Polypeptide ligase mutant and preparation method of polypeptide

Abstract

The invention provides a polypeptide ligase mutant and a preparation method of polypeptide. The polypeptide ligase mutant comprises: (a) a protein mutated on the basis of an amino acid sequence as shown in SEQ ID NO: 1, wherein mutation comprises mutation at an S307 site; or (b) protein which has more than 70% of homology with the amino acid sequence limited in (a) and has polypeptide ligase activity. The problem of poor activity of polypeptide ligase in the prior art can be solved, and the method is suitable for the field of polypeptide synthesis.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide synthesis, and in particular to a method for preparing a polypeptide ligase mutant and a polypeptide. Background Art

[0002] Peptides are biologically active compounds formed by multiple amino acids connected by peptide bonds, usually containing 10 to 100 amino acid residues and a molecular weight of less than 10,000. Compared with small molecule drugs and protein drugs, peptide drugs show higher activity and selectivity, fewer side effects on the human body, are more stable, and have low immunogenicity. Peptide drugs have been widely used in the treatment of vaccines, anti-cancer, endocrine and cardiovascular diseases. As of January 2023, there are about 180 peptide drugs on the market worldwide, showing great development potential.

[0003] Solid phase synthesis (SPPS) is the main technology for synthesizing non-natural peptides, but during the synthesis process, problems such as many elution impurities and difficult purification may be encountered. In addition, as the polypeptide chain lengthens, the yield of SPPS decreases exponentially, and multiple HPLC purifications are usually required to obtain high-purity products. For medium-length peptides, the total yield of SPPS is often less than 25%.

[0004] In order to synthesize longer polypeptides, researchers often split the polypeptide into several small fragments and then connect them. Existing connection methods are mainly divided into chemical and enzymatic methods. When connecting by chemical method, the C-terminus of the fragment is prone to racemization, and the connected amino acids need to be fully protected, which leads to poor solubility of the fragment and difficulty in purification. In contrast, enzymatic connection has no risk of racemization, the side chain does not need to be protected, and it is easier to purify. The Wells team modified a serine protease (Subtilisin) from Bacillus myloliquefaciens through protein engineering, and through mutations at two sites (S221C / P225A), created a polypeptide ligase (Subtiligase) that can connect C-terminal ester donor fragments and acyl acceptor fragments without N-terminal protection in aqueous solution. However, subtiligase has low ligation efficiency and instability, which limits its industrial application (Abrahmsen, Lars, et al. "Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution." Biochemistry 30.17 (2012): 4151-4159.). Enzypep has developed Omniligase-1, a peptide ligase with greater stability and a wider catalytic substrate spectrum, through a series of mutation studies.

[0005] Although peptide ligase products have been commercialized, they still face challenges in practical applications. These ligases are not very selective for specific amino acid residues, especially for non-natural amino acids and proline, resulting in limited catalytic activity for certain specific peptide drug fragments and insufficient stability under certain conditions. Therefore, the development of new peptide ligases to achieve efficient and stable connection of various peptide drug fragments has become an urgent and necessary task. Summary of the invention

[0006] The main purpose of the present invention is to provide a polypeptide ligase mutant and a method for preparing a polypeptide, so as to solve the problem of poor activity of polypeptide ligase in the prior art.

[0007] In order to achieve the above object, according to a first aspect of the present invention, a polypeptide ligase mutant is provided, the polypeptide ligase mutant comprising:

[0008] (a) a protein having a mutation based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes a mutation at the S307 site; or (b) a protein having a homology of more than 70% with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0009] Further, the mutation is selected from the S307C mutation and any one or more of the following mutations: the mutation is selected from the S307C mutation and any one or more of the following mutations: S107 mutates to S107T; S154 mutates to S154A; S186 mutates to S186Y; I192 mutates to I192V; S241 mutates to S241N; K272 mutates to K272L; F275 mutates to F275W; S290 mutates to S290G; L303 mutates to L303H; M308 mutates to M308P; T88 mutates to T88S or T88A; S337 mutates to S337I, S337Y, S337R or S337M; P311 mutates to P311A, P311N, P311Y , P311Q or P311G; H346 mutated to H346A, H346G, H346Y or H346P; S214 mutated to S214P, S214Y, S214A, S214F or S214V; N161 mutated to N161H, N162T, N161C, N161D or N161K; P140 mutated to P14 0A, P140G, P140V, P140R, P140K or P140D; G92 is mutated to G92A, G92S, G92C, G92R, G92H, G92T, G92F or G92M; N162 is mutated to N162T, N162V, N162R, N162E, N162Y, N162S or N162C.

