Polypeptide ligase mutants and methods of making polypeptides

Abstract

The application provides a polypeptide ligase mutant and a polypeptide preparation method. The polypeptide ligase mutant comprises: (a) a protein which is mutated based on the amino acid sequence shown in SEQ ID NO: 1, and the mutation comprises a mutation at a S307 site; or (b) a protein which has more than 70% homology with the amino acid sequence defined in (a) and has polypeptide ligase activity. The polypeptide ligase activity in the prior art can be improved, and the polypeptide ligase mutant 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 bioactive compounds composed of multiple amino acids linked by peptide bonds. They typically contain 10 to 100 amino acid residues and have a molecular weight of less than 10,000. Compared to small molecule drugs and protein-based drugs, peptide drugs exhibit higher activity and selectivity, have fewer side effects, are more stable, and have low immunogenicity. Peptide drugs have been widely used in vaccines, anti-cancer, endocrine, and cardiovascular disease treatments. As of January 2023, approximately 180 peptide drugs have been marketed worldwide, demonstrating enormous potential for development.

[0003] Solid-phase synthesis (SPPS) is the primary technique for synthesizing non-natural peptides. However, the synthesis process can be plagued by numerous elution impurities and purification difficulties. Furthermore, SPPS yields decrease exponentially with peptide chain length, often requiring multiple HPLC purifications to obtain a high-purity product. For peptides of medium length, 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. During chemical connection, 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 racemization risk, 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 that do not require N-terminal protection in aqueous solution. However, subtiligase has low ligation efficiency and instability, limiting 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.). Through a series of mutation studies, Enzypep developed Omniligase-1, a peptide ligase with improved stability and a broader substrate spectrum.

[0005] Despite the commercialization of existing peptide ligases, practical applications still face challenges. These ligases lack selectivity for specific amino acid residues, particularly unnatural amino acids and proline, resulting in limited catalytic activity for certain peptide drug fragments and insufficient stability under certain conditions. Therefore, the development of novel peptide ligases that can achieve efficient and stable ligation 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 the polypeptide, so as to solve the problem of poor activity of the 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 of SEQ ID NO: 1, wherein the mutation includes a mutation at position S307; or (b) a protein having greater than 70% homology to the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0009] Furthermore, 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] Furthermore, 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 proteins having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0012] In order to achieve the above object, according to a 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 to the above DNA molecule.

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

[0015] Furthermore, 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, which comprises: 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 chains contain 5 to 30 amino acids. Preferably, the amino acids in the substrate peptide chains contain unnatural 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 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 shown in SEQ ID NO: 4; 2) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 28; 3) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 29; 4) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 30; 5) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is 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 accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The drawings illustrate one illustrative embodiment of the application and, together with the description, serve to explain the application. In the drawings:

[0021] Figure 1 A schematic diagram showing the ligation of 5-peptide substrates according to embodiment 4 of the present application is shown.

[0022] Figure 2 A schematic diagram showing the catalytic results of polypeptide ligase mutants according to embodiment 7 of the present application on substrates with different amino acids at position P1 is shown. DETAILED DESCRIPTION

[0023] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

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

[0025] In a first typical embodiment of the present application, a polypeptide ligase mutant is provided, which comprises: (a) a protein mutated based on the amino acid sequence shown in SEQ ID NO: 1, the mutation including a mutation at position S307; 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 peptide bond synthesis. Subsequently, the inventors of this application obtained the three-dimensional structure of the above-mentioned protease through methods such as homology modeling and AI prediction, and mutated the key catalytic site serine near the active center to cysteine. The ligation activity of the enzyme after single-point mutation was tested using 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)), and analyzed by HPLC after the reaction was completed. The results showed that in about 10% of the mutants, in addition to the hydrolysis product, a product 10 peptide was also detected. This indicates that after a single point mutation, some enzymes retain partial hydrolysis activity and also exhibit ligation activity. However, after the remaining approximately 90% of the enzymes undergo single point mutation, in addition to no ligation activity, the hydrolysis activity is also significantly reduced. This suggests that the key catalytic site serine near the active center is crucial for protease catalysis. However, 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 from Bacillus gobiensis (SEQ ID NO: 1), which showed high activity during the polypeptide ligation reaction. They further modified its active pocket to improve its activity and substrate selectivity, and obtained 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 comprises 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.

