Orthogonal aminoacyl tRNA synthetase mutants and uses thereof

Abstract

This invention relates to an orthocyanin-tRNA synthetase mutant and its applications, belonging to the field of biotechnology. This invention obtained orthocyanin-tRNA synthetase mutants through continuous evolution of the orthocyanin-tRNA synthetase with the amino acid sequence shown in SEQ ID NO.1. Based on the orthocyanin-tRNA synthetase with the amino acid sequence shown in SEQ ID NO.1, this invention, through continuous evolution, discovered a series of highly efficient mutants. Compared with the wild-type orthocyanin-tRNA synthetase, the obtained mutants significantly improve the efficiency of introducing non-natural amino acids. Applying them to the introduction of non-natural amino acids such as azidophenylalanine can also save experimental costs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an orthogonalyl tRNA synthetase mutant and its applications. Background Technology

[0002] Codon expansion (GCE) technology extends protein function by introducing unnatural amino acids (ncAAs) into proteins. The most crucial component is the orthogonal tRNA / orthogonal aminoacyl-tRNA synthetase pair. Orthogonal aminoacyl-tRNA synthetase can aminoacylate orthogonal tRNA with unnatural amino acids, and is a key factor in the efficiency of unnatural amino acid introduction. Currently used orthogonal aminoacyl-tRNA synthetases are mainly obtained through directed evolution, which is time-consuming, labor-intensive, and its efficiency cannot be guaranteed. Azide-phenylalanine (pAzF), as an unnatural amino acid containing an azido group, has significant applications in orthogonal biological reactions. Obtaining a highly efficient class of tyrosine-tRNA synthetases (MjTyrRS) through continuous evolution to improve the introduction efficiency of azidophenylalanine in codon expansion is of great significance. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the lack of an efficient orthogonalyl-tRNA synthetase in the prior art.

[0004] To address the aforementioned technical problems, this invention provides an orthogonal tRNA synthetase mutant and its applications.

[0005] The first objective of this invention is to provide a series of orthocyanin-tRNA synthetase mutants obtained through sequential evolution of the orthocyanin-tRNA synthetase mutant RS.2.t1. The orthocyanin-tRNA synthetase mutant RS.2.t1 shown in SEQ ID NO. 1 is subjected to any one or more of the following mutations:

[0006] (1) The aspartic acid at position 54 is mutated to tyrosine (D54Y).

[0007] (2) The leucine at position 55 is mutated to glutamic acid (L55E);

[0008] (3) The leucine at position 73 is mutated to glutamic acid (L73E).

[0009] (4) The lysine at position 90 is mutated to phenylalanine (K90F).

[0010] (5) The valine at position 168 is mutated to glutamine (V168Q).

[0011] (6) The proline at position 185 is mutated to glutamine (P185Q).

[0012] (7) The isoleucine at position 191 is mutated to valine (I191V).

[0013] (8) The tyrosine at position 230 is mutated to histidine (Y230H);

[0014] (9) The alanine at position 224 is mutated to arginine (A224R);

[0015] (10) The lysine at position 225 is mutated to aspartic acid (K225D);

[0016] (11) Glutamate at position 250 is mutated to methionine (E250M).

[0017] Furthermore, SEQ ID NO.1:

[0018] MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTYMLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPI MQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL.

[0019] A second objective of this invention is to provide a gene encoding the above-mentioned orthogonal tRNA synthetase mutant.

[0020] A third objective of this invention is to provide a recombinant plasmid carrying the aforementioned gene.

[0021] A fourth object of the present invention is to provide a recombinant cell containing the above-described recombinant plasmid, wherein the recombinant cell comprises bacteria.

[0022] A fifth object of the present invention is to provide the use of the above-mentioned orthocyanin tRNA synthetase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid, or the above-mentioned recombinant cell in the preparation of a protein containing azidophenylalanine.

[0023] A sixth objective of the present invention is to provide a method for introducing azidophenylalanine into a target protein, the method comprising adding an expression system containing the target protein and the above-mentioned orthocyanin tRNA synthetase mutant to a system containing azidophenylalanine for reaction, wherein the codon encoding the target protein contains an amber stop codon.

[0024] Furthermore, the expression system is induced by anhydrous tetracycline.

[0025] A seventh objective of this invention is to provide a method for characterizing an orthocyanin-tRNA synthetase mutant, wherein the method involves co-expressing a green fluorescent protein and an orthocyanin-tRNA synthetase mutant in the same expression system, wherein the green fluorescent protein contains an amber stop codon.

[0026] Furthermore, the codon encoding the 16th amino acid in the green fluorescent protein is replaced with an amber stop codon.

[0027] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0028] This invention, through continuous evolution of orthocyanin-tRNA synthetase, yields an orthocyanin-tRNA synthetase mutant that significantly improves the efficiency of introducing non-natural amino acids compared to the wild-type orthocyanin-tRNA synthetase. When applied to the introduction of non-natural amino acids such as azidophenylalanine, it saves experimental costs while achieving efficient introduction. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This is a structural diagram of the orthogonal tRNA synthetase mutant of the present invention;

[0031] Figure 2 This invention relates to a linear plasmid for evolving orthogonal aminoacyl-tRNA synthetase;

[0032] Figure 3 This refers to the non-natural amino acid infiltration efficiency of the orthogonal tRNA synthetase mutant of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] Recombinant strain seed culture and fermentation:

[0035] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10.

[0036] Antibiotic concentrations (mg / L): Kanamycin 50, Spectinomycin 50, Tetracycline 50.

[0037] Inducer concentration (μM): Anhydrous tetracycline: 5.