[0010] Further, the mutation includes any one or more of the following amino acid mutations: S307C, S307C+S107T, S307C+S337I, S307C+G92A, S307C+P140D, S307C+S154A, S307C+N161D, S307C+N162T, S307C+S186Y, S307C+I192V, S307C+S214P, S307C+S241N, S307C+K272L, S307C+F275W, S307C+S 290G, S307C+L303H, S307C+M308P, S307C+P311A, S307C+P311G, S307C+P311Y, S307C+H346Y, S307C+T88S, S307C+T8 8A, S307C+S107T+S337I, S307C+S107T+S337Y, S307C+S107T+S337R, S307C+S107T+S337M, S307C+S107T+G92A, S307C +S107T+G92S, S307C+S107T+G92C, S307C+S107T+N162T, S307C+S107T+N162V, S307C+S107T+N162R, S307C+S107T+N 162E, S307C+S107T+P311A, S307C+S107T+P311N, S307C+S107T+P311Q, S307C+S107T+P311G, S307C+S107T+S337I+H 346A, S307C+S107T+S337I+H346G, S307C+S107T+S337I+H346Y, S307C+S107T+S337I+H346P, S307C+S107T+S337I+P 311A, S307C+S107T+S337I+P311N, S307C+S107T+S337I+G92A, S307C+S107T+S337I+G92R, S307C+S107T+S337I+G92H , S307C+S107T+S337I+P311A+G92T, S307C+S107T+S337I+P311A+G92A, S307C+S107T+S337I+P311A+G92F , S307C+S107T+S337I+P311A+G92M, S307C+S107T+S337I+P311A+N162T, S307C+S107T+S337I+P311A+N162Y,S307C+S107T+S337I+P311A+N162S, S307C+S107T+S337I+P311A+N162C, S307C+S107T+S337I+P311A+N161H, S307C+S107T+S337I+P311A+N161C, S307C+S107T+S337I+P311A+N161D, S307C+S107T+S337I+P311A+N161K, S307C+S107T+S337I+P311A+G92A+P140A, S307C+S 107T+S337I+P311A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P140V, S307C+S107T+S337I+P311A+G92A+P140R, S307C +S107T+S337I+P311A+G92A+P140K, S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S337I+P311A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T+S337I+P311A+G92A+S214F or S307C+S107T+S337I+P311A+G92A+S214V.

[0011] Furthermore, the polypeptide ligase mutant includes a protein having a homology of more than 75%, more than 80%, more than 85%, more preferably more than 95%, and even more preferably more than 99% with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0012] In order to achieve the above object, according to the second aspect of the present invention, a DNA molecule is provided, which encodes the above polypeptide ligase mutant.

[0013] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected with the above DNA molecule.

[0014] In order to achieve the above object, according to the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or the above recombinant plasmid; the host cell is not an animal or plant variety.

[0015] Further, the host cell includes a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell includes a yeast cell; preferably, the yeast cell includes Pichia pastoris; preferably, the prokaryotic cell includes Escherichia coli or Bacillus subtilis; preferably, Escherichia coli includes BL21 (DE3); preferably, Bacillus subtilis includes WB600.

[0016] In order to achieve the above object, according to the fifth aspect of the present invention, a method for preparing a polypeptide is provided, the method comprising: using the above polypeptide ligase mutant to catalyze the binding of substrate peptide chains to prepare the polypeptide.

[0017] Furthermore, the number of substrate peptide chains includes 2 to 3. Preferably, the number of substrate peptide chains is 2; preferably, the substrate peptide chain contains 5 to 30 amino acids. Preferably, the amino acids in the substrate peptide chain contain non-natural amino acids.

[0018] Further, when the number of substrate peptide chains is 2, the substrate peptide chains include a first substrate peptide chain and a second substrate peptide chain; preferably, the combination of the first substrate peptide chain and the second substrate peptide chain is selected from any one or more of the following groups: 1) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NOs: 2 to 3 or SEQ ID NOs: 5 to 23, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 4; 2) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 28; 3) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 29; 4) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 30; 5) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: NO: 31; 6) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 32, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 33; 7) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 34, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 35; preferably, the C-terminus of the first substrate peptide chain contains an acyl group.

[0019] By applying the technical solution of the present invention, the above-mentioned polypeptide ligase mutant has higher activity than the polypeptide ligase in the prior art, broadens the substrate spectrum of the polypeptide ligase, and can realize the connection of different polypeptide fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0021] Figure 1 A schematic diagram of the connection of 5 peptide substrates in Example 4 of the present invention is shown.

[0022] Figure 2 A schematic diagram showing the catalytic results of the polypeptide ligase mutant according to Example 7 of the present invention on substrates with different amino acids at the P1 position. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] As mentioned in the background art, the peptide ligase in the prior art has low activity, so the selectivity for specific amino acid residues is poor, the substrate spectrum is narrow, the catalytic activity for certain specific peptide drug fragments is limited, and the stability is insufficient. Therefore, in this application, the inventors tried to develop a new peptide ligase mutant, and based on this, proposed a series of protection schemes of this application.

[0025] In a first typical embodiment of the present application, a polypeptide ligase mutant is provided, which polypeptide ligase mutant includes: (a) a protein mutated based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes a mutation at the S307 site; or (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0026] In the early stage of this application, an enzyme library containing 200 proteases was constructed. By testing the activity of these proteases using different polypeptide substrates, it was found that these enzymes only had hydrolysis activity and could not be used for the synthesis of peptide bonds. Subsequently, the inventors of this application obtained the three-dimensional structure of the above-mentioned protease by homology modeling and AI prediction, and mutated the key catalytic site serine near the active center to cysteine, and used 5-peptide substrates (such as Ac-Ala-Asp-Ser-Lys-Leu-O-Cam-Leu-OH (SEQ ID NO: 36) and H-Ala-Leu-Arg-His-Glu-NH2 (SEQ ID NO: 37)) to test the ligation activity of the enzyme after the single-point mutation, and analyzed it by HPLC after the reaction was completed. The results showed that in about 10% of the mutants, in addition to the hydrolysis product, the product 10 peptide was also detected. This shows that after a single-point mutation, some enzymes retain some hydrolysis activity and also show ligation activity. However, after the single-point mutation of the remaining about 90% of the enzymes, in addition to no ligation activity, the hydrolysis activity was significantly reduced. This suggests that the key catalytic site serine near the active center is essential for protease catalysis, but simply mutating it to cysteine ​​to obtain ligation activity is not applicable to most proteases.

[0027] Through the above tests, the inventors of the present application screened out a single-point mutant of the protease derived from Bacillus gobiensis (SEQ ID NO: 1), which had high activity during the polypeptide ligation reaction, and further modified its active pocket to improve its activity and substrate selectivity, thereby obtaining a polypeptide ligase with improved amino acid residue selectivity containing at least an amino acid mutation at the S307 site.