[0031] The ligation efficiency of peptide ligases is related to the six substrate recognition pockets (S4-S1, S1', and S2') in the enzyme's active center. 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 ligation of peptide substrates, if amino acids that affect catalytic efficiency (such as histidine, glutamic acid, lysine, aspartic acid, and some unnatural amino acids) are present at the P4 and P1 positions of the peptide substrate, its catalytic activity will be reduced. This limits its application in the catalytic synthesis of peptide drugs. Therefore, Omnilgase-1 has a narrow substrate spectrum (Li, Ruifeng, et al. "Traceless enzymatic protein synthesis without 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 modifies the protease derived from Bacillus gobiensis (SEQ ID NO: 1) to obtain a mutant with the above-mentioned mutation combination, which shows better acceptance of amino acids and non-natural amino acids at the P1 position of the polypeptide substrate and has 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. In addition, 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 comprises 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 were all experimentally explored in the examples of this application, and compared to 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 effect on the catalytic ability of the enzyme is small, 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 to the above amino acid sequence and the same catalytic activity.

[0035] The term "identity" used herein refers to the "homology" between amino acid sequences, that is, the total ratio of identical amino acid residues in an 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%, or more than 99% (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 of the protein provided by sequence (a), are homologous proteins obtained by amino acid mutations.

[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 refer to the fact that amino acids with similar properties will have similar effects when substituted with each other. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" 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] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

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

[0043] Those 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 publicly available artificial intelligence model that can predict the conformation of protein complexes. Its predictions of protein 3D structures are very close to those observed in real experiments using instruments such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thus guiding the study of protein structure and 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 ligated 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, ribosomes, tRNA, etc. to obtain the polypeptide ligase mutant.

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

[0048] In a fourth typical embodiment of the present application, a host cell containing the above-mentioned DNA molecule or the above-mentioned recombinant plasmid is provided; 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] Utilizing the above-mentioned 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. Utilizing existing technology, the host cells are subjected to protein purification by crushing, crude enzyme catalysis after crushing, or other methods to obtain polypeptide ligase mutants, and subsequent catalysis of substrate nucleosides is performed. The host cells are non-plant or animal-derived host cells. The examples of the present application verify that the polypeptide ligase mutants of the present application can be expressed and a high-purity enzyme solution can be obtained using any of the above-mentioned host cells.

[0051] In a fifth typical embodiment of the present application, a method for preparing a polypeptide is provided, wherein the polypeptide ligase mutant is used to catalyze the binding of substrate peptide chains to prepare the polypeptide.

[0052] In a preferred embodiment, the number of substrate peptide chains is 2 to 3. The number of substrate peptide chains is 2; preferably, the substrate peptide chains contain 5 to 30 amino acids. Preferably, the amino acids in the substrate peptide chains include unnatural amino acids.

[0053] In a preferred embodiment, 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 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 shown in SEQ ID NO: 4; 2) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 28; 3) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 29; 4) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 30; 5) the amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is 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 half of the substrate in the peptide ligation reaction, while the second substrate peptide chain refers to the lower half of the substrate in the peptide ligation reaction. In the ligation reaction, because the C-terminus of the first substrate peptide chain contains a protective ester acyl group, it can also be called the acyl donor, and the second substrate peptide chain can be called the acyl acceptor.

[0055] The beneficial effects of the present application will be further explained in detail below with reference to 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) from Bacillus gobiensis (SEQ ID NO: 1) was cloned into the Escherichia coli-Bacillus subtilis shuttle expression vector pBE-S between the MLu I and Bam HI restriction enzyme sites. The recombinant expression plasmid was then transformed into Bacillus subtilis WB600 using 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). Transformants were selected on LB plates containing 50 μg / mL kanamycin sulfate.

[0060] Positive transformants were transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C, 200 rpm, for 16 h. The culture was then inoculated with 1% (v / v) of the inoculum into 500 mL of Terrific broth medium (12 g / L tryptone, 24 g / L yeast extract, 0.4% glycerol) and cultured at 37°C, 200 rpm, for 48 h. After fermentation, the supernatant was collected by centrifugation at 4°C, 8000 rpm, 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 washed 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 exchanged with an ultrafiltration tube to remove imidazole and salts, and stored at -20°C for 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 approximately 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 expression vector pET28a (+) between the restriction enzyme sites Nco I and BamH I and transformed into Escherichia coli BL21 (DE3) competent cells. The bacteria were cultured on LB plates containing 50 μg / mL kanamycin sulfate at 37°C for 16 hours, picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate. The bacteria were 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 incubated at 18°C ​​for 17 h before collecting the bacterial sludge.

