A pyrrolysine-tRNA synthetase mutant
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NINGBO INST OF MARINE MEDICINE PEKING UNIV
- Filing Date
- 2026-03-19
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256275A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a pyrrolidone-lysine-tRNA synthetase mutant, belonging to the field of enzyme engineering technology. Background Technology
[0002] With the development of science, technology, and medicine, there is an urgent need to design more complex proteins to meet research needs, requiring a greater diversity of amino acids to provide richer chemical properties and spatial structures. Directly inserting non-natural amino acids during natural translation by altering the codon-amino acid correspondence is a commonly used method. This method requires a set of orthogonal blank codons, aminoacyl-tRNA synthetase, tRNA, and non-natural amino acids. The aminoacyl-tRNA synthetase recognizes the target non-natural amino acid and inserts it into the target site. Currently, aaRS enzymes developed for site-directed insertion of non-natural amino acids include EcTyrRS, EcLeuRS, and PylRS. However, EcTyrRS and EcLeuRS are derived from *E. coli*, making direct application within *E. coli* difficult. PylRS enzymes commonly use *Methanococcus pasteurellii* as a source. Methanosarcina barkeri ), Methanosarcina mazei, etc.
[0003] Pyrrolysine-tRNA ligase (PylRS, EC 6.1.1.26) is a commonly used tool for site-directed insertion of non-natural amino acids. Its natural substrate, pyrrolysine, is a lysine derivative, and the non-natural amino acids it can insert are also mostly lysine derivatives, such as BOCK, AlocK, and NAEK. NAEK, in particular, is of significant value in protein labeling. Its unique azide group can bind to alkyne groups via click chemistry, enabling efficient, stable, and specific labeling of proteins, thereby facilitating the localization, quantification, and functional studies of proteins.
[0004] However, there are currently few types of PylRS enzymes available for use in E. coli, and their insertion efficiency is difficult to meet production needs. This greatly limits the application scope of non-natural amino acid insertion in E. coli. In order to increase the yield of target proteins and expand the application scope of non-natural amino acid insertion, it is urgent to improve the insertion efficiency of PylRS enzymes. Summary of the Invention
[0005] To address the shortcomings of the prior art, this invention provides a pyrrolidone-tRNA synthetase pair mutant, aiming to solve the problem of low PylRS enzyme insertion efficiency.
[0006] The first technical solution provided by this invention is a pyrrolysine-tRNA synthetase mutant, wherein the pyrrolysine-tRNA synthetase mutant is formed by any one of the following mutations to the pyrrolysine-tRNA synthetase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The 44th position of aspartic acid is mutated to glycine, the 275th position of tyrosine is mutated to alanine, the 278th position of leucine is mutated to alanine, the 282nd position of aspartic acid is mutated to serine, the 291st position of isoleucine is mutated to alanine and / or the 353rd position of tyrosine is mutated to phenylalanine. (2) Delete five amino acids from alanine at position 107 to serine at position 111.
[0007] In some embodiments, the pyrrolidone-tRNA synthetase mutant is a parent pyrrolidone-tRNA synthetase with the amino acid sequence shown in SEQ ID NO.1 having the aspartic acid at position 44 mutated to glutamine and the leucine at position 278 mutated to asparagine.
[0008] The second technical solution provided by the present invention is a gene encoding the pyrrolidone-tRNA synthetase mutant described in the first technical solution.
[0009] The third technical solution provided by the present invention is an orthogonal tRNA mutant gene, wherein the orthogonal tRNA mutant gene is a gene in which adenine (A) at position 76 of the orthogonal tRNA parent with a nucleotide sequence as shown in SEQ ID NO.3 is mutated to thymine (T).
[0010] The fourth technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution and / or the mutated gene described in the third technical solution.
[0011] In some embodiments, the recombinant vector uses pET series, Duet series, or PUC series plasmids as expression vectors.
[0012] The fifth technical solution provided by the present invention is a recombinant microbial cell expressing the pyrrolidone-tRNA synthetase mutant described in the first technical solution, or containing the gene described in the second technical solution or the mutant gene described in the third technical solution, or transformed with the recombinant vector described in the fourth technical solution.