[0028] SEQ ID NO: 1: MESTSLKSKDYIVGFKSSEIGAMSNEVTVSKAGGKLEKQFSIINAAKA TLTDKAVKDLKNNPAVAYIEEDHIATAYEQAYKLKPSAQTVPYGIPHIKADRVQAQGYSGGGVKVAVLDTGIDASHEDLNVVGGASFVPSEPSPYSDGNGHGTHVSGTVAALNNTTGVLGVAPDASLYAVKVLDSAGSGSYSGIVSGIEWATANGMD VINMSLGGSSGSKALKQAVDNAYANDVVVVAAAGNSGSSGGRVNTIGYPAKYSSVIAVGAVDSNNKKAYFSSVGDELEVMAPGVSVQSTLPGNQYTELDGTSMASPHVAGAAALIKSKHPDLSASQIRQRLSDTADYLGDHFYYGNGVINVEAAAN.

[0029] In a preferred embodiment, the mutation is selected from the S307C mutation and any one or more of the following mutations: the mutation is selected from the S307C mutation and any one or more of the following mutations: S107 mutates to S107T; S154 mutates to S154A; S186 mutates to S186Y; I192 mutates to I192V; S241 mutates to S241N; K272 mutates to K272L; F275 mutates to F275W; S290 mutates to S290G; L303 mutates to L303H; M308 mutates to M308P; T88 mutates to T88S or T88A; S337 mutates to S337I, S337Y, S337R or S337M; P311 mutates to P311A, P311N, P311A 11Y, P311Q or P311G; H346 mutated to H346A, H346G, H346Y or H346P; S214 mutated to S214P, S214Y, S214A, S214F or S214V; N161 mutated to N161H, N162T, N161C, N161D or N161K; P140 mutated to P 140A, P140G, P140V, P140R, P140K or P140D; G92 is mutated to G92A, G92S, G92C, G92R, G92H, G92T, G92F or G92M; N162 is mutated to N162T, N162V, N162R, N162E, N162Y, N162S or N162C.

[0030] In a preferred embodiment, the mutation includes any one or more of the following amino acid mutations: S307C, S307C+S107T, S307C+S337I, S307C+G92A, S307C+P140D, S307C+S154A, S307C+N161D, S307C+N162T, S307C+S186Y, S307C+I192V, S307C+S214P, S307C+S241N, S307C+K272L, S307C+F275W, S307C+S290G, S307C +L303H, S307C+M308P, S307C+P311A, S307C+P311G, S307C+P311Y, S307C+H346Y, S307C+T88S, S307C+T88A, S307C+S107T+S33 7I, S307C+S107T+S337Y, S307C+S107T+S337R, S307C+S107T+S337M, S307C+S107T+G92A, S307C+S107T+G92S, S307C+S107T+G9 2C, S307C+S107T+N162T, S307C+S107T+N162V, S307C+S107T+N162R, S307C+S107T+N162E, S307C+S107T+P311A, S307C+S107T +P311N, S307C+S107T+P311Q, S307C+S107T+P311G, S307C+S107T+S337I+H346A, S307C+S107T+S337I+H346G, S307C+S107T+S3 37I+H346Y, S307C+S107T+S337I+H346P, S307C+S107T+S337I+P311A, S307C+S107T+S337I+P311N, S307C+S107T+S337I+G92A , S307C+S107T+S337I+G92R, S307C+S107T+S337I+G92H, S307C+S107T+S337I+P311A+G92T, S307C+S107T+S337I+P311A+G92A, S307C+S107T+S337I+P311A+G92F, S307C+S107T+S337I+P311A+G92M, S307C+S107T+S337I+P311A+N162T, S307C+S107T+S337I+P311A+N162Y,S307C+S107T+S337I+P311A+N162S, S307C+S107T+S337I+P311A+N162C, S307C+S107T+S337I+P311A+N161H, S307C+S107T+S337I+P311A+N161 C. S307C+S107T+S337I+P311A+N161D, S307C+S107T+S337I+P311A+N16 1K、S307C+S107T+S337I+P311A+G92A+P140A、S307C+S107T+S337I+P31 1A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P140V, S307C+S107 T+S337I+P311A+G92A+P140R, S307C+S107T+S337I+P311A+G92A+P140 K. S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S337I+P311 A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T +S337I+P311A+G92A+S214F or S307C+S107T+S337I+P311A+G92A+S214V.

[0031] The connection efficiency of the peptide ligase is related to the six substrate recognition pockets (S4-S1, S1' and S2') in the active center of the enzyme. S4-S1 corresponds to the amino acids that recognize the acyl donor P4-P3-P2-P1, and S1' and S2' correspond to the amino acids that recognize the acyl acceptor P1'-P2'. When the peptide ligase Omnilgase-1 in the prior art catalyzes the connection of peptide substrates, when there are amino acids that affect the catalytic efficiency at the positions of P4 and P1 of the peptide substrate (such as histidine, glutamic acid, lysine, aspartic acid and some non-natural amino acids, etc.), its catalytic activity will be reduced. This limits its application in the catalytic synthesis of peptide drugs. Therefore, the substrate spectrum of Omnilgase-1 is narrow (Li, Ruifeng, et al. "Traceless enzymatic protein synthesiswithout ligation sites constraint." National Science Review 5 (2022): 103-113.), which limits its application in the catalytic synthesis of peptide drugs.