[0065] Weigh the bacterial sludge, add lysis buffer (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 take the supernatant for purification. The specific process is: the sample is loaded at a flow rate of 2mL / min, then rinsed with buffer A (25mM Tricine, pH7.5, 0.5MNaCl, 20mM imidazole) until the unbound protein is completely eluted, followed by a linear gradient of imidazole elution for 5 column volumes (imidazole concentration increased from 20mM to 50mM), and then the target protein is eluted at 200mM. The affinity-purified protein is further centrifuged and exchanged with an ultrafiltration tube to remove imidazole and salts, and placed at -20℃ for 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 ligation.

[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 Pichia pastoris expression vector pPICZ A between the restriction enzyme sites EcoR I and Not I. After obtaining the recombinant expression plasmid, the recombinant expression plasmid was linearized at 37°C using Sac I restriction endonuclease. After agarose electrophoresis confirmed 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 transformants were picked and transferred to 10 mL of 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 and 200 rpm for 18 h, then transferred to 100 mL BMGY liquid medium at a 1% (v / v) inoculum, and cultured further at 30°C and 200 rpm for 36 h.

[0069] At room temperature, the bacterial cells were collected by centrifugation at 1500-3000 g 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 methanol was added every 24 h to induce expression at a final concentration of 5 g / L.

[0070] After 5 days of fermentation, the fermentation supernatant was collected by centrifugation at 4°C and 8000rpm for 10 minutes, the precipitate was discarded, and the fermentation supernatant was used for subsequent purification. The specific process is: the sample is loaded at a flow rate of 2mL / min, and then washed with buffer A (20Mm KPB, pH7.5, 0.5M NaCl, 20mM imidazole) until the unbound protein is completely eluted, followed by a linear gradient of imidazole elution for 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 using an ultrafiltration tube to replace the liquid, remove imidazole and salt, and store at -20°C for use.

[0071] The protein concentration was determined by the Bradford method and the purity was analyzed by 12% separating gel SDS-PAGE. The resulting 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 used for peptide fragment ligation.

[0072] The other polypeptide ligase mutants of the present application can be obtained by using any one of the expression and purification methods of Examples 1-3 to obtain an enzyme solution of the mutant, which is used to catalyze the ligation of polypeptide substrates.

[0073] Example 4

[0074] In this example, the protease derived from Bacillus gobiensis (SEQ ID NO: 1) is modified by mutating the key catalytic site serine near the active center to cysteine, and the mutant S307C and Omnilgase-1 are subjected to ligation reaction test for activity using 5-peptide (acyl donor P1 position is non-natural amino acid Aib, alpha-amino isobutyric acid) as substrate. The reaction system is as follows:

[0075] 1 mL reaction system includes: 2.5 mM 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), 3.75 mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), enzyme 0.5 mg / mL, reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction is carried out at 25°C for 4 h, followed by adding one volume of absolute ethanol to terminate the reaction, and after thorough shaking and mixing, centrifuging at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which is subjected to HPLC detection of conversion rate. The specific results are shown in Table 1.

[0076] Table 1

[0077]

[0078] Note: In Table 1, * represents a conversion rate less than 0.1%, ** represents a conversion rate greater than or equal to 0.1% and less than 10% (not including 10%), **** represents a conversion rate greater than or equal to 30% and less than 50% (not including 50%), ***** represents a conversion rate greater than or equal to 50% and less than 70% (not including 70%), and ****** represents a conversion rate greater than or equal to 70% and less than 90% (not including 90%). Acyl donor refers to the ester acyl group at the C-terminus of the first half of the substrate in the ligation reaction; acyl acceptor refers to the second half of the substrate in the ligation reaction.

[0079] The polypeptide ligation reaction scheme in this example is shown in Figure 1 Figure 1 ​Where 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 for 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 known to those skilled in the art that 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 mode and connection purpose of the same groups in the sequence of the remaining substrate peptide chains of this application are the same as described here.

[0080] Both the peptide ligase mutant S307C and Omniligase-1 in this example catalyzed ligation reactions in which the acyl donor did not contain an unnatural amino acid, and exhibited high conversion rates. However, in reactions in which the acyl donor contained the unnatural amino acid Aib at the P1 position, S307C exhibited approximately ten times the catalytic activity of Omniligase-1. This demonstrates that the Bacillus gobiensis mutant possesses excellent catalytic potential for ligation reactions in which the unnatural amino acid Aib is present at the P1 position.