[0038] Culture conditions: Seeds cultured at 37℃ and 220 rpm for 12 h were transferred to fermentation medium at an inoculum rate of 0.1% and cultured at 37℃ and 220 rpm for 12 h. Antibiotics and inducers were added according to the culture objectives.

[0039] Continuous passage: Add 1000 μL of fermentation medium, along with the corresponding antibiotics and inducers, to each well of a 24-well plate. After inoculation at a rate of 0.1%, subculture at a rate of 0.1% every 12 hours, which is considered as 10 generations.

[0040] Continuous evolution: Under appropriate culture conditions, continuous passage will generate a mutant library in the fermentation broth. With the help of a suitable screening strategy, the target mutant can be obtained.

[0041] Characterization of mutants: Ortho-aquamoyl-tRNA synthetase mutants were obtained by expressing the promoter induced by anhydrous tetracycline, and fluorescence values ​​and OD were measured with and without the addition of non-natural amino acids. 600 .

[0042] The previously reported orthocyanin-tRNA synthetase mutant RS.2.t1, which can utilize non-natural amino acids, was published in the paper “Amiram, M., Haimovich, A., Fan, C. et al. Evolution of translationmachinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol 33, 1272–1279 (2015). https: / / doi.org / 10.1038 / nbt.3372.”

[0043] Example 1: Expression of orthocyanin-tRNA mutant

[0044] The orthocyanin-tRNA synthetase mutant and green fluorescent protein were encoded into the same expression vector. The orthocyanin-tRNA synthetase mutant was induced by anhydrous tetracycline, and the 16th amino acid of the green fluorescent protein was replaced with the amber stop codon (TAG) and constitutively expressed. The plasmid was named pGCE-N, where N refers to the mutant code.

[0045] Example 2: Obtaining Orthocyanin-tRNA Mutants

[0046] All orthocyanin-tRNA synthetase mutants were obtained through continuous evolution based on orthogonal DNA replication. Specifically, the starting mutant orthocyanin-tRNA synthetase RS.2.t1 was cloned into a linear plasmid in E. coli, and mutations were continuously induced into the linear plasmid during passage. After a period of mutation accumulation, the mutants were selected.

[0047] Example 3: Characterization of orthocyanin-tRNA mutants

[0048] Single colonies carrying pGCE-N were picked from resistance plates and inoculated into liquid LB medium containing the corresponding resistance. After incubation at 37°C and 220 rpm for 12 h, they were transferred at 1% volume to characterization medium containing non-natural amino acids. The characterization medium contained the corresponding resistance and an inducer. After incubation at 37°C and 220 rpm for 6 h, the fluorescence value and OD were measured. 600 Efficiency refers to the fluorescence / OD ratio of wild-type GFP. 600 100% fluorescence / OD of GFP containing the TAG stop codon 600 The proportion of wild-type GFP to wild-type GFP, which refers to GFP fluorescent protein that does not contain the TAG stop codon.

[0049] Table 1. Efficiency of introducing non-natural amino acids into different mutants

[0050]

[0051] Comparative Example 1: Reported orthogonalyl-tRNA synthetase mutants that can utilize non-natural amino acids

[0052] The previously reported axoyl-tRNA synthetase mutant RS.2.t1, which can utilize non-natural amino acids, and green fluorescent protein were encoded into the same expression vector. The axoyl-tRNA synthetase mutant was induced by anhydrous tetracycline, and the 16th amino acid of green fluorescent protein was replaced with an amber stop codon (TAG) and constitutively expressed. The plasmid was named pGCE-RS.2.t1.

[0053] As described in Example 2, pGCE-RS.2.t1 was continuously evolved.

[0054] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A mutant of orthocyanin-tRNA synthetase, characterized in that, The orthocyanin-tRNA synthetase mutant is any one or more of the following mutations performed on the orthocyanin-tRNA synthetase with the amino acid sequence shown in SEQ ID NO.1: (1) The aspartic acid at position 54 is mutated to tyrosine; (2) The leucine at position 55 is mutated to glutamic acid; (3) The leucine at position 73 is mutated to glutamic acid; (4) The lysine at position 90 is mutated to phenylalanine; (5) The valine at position 168 is mutated to glutamine; (6) The proline at position 185 is mutated to glutamine; (7) The isoleucine at position 191 is mutated to valine; (8) The tyrosine at position 230 is mutated to histidine; (9) The alanine at position 224 is mutated to arginine; (10) The lysine at position 225 is mutated to aspartic acid; (11) Glutamate at position 250 is mutated to methionine.

2. The gene encoding the orthogonal tRNA synthetase mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. Recombinant cells containing the recombinant plasmid as described in claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells include bacteria.

6. The use of the orthogonal tRNA synthetase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 in the preparation of a protein containing azidophenylalanine.

7. A method for introducing azidophenylalanine into a target protein, characterized in that, The method involves adding an expression system containing the target protein and the orthocyanin tRNA synthetase mutant of claim 1 to a system containing azidophenylalanine for reaction, wherein the codon encoding the target protein contains an amber stop codon.

8. The method according to claim 7, characterized in that, The expression system is induced by tetracycline.

9. A method for characterizing orthogonal aminoacyl-tRNA synthetase mutants, characterized in that, The method involves co-expressing green fluorescent protein and the orthogonal tRNA synthetase mutant of claim 1 in the same expression system, wherein the green fluorescent protein contains an amber stop codon.

10. The method according to claim 9, characterized in that, The codon encoding the 16th amino acid in the green fluorescent protein has been replaced with an amber stop codon.

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