[0032] The present application transforms the protease derived from Bacillus gobiensis (SEQ ID NO: 1) to obtain a mutant of the above-mentioned mutation combination, which shows better acceptance of the amino acid and non-natural amino acid at the P1 position of the polypeptide substrate, and has a higher catalytic efficiency than Omnilgase-1. Moreover, when the polypeptide ligase mutant obtained in the present application is used in the ligation reaction of semaglutide and liraglutide, the product purity and yield can be maintained at a high level, which indicates that the polypeptide ligase mutant of the present application has higher activity, a wider substrate spectrum, and higher selectivity for substrate amino acids, and the polypeptide ligase of the present application is more stable than Omnilgase-1 and can be used in the synthesis of polypeptide drugs.

[0033] In a preferred embodiment, the polypeptide ligase mutant includes a protein having more than 75%, more than 80%, more than 85%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0034] The above amino acid mutations are all experimentally explored in the examples of this application, and compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, they all have the activity of catalyzing the binding of the substrate peptide chain to obtain the target polypeptide. The above mutation sites are all mutations made around the amino acid active site, and such mutations can improve the binding ability and / or catalytic ability of the mutant with the substrate. For mutations far away from the active site, the catalytic ability of the enzyme is less affected, so it is possible to obtain proteins with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or more homology with the above amino acid sequence and the same catalytic activity.

[0035] The homology in this specification refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0036] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those provided by the sequence (a), are homologous proteins obtained by amino acid mutation.

[0037] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0038] Substitution and replacement rules. Generally speaking, the effects of replacing amino acids with similar properties are similar. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0039] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0040] The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

[0041] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0042] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0043] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0044] The "AlphaFold2-Multimer" used in this application is a public artificial intelligence model that can predict the conformation of protein complexes. The prediction of protein three-dimensional structure can be very close to the level observed by cryo-electron microscopes and other equipment in real experiments. It can obtain a more realistic protein structure, thereby guiding the exploration of protein structure and protein activity.

[0045] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned polypeptide ligase mutant.

[0046] The DNA can encode the polypeptide ligase mutant and can be connected to a recombinant vector to form a circular DNA. Both the DNA and the recombinant vector can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the polypeptide ligase mutant.

[0047] In a third typical embodiment of the present application, a recombinant plasmid connected with the above-mentioned DNA molecule is provided.

[0048] In a fourth typical embodiment of the present application, a host cell is provided which contains the above-mentioned DNA molecule or the above-mentioned recombinant plasmid; the host cell is not an animal or plant species.

[0049] In a preferred embodiment, the host cell includes a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell includes a yeast cell; preferably, the yeast cell includes Pichia pastoris; preferably, Pichia pastoris includes X33; preferably, the prokaryotic cell includes Escherichia coli or Bacillus subtilis; preferably, Escherichia coli includes BL21 (DE3); preferably, Bacillus subtilis includes WB600.

[0050] By using the above host cells, the recombinant vector can be replicated in the host cells, and the DNA molecules carried on the recombinant vector can also be transcribed and translated to obtain a large number of polypeptide ligase mutants. By using the prior art, the host cells are crushed for protein purification, crushed and then catalyzed by crude enzymes or other methods to obtain polypeptide ligase mutants, and subsequent catalysis of substrate nucleosides is performed. The host cell is a host cell of non-plant or animal origin. The present application embodiment verifies that the polypeptide ligase mutant of the present application can be expressed and a high-purity enzyme solution can be obtained by using any of the above host cells.

[0051] In a fifth typical embodiment of the present application, a method for preparing a polypeptide is provided, wherein the polypeptide is prepared by utilizing the above-mentioned polypeptide ligase mutant to catalyze the binding of substrate peptide chains.

[0052] In a preferred embodiment, the number of substrate peptide chains includes 2 to 3. The number of substrate peptide chains is 2; preferably, the substrate peptide chain contains 5 to 30 amino acids. Preferably, the amino acids in the substrate peptide chain include non-natural amino acids.

[0053] In a preferred implementation, when the number of substrate peptide chains is 2, the substrate peptide chains include a first substrate peptide chain and a second substrate peptide chain; preferably, the combination of the first substrate peptide chain and the second substrate peptide chain is selected from any one or more of the following groups: 1) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NOs: 2-3 or SEQ ID NOs: 5-23, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 4; 2) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 28; 3) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 29; 4) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 30; 5) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: NO: 31; 6) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 32, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 33; 7) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 34, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 35; preferably, the C-terminus of the first substrate peptide chain contains an acyl group.

[0054] The first substrate peptide chain refers to the upper substrate in the polypeptide ligation reaction, and the second substrate peptide chain refers to the lower substrate in the polypeptide ligation reaction. In the ligation reaction, since the C-terminus of the first substrate peptide chain contains a protective ester acyl group, it can also be called an acyl donor, and the second substrate peptide chain can be called an acyl acceptor.

[0055] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0056] Unless otherwise specified, all reagents in the examples of this application are conventional commercially available products.

[0057] Example 1

[0058] Expression and purification of peptide ligase mutants in Bacillus subtilis:

[0059] The mutant S307C gene (N-terminal or C-terminal His tag) derived from Bacillus gobiensis (SEQ ID NO: 1) was cloned into the restriction enzyme sites MLu I and BamHI of the Escherichia coli-Bacillus subtilis shuttle expression vector pBE-S. After obtaining the recombinant expression plasmid, it was transformed into the Bacillus subtilis WB600 strain by the Spizizen method (Spizizen J. Transformation of biochemically deficient strain of B. subtilis by deoxyribonucleate. Proceeding of National Academy of Science USA, 1958, 44: 1072-1078.), and the transformants were selected on LB plates containing 50 μg / mL kanamycin sulfate.