[0013] In some embodiments, the microbial cells include, but are not limited to, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pichia pastoris.
[0014] The seventh technical solution provided by the present invention is a method for expressing a recombinant protein with site-directed insertion of non-natural amino acids. The method involves site-directed insertion using the pyrrolidone-tRNA synthetase mutant described in the first technical solution, the gene described in the second technical solution, the mutant gene described in the third technical solution, the recombinant vector described in the fourth technical solution, or the recombinant microbial cell described in the fifth technical solution.
[0015] In some embodiments, the non-natural amino acids include, but are not limited to, NAEK, BOCK, and AlocK.
[0016] In some embodiments, the non-natural amino acid is Nε-2-Azidoethyloxycarbonyl-L-lysine (NAEK), a non-natural amino acid containing an azide group.
[0017] In some embodiments, the recombinant protein is IL-2 or EGFP.
[0018] The eighth technical solution provided by the present invention is a method for increasing the yield of recombinant protein expression in Escherichia coli, which uses the pyrrolidone-tRNA synthetase mutant described in the first technical solution, or the gene described in the second technical solution, or the mutant gene described in the third technical solution, or the recombinant vector described in the fourth technical solution, or the recombinant microbial cell described in the fifth technical solution to insert non-natural amino acids into the recombinant protein at specific sites.
[0019] In some embodiments, the non-natural amino acids include, but are not limited to, NAEK, BOCK, and AlocK.
[0020] In some embodiments, the non-natural amino acid is Nε-2-Azidoethyloxycarbonyl-L-lysine (NAEK), a non-natural amino acid containing an azide group.
[0021] In some embodiments, the recombinant protein is IL-2 or EGFP.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: The pyrrolidone-tRNA synthetase pair described in this invention can be used for site-directed insertion of non-natural amino acids. The enzyme activity, non-natural amino acid insertion efficiency, and target protein yield of the mutant are significantly improved compared with the wild type. Among them, the relative fluorescence value of NAEK insertion in EGFP (Y39) is increased by 92.1% compared with the wild type, and the IL2 unit yield of NAEK insertion in IL2 (Y45) is increased by 70.6%. Attached Figure Description
[0023] Figure 1 The image shows the plasmid pET-MhPylRS-EGFP(Y39)-His.
[0024] Figure 2 The relative fluorescence values of pyrrolidone-tRNA synthetase for wild-type and mutant NAEK insertion in EGFP(Y39) are given.
[0025] Figure 3 The image shows the spectrum of plasmid pET-MhPylRS-IL2(Y45).
[0026] Figure 4 SDS-PAGE gel images of pyrrolidone-tRNA synthetase for wild-type and mutant NAEK insertion at IL2(Y45). Detailed Implementation
[0027] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0028] Test method: Take 200 μl of the sample to be tested into a 96-well plate with a black wall and a transparent bottom, and perform fluorescence and OD analysis using a multi-functional microplate reader. 600 The detection parameters are as follows.
[0029] Microplate reader fluorescence detection parameters: Detect fluorescence, excitation wavelength 485 nm, emission wavelength 528 nm, bottom gain 70, plate shaking 30 s.
[0030] ELISA reader OD 600 Detection parameters: Detection of absorbed light, wavelength 600 nm, vibration plate 30 s.
[0031] Raw materials used in the examples: LB medium: 10 g / L peptone -1 Yeast extract 5 g·L -1 NaCl 10 g·L -1 .
[0032] 2×YT fermentation medium: peptone 16 g·L -1 Yeast extract 10 g·L -1 NaCl 5 g·L -1 .
[0033] High-density medium: 12 g·L -1 Yeast extract 8 g·L -1 NaCl 0.5 g·L -1 Glycerol 20 g·L -1, KH2PO42 g·L -1 K2HPO4·3H2O 8 g·L -1 (NH4)2SO4 2 g·L -1 MgSO4 2g·L -1 Citric Acid·H2O 1 g·L -1 .
[0034] The sources and item numbers of all the above raw materials are shown in Table 1.