[0081] Example 5

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

[0083] A 1 mL reaction system contained: 5 mM acyl donor Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH (SEQ ID NO: 2), 3.75 mM acyl acceptor H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4), enzyme concentration reduced to 0.05 mg / mL, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was incubated at 25°C for 2 h, followed by termination by the addition of one volume of anhydrous ethanol. After thorough vortexing, the reaction was centrifuged at 8000 rpm for 1 min. The supernatant aqueous phase was collected and analyzed for conversion by HPLC.

[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 enhanced 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. Enzyme solutions were prepared from the mutants according to the methods described in Examples 1, 2, or 3. The enzyme solutions were treated at 70°C for one hour, and then the mutants were tested for activity using the following reaction system.

[0093] A 1 mL reaction system contained: 2.5 mM acyl donor Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH (SEQ ID NO: 2), 3.75 mM acyl acceptor H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4), 0.1 mg / mL enzyme (treated at 70°C for one hour), and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was allowed to proceed for 2 hours at 25°C, followed by termination by the addition of one volume of anhydrous ethanol. After thorough vortexing and centrifugation at 8000 rpm for 1 minute, the supernatant aqueous phase was collected and analyzed for conversion by HPLC. The stability test results of some mutants are shown in Table 3.

[0094] Table 3

[0095] Peptide ligase mutants 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 after the enzyme solution is treated at 70°C for 1 hour, divided by the conversion rate when the enzyme solution is directly used in the reaction without high-temperature treatment, multiplied by 100%. * represents a residual activity greater than or equal to 10% and less than 30% (excluding 30%), ** represents a residual activity greater than or equal to 30% and less than 50% (excluding 50%), *** represents a residual activity greater than or equal to 50% and less than 70% (excluding 70%), and **** represents a 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 its catalytic activity against 5 peptide acyl donors with different amino acids at the P1 position. The acyl donor was 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 NOs: 5-23, respectively), and the acyl acceptor was H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4). The reaction system was as follows:

[0101] A 1mL reaction system contains: 5mM acyl donor, 7.5mM acyl acceptor, 0.05mg / mL enzyme, and reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). Incubate at 25°C for 2 hours. Terminate the reaction by adding one volume of anhydrous ethanol, thoroughly shaken, and centrifuged at 8000rpm for 1 minute. The supernatant aqueous phase was collected and analyzed for conversion by HPLC.

[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 containing the unnatural amino acid Aib at the P1 position as the target substrate, and the resulting mutants 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: 8+8, 12+12, 15+15, and 20+18 (the numbers represent the lengths of the acyl donor and acyl acceptor, respectively). The reaction system is shown below:

[0106] A 1 mL reaction system contained 2.5 mM acyl donor, 3.75 mM acyl acceptor, 0.1 mg / mL enzyme, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was allowed to proceed for 2 h at 25°C. The reaction was then terminated by adding one volume of anhydrous ethanol, thoroughly shaken, and centrifuged at 8000 rpm for 1 min. The supernatant aqueous phase was collected and analyzed for conversion by HPLC. The results are shown in Table 4.

[0107] Table 4

[0108]

[0109]

[0110] Note: In Table 4, S / H 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 a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0113] In a 200 mL four-necked flask, 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) and 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 The reaction mixture was stirred at 25°C for 2 h, followed by a 50 mL volume of 50 mL (Tricine, pH 8.0, 0.8 mg / mL TCEP), and the pH was adjusted to 7.8-8.2. The reaction was stirred at 25°C for 2 h. HPLC analysis confirmed complete reaction of the donor substrate with a conversion of 93% (no product isomers were detected). The S / H ratio was 44. The system was then acidified to pH 1-2, and the protein was denatured. The supernatant aqueous phase was obtained by centrifugation at 8000 rpm for 10 min, which was then purified by preparative HPLC and lyophilized to obtain the product, semaglutide.

[0114] HPLC and Q-NMR detection showed that the product purity 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 flask, 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), 0.01 mg / mL mutant (S307C+S107T+S337I+P311A+G92A+S214A) enzyme solution based on SEQ ID NO: 1, buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP) were added to a total volume of 50 mL, pH was adjusted to 7.8-8.2, and the reaction was stirred at 25°C for 3 h. HPLC detection showed that the donor substrate was completely reacted, the conversion rate was 91% (no product isomer was detected), and S / H was 43. Then the system was acidified to pH 1-2, and the denatured protein was obtained. The system was centrifuged at 8000 rpm for 10 min to obtain the supernatant aqueous phase, which was then purified by preparative HPLC and then lyophilized to obtain the product liraglutide.