[0060] Positive transformants were picked into 5 mL LB liquid medium containing 50 μg / mL kanamycin sulfate, cultured at 37°C 200 rpm for 16 h, and then transferred to 500 mL TB medium (Terrific broth medium, 12 g / L tryptone, 24 g / L yeast powder, 0.4% glycerol) at a 1% (v / v) inoculation amount, and cultured at 37°C 200 rpm for 48 h. After the fermentation was completed, the fermentation supernatant was collected by centrifugation at 4°C and 8000 rpm for 10 min, and the precipitate was discarded.

[0061] The fermentation broth was purified after being treated with a 10KDa membrane package. Specific process: the sample was loaded at a flow rate of 2mL / min, and then rinsed with buffer A (25mM Tricine, pH7.5, 0.5M NaCl, 20mM imidazole) until the unbound protein was completely eluted, followed by elution of the impurities with a linear gradient of imidazole for 5 column volumes (the imidazole concentration was increased from 20mM to 50mM), and then the target protein was eluted at 200mM. The affinity-purified protein was further centrifuged and replaced with an ultrafiltration tube to remove imidazole and salt, and stored at -20°C for standby use. The protein concentration was determined by the Bradford method, and the purity was analyzed by 12% separation gel SDS-PAGE. Finally, an aqueous solution (50mM Tricine, 0.1M NaCl, pH8.0) containing about 2mg / mL (purity greater than 90%) of the obtained enzyme was obtained for polypeptide fragment connection.

[0062] Example 2

[0063] Expression and purification of peptide ligase in E. coli:

[0064] The mutant S307C gene (N-terminal or C-terminal His tag) from Bacillus gobiensis (SEQ ID NO: 1) was cloned into the restriction enzyme cutting site Nco I-BamH I of the expression vector pET28a (+) and transformed into the competent Escherichia coli BL21 (DE3). The strain was cultured on an LB plate containing 50 μg / mL of kanamycin sulfate at 37°C for 16 hours, and then picked and inoculated into LB liquid medium containing 50 μg / mL of kanamycin sulfate. The strain was cultured at 37°C until the OD 600 When the concentration was about 0.8, 0.1 mM IPTG was added, and the culture was continued at 18°C ​​for 17 h before collecting the bacterial sludge.

[0065] Weigh the bacterial mud, add lysis solution (50mM Tris-HCl, 500mM NaCl, 0.1% Triton, pH 8.0), use an ultrasonic disruptor to lyse the bacteria, centrifuge the lysate of the bacteria (12000rpm for 20 minutes), and purify the supernatant. The specific process is: the sample is loaded at a flow rate of 2mL / min, and then rinsed with buffer A (25mM Tricine, pH7.5, 0.5MNaCl, 20mM imidazole) until the unbound protein is completely eluted, followed by eluting the impurity protein with a linear gradient imidazole for 5 column volumes (imidazole concentration is increased from 20mM to 50mM), and then eluting the target protein at 200mM. The affinity-purified protein is further centrifuged and replaced with an ultrafiltration tube to remove imidazole and salt, and placed at -20°C for standby use. The protein concentration is determined by the Bradford method, and the purity is analyzed by 12% separation gel SDS-PAGE. Finally, an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing about 5 mg / mL (purity greater than 90%) of the obtained enzyme was obtained and used for polypeptide fragment connection.

[0066] Example 3

[0067] Expression and purification of peptide ligase mutants in yeast:

[0068] The mutant S307C gene (N-terminal or C-terminal His tag) from Bacillus gobiensis (SEQ ID NO: 1) was cloned into the restriction enzyme sites EcoR I and Not I of the Pichia pastoris expression vector pPICZ A. After obtaining the recombinant expression plasmid, the recombinant expression plasmid was linearized at 37°C with Sac I restriction endonuclease. After agarose electrophoresis verified that the enzyme digestion was complete, the linearized plasmid was recovered. The linearized plasmid was transformed into the Pichia pastoris X33 strain by electroporation, spread on a low-salt YPD plate containing 100 mg / mL bleomycin, and cultured at 30°C for 3 days. The transformant was picked into 10 mL BMGY liquid medium (10 g / L yeast powder, 20 g / L tryptone, 100 mM potassium phosphate pH 6.0, 13.4 g / L YNB, 4×10 -4 g / L biotin, 10 g / L glycerol), cultured at 30°C 200 rpm for 18 h, then transferred to 100 mL BMGY liquid culture medium with a 1% (v / v) inoculum, and cultured further at 30°C 200 rpm for 36 h.

[0069] At room temperature, the bacterial cells were collected by centrifugation at 1500-3000g and BMMY liquid medium (10 g / L yeast powder, 20 g / L tryptone, 100 mM potassium phosphate pH 6.0, 13.4 g / L YNB, 4×10 -4 g / L biotin, 5g / L methanol) to resuspend the bacterial cells to OD 600 ≈1.0, cultured at 30°C, 200 rpm, and added with methanol at a final concentration of 5 g / L every 24 h to induce expression.

[0070] After 5 days of fermentation, the supernatant of the fermentation broth was collected by centrifugation at 4°C and 8000rpm for 10min, the precipitate was discarded, and the supernatant of the fermentation broth was used for subsequent purification. The specific process is: the sample is loaded at a flow rate of 2mL / min, and then rinsed with buffer A (20Mm KPB, pH7.5, 0.5M NaCl, 20mM imidazole) until the unbound protein is completely eluted, followed by a linear gradient imidazole elution of 5 column volumes (imidazole concentration increased from 20mM to 100mM), and then the target protein is eluted at 200mM. The affinity-purified protein is further centrifuged and replaced with an ultrafiltration tube to remove imidazole and salt, and stored at -20°C for use.

[0071] The protein concentration was determined by Bradford method and the purity was analyzed by 12% separation gel SDS-PAGE. Finally, an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing 2 mg / mL (purity greater than 90%) of the obtained enzyme was obtained for polypeptide fragment connection.