[0035] Table 1 Source of Raw Materials
[0036] Example 1: Construction of mutant plasmid using pyrrolidone-tRNA synthetase expressing EGFP(Y39) by Figure 1 The laboratory-preserved plasmid pET-MhPylRS-EGFP(Y39)-His (nucleotide sequence shown in SEQ ID NO. 6) was used as a template. Full plasmid PCR amplification was performed using primers from Table 2. The PCR system and PCR conditions are shown in Table 3. The amino acid sequence of pyrrolidone-tRNA synthetase (MhPylRS) is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 4.
[0037] Table 2 Single-point mutation primers
[0038] Table 3. PCR amplification reaction system for whole plasmids
[0039] Table 4. Reaction conditions for whole plasmid PCR amplification
[0040] The PCR products were purified and transformed into DH5α competent cells. The cells were sequenced and plasmids were extracted to obtain a series of recombinant plasmids expressing EGFP(Y39) and containing pyrrolidone-tRNA synthetase mutants. The mutants included D44G, Y275A, L278A, L278N, D282S, I291A, Y353F, DEL107-111, and tRNA / A76T.
[0041] Using the recombinant plasmid template that expresses EGFP(Y39) and contains the L278N mutant obtained above, full plasmid PCR amplification was performed using the primers in Table 5. The PCR system is shown in Table 3, and the PCR conditions are shown in Table 4.
[0042] Table 5 Combination Mutant Primers
[0043] The PCR product was purified and transformed into DH5α competent cells. The cells were sequenced and plasmids were extracted to obtain a recombinant plasmid containing the pyrrolidone-tRNA synthetase mutant D44Q / L278N. The amino acid sequence of the mutant D44Q / L278N is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.5.
[0044] Example 2: Method and yield of pyrrolidone-tRNA synthetase for inserting NAEK into EGFP(Y39) in wild-type and mutant strains. The pyrrolidone-lysine-tRNA synthetase pair from Example 2 was transformed into BL21(DE3) cells for wild-type and various mutant recombinant plasmids, respectively. Single colonies were picked and cultured in 5 mL LB tubes containing 100 μg / mL spectinomycin at 37°C and 220 r / min for 12 h. The bacterial culture was then measured to the final OD value. 600 0.2 Inoculate into a 50 mL 2YT medium shake flask containing 100 μg / mL spectinomycin and 2 mmol / L NAEK, and incubate at 37°C on a shaker at 220 r / min until growth reaches approximately OD. 600 At 2 o'clock, 0.4 mmol / L IPTG was added for overnight induction.
[0045] Collect the induced bacterial culture, dilute it 5 times, and measure its fluorescence value using an ELISA reader, as shown in Table 6. Figure 2 As shown in the results, the relative fluorescence value of EGFP(Y39) for the wild-type pyrrolidone-tRNA synthetase pair was 27521, while the relative fluorescence value of EGFP(Y39) for the mutant D44Q / L278N was 52868, which was 92.1% higher than that of the wild-type. All other mutants showed an increase of more than 18% compared to the wild-type.
[0046] Table 6. Relative fluorescence values of each mutant strain
[0047] Example 3: Construction of mutant plasmid using pyrrolidone-tRNA synthetase expressing IL2 (Y45) by Figure 3Using the laboratory-preserved plasmid pET-MhPylRS-IL2(Y45) (nucleotide sequence shown in SEQ ID NO.7) as a template, full-plasmid PCR amplification was performed using primers P7 and P8 from Table 2. The PCR system is shown in Table 3, and the PCR conditions are shown in Table 4. The PCR products were purified and transformed into DH5α competent cells. The sequences were then sequenced, and the plasmids were extracted to obtain a series of recombinant plasmids expressing IL2(Y45) and containing the pyrrolidone-tRNA synthetase pair mutant L278N.
[0048] Using the recombinant plasmid template obtained above that expresses IL2(Y45) and contains the L278N mutant, full-plasmid PCR amplification was performed using primers listed in Table 5. The PCR system is shown in Table 3, and the PCR conditions are shown in Table 4. The PCR product was purified and transformed into DH5α competent cells, sequenced, and the plasmid was extracted to obtain the recombinant plasmid expressing IL2(Y45) and containing the pyrrolidone-tRNA synthetase-peptide mutant D44Q / L278N.
