa molecular weight cutoff) and replaced with DPBS (pH 7.5) buffer.

[0080] The concentrated protein samples were subjected to SDS-PAGE electrophoresis and mass spectrometry identification. SDS-PAGE results showed that, after induced expression, the AGGA codon and TAG double codon could be successfully decoded to obtain the full-length myoglobin protein. Figure 4 Mass spectrometry analysis revealed that the purified Myo-wt protein had a molecular weight of 18355.9 Da. Figure 5 The molecular weight is basically consistent with the theoretical molecular weight of 18355.1; the molecular weight of Myo-S4Sep protein is 18435.9 Da. Figure 6 The molecular weight is basically consistent with the theoretical molecular weight of 18435.1; the molecular weight of the purified Myo-S4TAG-K99Pmp protein is 18549.0 Da. Figure 7 The result is basically consistent with the theoretical molecular weight of 18484.1, indicating that the Myo-S4TAG-K99Pmp protein, which simultaneously encodes phosphorylated serine (Sep) and phosphorylated tyrosine analogue (Pmp), was successfully expressed.

[0081] In summary, this invention successfully encodes and synthesizes proteins containing both site-directed phosphorylated serine and phosphorylated tyrosine, two post-translational modified amino acids, within living cells using synthetic biology techniques. Furthermore, the method of this invention can also be widely applied to introduce three or more phosphorylated amino acids into proteins, providing a simple and feasible reference for the study of phosphorylated proteins.

Claims

1. A method for preparing a protein, wherein the protein has phosphorylated serine and phosphorylated tyrosine at specific positions, the method comprising: (i) Provide: (a) M maripaludis aminoacyl-tRNA synthetase / tRNA orthogonal pair that can recognize TAG codons encoding phosphorylated serine; (b) Methanocaldococcus janaschii (Mj.) aminoacyl-tRNA synthetase / tRNA orthogonal pair that can recognize AGGA codons encoding phosphorylated tyrosine; (c) Nucleic acids that encode proteins containing phosphorylated serine and phosphorylated tyrosine codons at specific positions; (d) Ortho-Ribo / Ortho-RBS improves the decoding efficiency of phosphorylated tyrosine; (e) Components that enhance the efficiency of phosphorylated serine aminoacylation; (ii) Construct recombinant vector 1, in which Ortho-RBS is located upstream of the protein nucleic acid sequence to drive the expression of the protein gene; (iii) Constructing a recombinant vector 2, wherein the recombinant vector 2 contains nucleic acid sequences of aminoacyl-tRNA synthetase / tRNA orthogonal pairs that can recognize phosphorylated serine and phosphorylated tyrosine respectively, and elements that enhance the acylation efficiency of phosphorylated serine; (iv) Recombinant vector 1 from step (ii) and recombinant vector 2 from step (iii) were co-transformed into E. coli cells, and K-Pmp amino acid induction was added to obtain proteins with phosphorylated serine and phosphorylated tyrosine at specific positions, respectively.

2. The method of claim 1, wherein the orthogonal pair of TAG codon-encoding phosphorylated serine is SepRS / Sep tRNA CUA ; preferably, the SepRS has a nucleic acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence as set forth in SEQ ID NO: 13, and the Sep tRNA CUA has a nucleic acid sequence as set forth in SEQ ID NO:

14.

3. The method of claim 1, wherein the orthogonal pair of the recognition AGGA codon encoding phosphorylated tyrosine is CMFRSq / Mj.Tyr tRNA UCCU Preferably, the CMFRSq has a nucleic acid sequence as shown in SEQ ID NO:1 or an amino acid sequence as shown in SEQ ID NO:2, and the Mj.Tyr tRNA UCCU It has a nucleic acid sequence as shown in SEQ ID NO:

3.

4. The method of claim 1, wherein the Ortho-RBS that improves the decoding efficiency of AGGA codon-encoded phosphorylated tyrosine has a nucleic acid sequence as shown in SEQ ID NO:

7.

5. The method of claim 1, wherein the Ortho-Ribo that enhances phosphorylated tyrosine decoding efficiency has a nucleic acid sequence as shown in SEQ ID NO:

6.

6. The method of claim 1, wherein the element for enhancing the efficiency of phosphorylated serine aminoacylation is an elongation factor EF-Sep, wherein EF-Sep has a nucleic acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence as shown in SEQ ID NO:

16.

7. The protein prepared by the method according to any one of claims 1-6.

8. The protein of claim 9 has the amino acid sequence shown in SEQ ID NO:

19.

9. A nucleic acid encoding the protein as described in claim 7.

10. The encoding nucleic acid of claim 9 has the sequence shown in SEQ ID NO:

11.

11. A Methanocaldococcus janaschii (Mj.) aminoacyl-tRNA synthetase / tRNA orthogonal pair capable of recognizing phosphorylated tyrosine AGGA codons, wherein the orthogonal pair is CMFRSq / Mj.Tyr tRNA UCCU Preferably, the CMFRSq has a nucleic acid sequence as shown in SEQ ID NO:1 or an amino acid sequence as shown in SEQ ID NO:2, and the Mj.Tyr tRNA UCCU It has a nucleic acid sequence as shown in SEQ ID NO:

3.

12. An M maripaludis aminoacyl-tRNA synthetase / tRNA orthogonal pair capable of recognizing phosphorylated serine codons, wherein the orthogonal pair SepRS / Sep tRNA CUA Preferably, the SepRS has a nucleic acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence as shown in SEQ ID NO: 13, and the Sep tRNA CUA It has a nucleic acid sequence as shown in SEQ ID NO:

14.

13. An aminoacyl-tRNA synthetase / tRNA orthogonal pair that can simultaneously introduce phosphorylated serine and phosphorylated tyrosine at different sites in a protein, wherein the orthogonal pair comprises the CMFRSq / Tyr tRNA that recognizes the phosphorylated tyrosine AGGA codon as described in claim 11. UCCU Orthogonal pairs, and the SepRS / Sep tRNA for recognizing phosphorylated serine codons as described in claim 12. CUA Orthogonal pair.

14. The Ortho-Ribo / Ortho-RBS with improved phosphorylated tyrosine decoding efficiency according to any one of claims 1-6.

15. The element for improving the efficiency of phosphorylated serine aminoacylation according to any one of claims 1-6.

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