[0072] The other polypeptide ligase mutants of the present application can be expressed and purified by any of the methods in Examples 1 to 3 to obtain enzyme solutions of the mutants, which are used to catalyze the connection of polypeptide substrates.

[0073] Example 4

[0074] In this example, the protease from Bacillus gobiensis (SEQ ID NO: 1) was modified, the key catalytic site serine near the active center was mutated to cysteine, and the mutant S307C and Omnilgase-1 were subjected to a ligation reaction to test the activity using a pentapeptide (acyl donor P1 position is non-natural amino acid Aib, α-aminoisobutyric acid) as a substrate. The reaction system is as follows:

[0075] 1mL reaction system includes: 2.5mM acyl donor Ac-Asp-Aib-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 2) or Ac-Asp-Phe-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 3), 3.75mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), 0.5mg / mL enzyme, reaction buffer (0.1M Tricine, pH8.0, 0.8mg / mL TCEP). The reaction time is 4h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, the supernatant water phase is obtained by centrifugation at 8000rpm for 1min, and the conversion rate is detected by HPLC. The specific results are shown in Table 1.

[0076] Table 1

[0077]

[0078] Note: In Table 1, * represents a conversion rate of less than 0.1%, ** represents a conversion rate of greater than or equal to 0.1% and less than 10% (excluding 10%), **** represents a conversion rate of greater than or equal to 30% and less than 50% (excluding 50%), ***** represents a conversion rate of greater than or equal to 50% and less than 70% (excluding 70%), and ****** represents a conversion rate of greater than or equal to 70% and less than 90% (excluding 90%). Acyl donor refers to the substrate with an ester acyl group at the C-terminus in the first half of the ligation reaction; acyl acceptor refers to the substrate in the second half of the ligation reaction.

[0079] The schematic diagram of the polypeptide ligation reaction in this example is as follows Figure 1 As shown, Figure 1Where P1~P4 and P1'~P2' refer to the amino acid sites of the above-mentioned acyl donor and acyl acceptor, respectively. In the sequence of the above-mentioned acyl donor, "Ac" represents the acetyl group connected to the N-terminus of the 5-peptide substrate, and "O-Cam" represents the carboxamide methyl ester group connected to the C-terminus of the 5-peptide substrate. After the "O-Cam", a "Leu-OH" group is also connected, which is a leucine + hydroxyl group. The purpose of connecting the acetyl group at the N-terminus is to prevent the polypeptide ligase from self-ligating; the "O-Cam" group is a protective group at the C-terminus of the polypeptide substrate, and the "Leu-OH" group is a group connected after the "O-Cam" group. The "Leu" connected after "O-Cam" is an amino acid residue that is known to those skilled in the art and can improve the ligation efficiency in the ligation reaction. The specific structure of "O-Cam-Leu-OH" is as follows Figure 1 As shown in , the connection method and connection purpose of the same groups in the sequence of the remaining substrate peptide chains of the present application are the same as described here.

[0080] The polypeptide ligase mutant S307C and Omniligase-1 in this example can both catalyze the ligation reaction without non-natural amino acids in the acyl donor and show a high conversion rate. However, in the reaction where the acyl donor P1 position is the non-natural amino acid Aib, the catalytic activity of S307C is about several dozen times that of Omniligase-1. This indicates that the mutant derived from Bacillus gobiensis has excellent catalytic potential in the ligation reaction where the non-natural amino acid Aib exists at the P1 position.

[0081] Example 5

[0082] The mutant S307C from Bacillus gobiensis (SEQ ID NO: 1) was further subjected to enzyme evolution, with Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH as the acyl donor and H-Ala-Tyr-Gln-Lys-Ser-NH2 as the acyl acceptor, and the evolved mutant was tested for activity by ligation reaction. The reaction system is as follows:

[0083] 1mL reaction system includes: 5mM acyl donor Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH (SEQ ID NO: 2), 3.75mM acyl acceptor H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4), enzyme amount reduced to 0.05mg / mL, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation is performed at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC for detection of conversion rate.

[0084] After primary and secondary screening, the following mutants were found to have improved activity. The specific results are shown in Table 2.

[0085] Table 2

[0086]

[0087]

[0088] Note: In Table 2, * represents a conversion rate less than 0.1% (excluding 0.1%), ** represents a conversion rate greater than or equal to 0.1% and less than 10% (excluding 10%), *** represents a conversion rate greater than or equal to 10% and less than 30% (excluding 30%), **** represents a conversion rate greater than or equal to 30% and less than 50% (excluding 50%), ***** represents a conversion rate greater than or equal to 50% and less than 70% (excluding 70%), and ****** represents a conversion rate greater than or equal to 70% and less than 90%.

[0089] In this example, the single point mutation S307C was used as the parent for evolution, and the P1 position was used as the substrate of Aib for screening and activity testing to obtain a series of polypeptide ligase mutants with improved activity.

[0090] Example 6

[0091] Enzyme stability test:

[0092] Stability tests were performed on some of the mutants evolved in Example 5 to screen for mutants with improved stability. The mutants were prepared into enzyme solutions according to the method described in Example 1, 2 or 3, and the enzyme solutions were treated at 70° C. for one hour, and then the mutants were tested for activity according to the following reaction system.

[0093] 1mL reaction system includes: 2.5mM acyl donor Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH (SEQ ID NO: 2), 3.75mM acyl acceptor H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4), enzyme amount 0.1mg / mL (treated at 70℃ for one hour), reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation at 8000rpm for 1min is performed to obtain the supernatant aqueous phase, which is sent to HPLC for detection of conversion rate. The stability test results of some mutants are shown in Table 3.