[0118] The product purity was >98% and the yield was 73% by HPLC and Q-NMR detection.

[0119] As can be seen from the above description, the above-mentioned embodiments of the present application achieve the following technical effects: (1) Through enzyme evolution, a ligation enzyme with high polypeptide ligation activity is obtained, which shows good catalytic potential for the reaction of P1 position being non-natural amino acid Aib. (2) With 5-peptide with P1 position being Aib as the substrate, further enzyme evolution is carried out, and the activity of the obtained mutant is further improved. These mutants also show higher stability and catalytic ability to a wider substrate spectrum. (3) The obtained polypeptide ligation enzyme mutant can efficiently catalyze the ligation of polypeptide substrates of different lengths. (4) The polypeptide ligation enzyme mutant obtained in the present application can be used for efficient synthesis of various polypeptide drug APIs. (5) Compared with traditional chemical methods, enzyme methods do not require complex processes and steps, are more simple and mild to operate, do not cause racemization during the reaction process, do not generate isomers, have fewer impurities in the system, are simple to purify, and are more suitable for industrialized scale-up production.

[0120] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A polypeptide ligase mutant, characterized in that: The polypeptide ligase mutant is a protein having the following mutations based on the amino acid sequence shown in SEQ ID NO: 1: 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+P311 G. S307C+P311Y, S307C+H346Y, S307C+T88S, S307C+T88A, S307C+S107T+S 337I, S307C+S107T+S337Y, S307C+S107T+S337R, S307C+S107T+S337M, S3 07C+S107T+G92A, S307C+S107T+G92S, S307C+S107T+G92C, S307C+S107T+ N162T, S307C+S107T+N162V, S307C+S107T+N162R, S307C+S107T+N162E, S3 07C+S107T+P311A, S307C+S107T+P311N, S307C+S107T+P311Q, S307C+S10 7T+P311G, S307C+S107T+S337I+H346A, S307C+S107T+S337I+H346G, S307 C+S107T+S337I+H346Y, S307C+S107T+S337I+H346P, S307C+S107T+S337I +P311A, S307C+S107T+S337I+P311N, S307C+S107T+S337I+G92A, S307C+S 107T+S337I+G92R, S307C+S107T+S337I+G92H, S307C+S107T+S337I+P311 A+G92T, S307C+S107T+S337I+P311A+G92A, S307C+S107T+S337I+P311A+G 92F、S307C+S107T+S337I+P311A+G92M、S307C+S107T+S337I+P311A+N162 T、S307C+S107T+S337I+P311A+N162Y、S307C+S107T+S337I+P311A+N162S、S307C+S107T+S337I+P311A+N162C, S307C+S107T+S337I+P311A+N161H, S3 07C+S107T+S337I+P311A+N161C, S307C+S107T+S337I+P311A+N161D, S307C +S107T+S337I+P311A+N161K, S307C+S107T+S337I+P311A+G92A+P140A, S3 07C+S107T+S337I+P311A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P 140V, S307C+S107T+S337I+P311A+G92A+P140R, S307C+S107T+S337I+P311 A+G92A+P140K, S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S33 7I+P311A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T+S337I+P311A+G92A+S214F, or S307C+S107T+S337I+P311A+G92A+S214V.

2. A DNA molecule, characterized in that The DNA molecule encodes the polypeptide ligase mutant according to claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 2.

4. A host cell, characterized in that The host cell contains the DNA molecule according to claim 2 or the recombinant plasmid according to claim 3; the host cell is not an animal or plant species.

5. The host cell according to claim 4, characterized in that The host cell includes a eukaryotic cell or a prokaryotic cell.

6. The host cell according to claim 5, characterized in that The eukaryotic cells include yeast cells.

7. The host cell according to claim 6, characterized in that The yeast cells include Pichia pastoris.

8. The host cell according to claim 5, characterized in that The prokaryotic cell includes Escherichia coli or Bacillus subtilis.

9. The host cell according to claim 8, characterized in that The Escherichia coli includes BL21(DE3).

10. The host cell according to claim 8, characterized in that The Bacillus subtilis includes WB600.

11. A method for preparing a polypeptide, characterized in that: The preparation method comprises: utilizing the polypeptide ligase mutant according to claim 1 to catalyze the binding of substrate peptide chains to prepare the polypeptide.

12. The preparation method according to claim 11, characterized in that The number of substrate peptide chains includes 2 to 3.

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