[0049] Example 4: Method and yield of pyrrolidone-tRNA synthetase for inserting NAEK into IL2(Y45) in wild-type and mutant D44Q / L278N The pyrrolidone-lysine-tRNA synthetase pair from Example 3 was transformed into BL21(DE3) into wild-type and mutant D44Q / L278N recombinant plasmids, respectively. Single colonies were picked and placed in 5 mL LB tubes containing 100 μg / mL spectinomycin and cultured at 37°C and 220 r / min for 12 h. The bacterial culture in the tubes was then measured to the final OD value. 600 0.127 g was inoculated into a 50 mL high-density medium shake flask containing 100 μg / mL spectinomycin and 1 mmol / L NAEK, and cultured at 37°C on a shaker at 220 r / min until it grew to approximately OD. 600 At 8:00, 0.4 mmol / L IPTG was added for overnight induction.
[0050] Collect and concentrate the induced bacterial culture to OD. 600 5. Load 10 μl of sample for SDS-PAGE gel analysis and identification. The results are as follows: Figure 4 As shown in the figure. Semi-quantitative analysis results showed that the IL2(Y45) production of the mutant D44Q / L278N was increased by 70.6% compared with the wild type.
[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A pyrrolidone-tRNA synthetase mutant, characterized in that, The pyrrolidone-tRNA synthetase mutant is the parent pyrrolidone-tRNA synthetase with the amino acid sequence shown in SEQ ID NO.1, which is mutated by any of the following: (1) the aspartic acid at position 44 is mutated to glycine, the tyrosine at position 275 is mutated to alanine, the leucine at position 278 is mutated to alanine, the aspartic acid at position 282 is mutated to serine, the isoleucine at position 291 is mutated to alanine and / or the tyrosine at position 353 is mutated to phenylalanine; (2) the five amino acids from alanine at position 107 to serine at position 111 are deleted.
2. The pyrrolidone-tRNA synthetase mutant according to claim 1, characterized in that, The pyrrolidone-tRNA synthetase mutant is formed by mutating aspartic acid at position 44 to glutamine and leucine at position 278 to asparagine in the parent pyrrolidone-tRNA synthetase with the amino acid sequence shown in SEQ ID NO.
1.
3. A gene encoding the pyrrolidone-tRNA synthetase mutant according to any one of claims 1 to 2.
4. An orthogonal tRNA mutant gene, characterized in that, The orthogonal tRNA mutant gene is formed by mutating adenine (A) at position 76 of the orthogonal tRNA parent, whose nucleotide sequence is shown in SEQ ID NO.3, to thymine (T).
5. A recombinant vector carrying the gene of claim 3 and / or the mutated gene of claim 4.
6. A recombinant microbial cell expressing the pyrrolidone-tRNA synthetase mutant of any one of claims 1 to 2, or containing the gene of claim 3 or the mutant gene of claim 4, or transformed with the recombinant vector of claim 5.
7. A method for expressing a recombinant protein with site-directed insertion of non-natural amino acids, characterized in that, The method involves site-specific insertion using the pyrrolidone-tRNA synthetase mutant according to any one of claims 1-2, the gene according to claim 3, the mutant gene according to claim 4, the recombinant vector according to claim 5, or the recombinant microbial cell according to claim 6.
8. The method according to claim 7, characterized in that, The non-natural amino acids include, but are not limited to, NAEK, BOCK, and AlocK; the recombinant protein is IL-2 or EGFP.
9. A method for increasing the yield of recombinant proteins expressed in *Escherichia coli*, characterized in that, The method involves using the pyrrolidone-tRNA synthetase mutant according to any one of claims 1-2, or the gene according to claim 3, or the mutant gene according to claim 4, or the recombinant vector according to claim 5, or the recombinant microbial cell according to claim 6 to insert non-natural amino acids into the recombinant protein at specific sites.
10. The method according to claim 9, characterized in that, The non-natural amino acids include, but are not limited to, NAEK, BOCK, and AlocK; the recombinant protein is IL-2 or EGFP.