[0094] Table 3

[0095] Peptide ligase mutant Residual activity Omniligase-1 * S307C ** S307C+S107T ** S307C+S337I ** S307C+G92A ** S307C+P140D ** S307C+S107T+G92A *** S307C+S107T+G92S ** S307C+S107T+G92C *** S307C+S107T+N162T ** S307C+S107T+N162V ** S307C+S107T+N162R ** S307C+S107T+N162E ** S307C+S107T+S337I+P311A+N161D *** S307C+S107T+S337I+P311A+N161K ** S307C+S107T+S337I+P311A+G92A+P140G ** S307C+S107T+S337I+P311A+G92A+P140V ** S307C+S107T+S337I+P311A+G92A+P140K *** S307C+S107T+S337I+P311A+G92A+S214Y *** S307C+S107T+S337I+P311A+G92A+S214A ****

[0096] Note: The residual activity in Table 3 refers to the conversion rate of the enzyme solution treated at 70°C for 1 hour divided by the conversion rate of the enzyme solution directly reacted without high temperature treatment multiplied by 100%. Among them, * represents the residual activity greater than or equal to 10% and less than 30% (excluding 30%), ** represents the residual activity greater than or equal to 30% and less than 50% (excluding 50%), *** represents the residual activity greater than or equal to 50% and less than 70% (excluding 70%), and **** represents the residual activity greater than or equal to 70% and less than 90%.

[0097] The thermal stability of the polypeptide ligase mutant in this example is significantly improved compared to Omniligase-1.

[0098] Example 7

[0099] Substrate spectrum test:

[0100] The peptide ligase mutant S307C+S107T+S337I+P311A+G92A+S214A was used as a catalyst to test the catalytic ability of 5 peptide acyl donors with different amino acids at the P1 position. The acyl donor is Ac-His-Ala-Gly-Ser-Xxx-O-Cam-Leu-OH (Xxx refers to 20 different amino acids, including I, A (SEQ ID NO: 3), W, Y, V, K, Q, F, M, P, T, N, E, H, S, L, R, G, C and D, corresponding to SEQ ID NO: 5-23, respectively), and the acyl acceptor is H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4). The reaction system is as follows:

[0101] 1mL reaction system includes: 5mM acyl donor, 7.5mM acyl acceptor, 0.05mg / ml enzyme, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifuge at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC to detect the conversion rate.

[0102] The catalytic results of the above peptide ligase mutants for different amino acids at the P1 position are as follows Figure 2 shown.

[0103] In this example, enzyme evolution screening was performed using a pentapeptide with the non-natural amino acid Aib at the P1 position as the target substrate, and the mutants obtained exhibited a wider substrate adaptability at the P1 position.

[0104] Example 8

[0105] The peptide ligase mutant S307C+S107T+S337I+P311A+G92A+S214A was used as a catalyst to test the ligation reaction of substrate combinations of different lengths (as shown in Table 7), 8+8, 12+12, 15+15, 20+18 (the numbers represent the lengths of the acyl donor and the acyl acceptor, respectively), and the reaction system is as follows:

[0106] 1mL reaction system includes: 2.5mM acyl donor, 3.75mM acyl acceptor, enzyme amount 0.1mg / mL, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation is performed at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC to detect the conversion rate. The reaction results are shown in Table 4.

[0107] Table 4

[0108]

[0109]

[0110] Note: S / H in Table 4 refers to the ratio of the amount of enzyme synthesis product to the amount of enzyme hydrolyzed substrate.

[0111] Example 9

[0112] Enzymatic synthesis of semaglutide using 17-mer + 14-mer (acyl donor + acyl acceptor) fragments:

[0113] In a 200 mL four-necked bottle, 5 mM acyl donor His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Gly-Gln-O-Cam-Leu-OH (SEQ ID NO: 32), 7.5 mM acyl acceptor H-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-NH2 (SEQ ID NO: 33) were added, 0.01 mg / mL of the mutant (S307C+S107T+S337I+P311A+G92A+S214A) enzyme solution based on SEQ ID NO: 1 was added, and buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP) to a total volume of 50 mL, adjust pH = 7.8-8.2, stir at 25 ° C for 2 hours, HPLC detection of donor substrate reaction is complete, conversion rate 93% (no product isomers detected), S / H 44. Then adjust the system to pH = 1-2, denature the protein. The body is centrifuged at 8000 rpm for 10 minutes to obtain the supernatant water phase, which is then purified by preparative HPLC and then freeze-dried to obtain the product semaglutide.

[0114] Detection by HPLC and Q-NMR showed that the purity of the product was >98% and the yield was 75%.

[0115] Example 10

[0116] Enzymatic synthesis of liraglutide using a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0117] In a 200 mL four-necked bottle, 5 mM acyl donor His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-O-Cam-Leu-OH (SEQ ID NO: 34), 7.5 mM acyl acceptor H-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-NH2 (SEQ ID NO: 35) were added, 0.01 mg / mL of the mutant (S307C+S107T+S337I+P311A+G92A+S214A) enzyme solution based on SEQ ID NO: 1 was added, and buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP) to a total volume of 50 mL, adjust pH to 7.8-8.2, stir and react at 25°C for 3 h, HPLC detection shows that the donor substrate reaction is complete, the conversion rate is 91% (no product isomers are detected), S / H 43. Then the system is acidified to pH = 1-2 to denature the protein. The system is centrifuged at 8000 rpm for 10 min to obtain a supernatant aqueous phase, which is then purified by preparative HPLC and then freeze-dried to obtain the product liraglutide.

[0118] HPLC and Q-NMR detection showed that the product had a purity of >98% and a yield of 73%.

[0119] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: (1) Through enzyme evolution, a ligase with high polypeptide ligation activity is obtained, which shows good catalytic potential for the reaction of the non-natural amino acid Aib at the P1 position. (2) Taking the 5-peptide with Aib at the P1 position as the substrate, further enzyme evolution is carried out to obtain mutants with further improved activity. These mutants also show higher stability and catalytic ability for a wider substrate spectrum. (3) The obtained polypeptide ligase mutants can efficiently catalyze the synthesis of polypeptide substrates of different lengths. (4) The polypeptide ligase mutants obtained in this application can be used for the efficient synthesis of a variety of polypeptide drug APIs. (5) Compared with traditional chemical methods, the enzymatic method does not require complicated processes and steps, and the operation is simpler and gentler. No racemization will occur during the reaction process, no isomers will be generated, the system generates fewer impurities, and the purification is simple, which is more suitable for industrial scale-up production.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide ligase mutant, characterized in that: The polypeptide ligase mutants include: (a) a protein having a mutation based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation comprises a mutation at the S307 site; or (b) A protein having 70% or more homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

2. The polypeptide ligase mutant according to claim 1, characterized in that The mutation is selected from the group consisting of S307C mutation and any one or more of the following mutations: S107 mutated to S107T; S154 mutated to S154A; S186 mutated to S186Y; I192 mutated to I192V; S241 mutated to S241N; K272 mutated to K272L; F275 mutated to F275W; S290 mutated to S290G; L303 mutated to L303H; M308 mutated to M308P; T88 mutated to T88S or T88A; S337 mutation to S337I, S337Y, S337R, or S337M; P311 mutated to P311A, P311N, P311Y, P311Q, or P311G; H346 mutated to H346A, H346G, H346Y, or H346P; S214 mutation to S214P, S214Y, S214A, S214F, or S214V; N161 mutation is N161H, N162T, N161C, N161D, or N161K; P140 mutated to P140A, P140G, P140V, P140R, P140K, or P140D; G92 mutation is G92A, G92S, G92C, G92R, G92H, G92T, G92F or G92M; N162 mutation is N162T, N162V, N162R, N162E, N162Y, N162S or N162C.

3. The polypeptide ligase mutant according to claim 1, characterized in that The mutation includes any one or more of the following amino acid mutations: S307C、S307C+S107T、S307C+S337I、S307C+G92A、S307C+P140D、S307C+S154A、S307C+N161D、S307C+N162T、S307C+S186Y、S307C+I192V、S307C+S214P、S307C+S241N、S307C+K272L、S307C+F275W、S307C+S290G、S307C+L303H、S307C+M308P、S307C+P311A、S307C+P311G、S307C+P311Y、S307C+H346Y、S307C+T88S、S307C+T88A、S307C+S107T+S337I、S307C+S107T+S337Y、S307C+S107T+S337R、S307C+S107T+S337M、S307C+S107T+G92A、S307C+S107T+G92S、S307C+S107T+G92C、S307C+S107T+N162T、S307C+S107T+N162V、S307C+S107T+N162R、S307C+S107T+N162E、S307C+S107T+P311A、S307C+S107T+P311N、S307C+S107T+P311Q、S307C+S107T+P311G、S307C+S107T+S337I+H346A、S307C+S107T+S337I+H346G、S307C+S107T+S337I+H346Y、S307C+S107T+S337I+H346P、S307C+S107T+S337I+P311A、S307C+S107T+S337I+P311N、S307C+S107T+S337I+G92A、S307C+S107T+S337I+G92R、S307C+S107T+S337I+G92H、S307C+S107T+S337I+P311A+G92T、S307C+S107T+S337I+P311A+G92A、S307C+S107T+S337I+P311A+G92F、S307C+S107T+S337I+P311A+G92M、S307C+S107T+S337I+P311A+N162T、S307C+S107T+S337I+P311A+N162Y、S307C+S107T+S337I+P311A+N162S、S307C+S107T+S337I+P311A+N162C、S307C+S107T+S337I+P311A+N161H, S307C+S107T+S337I+P311A+N161 C. S307C+S107T+S337I+P311A+N161D, S307C+S107T+S337I+P311A+N16 1K、S307C+S107T+S337I+P311A+G92A+P140A、S307C+S107T+S337I+P31 1A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P140V, S307C+S107 T+S337I+P311A+G92A+P140R, S307C+S107T+S337I+P311A+G92A+P140 K. S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S337I+P311 A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T +S337I+P311A+G92A+S214F or S307C+S107T+S337I+P311A+G92A+S214V. , 4. The polypeptide ligase mutant according to any one of claims 1 to 3, characterized in that The polypeptide ligase mutant includes a protein having a homology of more than 75%, more than 80%, more than 85%, more preferably more than 95%, and further preferably more than 99% with the amino acid sequence defined in (a) and having polypeptide ligase activity.

5. A DNA molecule, characterized in that The DNA molecule encodes the polypeptide ligase mutant according to any one of claims 1 to 4.

6. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 5.

7. A host cell, characterized in that The host cell contains the DNA molecule according to claim 5 or the recombinant plasmid according to claim 6; the host cell is not an animal or plant species.

8. The host cell according to claim 7, characterized in that The host cell includes a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cell comprises a yeast cell; Preferably, the yeast cell comprises Pichia pastoris; Preferably, the prokaryotic cell comprises Escherichia coli or Bacillus subtilis; Preferably, the Escherichia coli comprises BL21(DE3); Preferably, the Bacillus subtilis includes WB600.

9. A method for preparing a polypeptide, characterized in that: The preparation method comprises: using the polypeptide ligase mutant according to any one of claims 1 to 4 to catalyze the binding of substrate peptide chains to prepare the polypeptide.

10. The preparation method according to claim 9, characterized in that: The number of the substrate peptide chains includes 2 to 3.

Images