Polypeptide ligase mutants and methods of making polypeptides

Abstract

The present application provides a polypeptide ligase mutant and a polypeptide preparation method. The polypeptide ligase mutant includes (a) a protein with a mutation based on the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes a mutation at the S297 site; or (b) a protein with more than 70% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity. The polypeptide ligase mutant can solve the problem of poor connection activity of the polypeptide ligase in the prior art and 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] A peptide is a biologically active compound composed of multiple amino acids linked by peptide bonds. It typically consists of 10 to 100 amino acid molecules, linked in the same way as proteins, with a relative molecular mass below 10,000. Compared to small molecule drugs and protein-based drugs, peptide drugs offer higher activity and selectivity, fewer side effects, greater stability, and lower immunogenicity. Peptide drugs are widely used in medical fields such as vaccines, anti-tumor, endocrine, and cardiovascular medicine. As of January 2023, approximately 180 peptide drugs have been marketed globally, showing broad development prospects.

[0003] Currently, solid-phase synthesis (SPPS) remains the primary method for conveniently obtaining non-natural peptides. However, the SPPS process presents challenges such as numerous eluted impurities and difficulties in preparation and purification. Furthermore, the yield of SPPS-synthesized peptides decreases exponentially with chain length, requiring two or more preparative HPLC runs to obtain a product of sufficient purity. For medium-length peptides, the total yield of SPPS is less than 25%.

[0004] In order to synthesize longer polypeptides, the polypeptide is usually divided into several fragments for connection. Existing polypeptide connection methods are divided into two categories: chemical method and enzymatic method. In chemical connection, the C-terminus of the fragment is prone to racemization (except Gly and Pro), and the amino acids of the connected fragments need to be fully protected, resulting in 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 easy to purify. The Wells team modified a serine protease (Subtilisin) derived from Bacillus myloliquefaciens through protein engineering, mutated it at two sites (S221C / P225A), and obtained a polypeptide ligase (Subtiligase) that can connect an acyl donor fragment with an ester at the C-terminus and an acyl acceptor fragment that does not require protection at the N-terminus in aqueous solution. However, subtiligase has low ligation efficiency and instability, which limits its application in industry (Abrahmsen, Lars, et al. "Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution." Biochemistry 30.17(2012):4151-4159.). Subsequently, Enzypep conducted a series of mutation studies and obtained a peptide ligase Omniligase-1 with further improved stability and catalytic substrate spectrum (Toplak, Ana, et al. "From Thiol-Subtilisin to Omniligase: Design and Structure of a Broadly Applicable Peptide Ligase." Computational and Structural Biotechnology Journal 19.10(2021).).

[0005] Although peptide ligases have been commercialized, they still face a number of challenges in practical application. First, their limited catalytic activity towards certain peptide drug fragments restricts their application in drug synthesis. Given these limitations, the development of new peptide ligases that can efficiently and stably connect a variety of peptide drug fragments is urgent and necessary. Summary of the Invention

[0006] The main purpose of the present invention is to provide a polypeptide ligase mutant and its application to solve the problem of poor ligation activity of polypeptide ligase in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a polypeptide ligase mutant is provided, comprising: (a) a protein mutated based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes a mutation at position S297; or (b) a protein having greater than 70% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0008] Further, the mutation is selected from a S297C mutation and any one or more of the following mutations: N194 to N194K; L195 to L195M; S206 to S206A; Y219 to Y219A; V223 to V223M; S258 to S258E; S259 to S259N; S165 to S165E; S320 to S320K; S185 to S185A; P205 to P205G; N294 to N294S; Y285 to Y285A; S237 to S237G; I281 to I281N; T179 to T179S; N163 to N163R, N163G; I183 to I183V or I183N; S238 to S238Q or S238A; S232 to S232N, S232Q or S232E; S280 to S280T, S280L or S280K; G295 to G295C, G295S or G295H; S287 to S287H, S287M or S287K; S287 to S287N, S287H, S287M or S287K; L202 to L202F, L202M, L202A or L202P; R262 to R262L, R262E, R262Q or R262Y; G84 to G84A, G84S, G84Q, G84D, G84S or G84P; K246 to K246A, K246D, K246I, K246L or K246Y; H302 to H302F, H302G, H302T, H302A or H302G; G137 to G137D, G137I, G137C, G137A or G137Y; S221 to S221A, S221R, S221W, S221Y or S221T; T209 to T209A, T209R, T209M, T209S or T209W; T289 to T289R, T289S, T289M, T289K or T289A; I183 to I183V, I183A, I183Q, I183M or I183V; G137 to G137I, G137C, G137D, G137A or G137Y; S177 to S177Q, S177H, S177R, S177K or S177G; K92 to K92A, K92G, K92D, K92E, K92S or K92N; T80 to T80A, T80P, T80S, T80D, T80F, T80G or T80R;Y282 mutates to Y282L, Y282G, Y282H, Y282M, Y282C or Y282AR325 mutates to R325A, R325F, R325G, R325Y, R325E or R325M; S314 mutates to S314N, S314K, S314C, S314R, S314Y or S314N; G242 mutates to G242T, G242A, G242Q, G242L, G242S, G242M or G24 2R; A113 is mutated to A113D, A113P, A113M, A113S, A113E, A113R, or A113T; R325 is mutated to R325N, R325A, R325G, R325Y, R325E, R325M, or R325D; N134 is mutated to N134F, N134D, N134G, N134T, N134R, N134P, N134E, N134L, N134S, N134I, or N134A. The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0009] Further, the mutation includes any one or more of the following amino acid mutations: S297C, S297C+K92N, S297C+T80S, S297C+G84A, S297C+L202F, S297C+K246A, S297C+R262L, S297C+H302F, S297C+N134D, S297C+N134F, S297C+G137D, S297C+N194K, S297C+L195M, S297C+S206A, S297C+Y219A, S297C+S221A, S297C+V223M, S297C+S238Q, S297C+G242Q, S297C+ K246A, S297C+S258E, S297C+S259N, S297C+Y282L, S297C+S287N, S297C+T2 89R, S297C+T289S, S297C+T289M, S297C+T289K, S297C+T289A, S297C+A113D , S297C+A113P, S297C+A113M, S297C+A113S, S297C+S314N, S297C+S314K, S 297C+S314C, S297C+S314R, S297C+S314I, S297C+S314Y, S297C+R325A, S297 C+R325F, S297C+R325G, S297C+R325Y, S297C+R325E, S297C+R325M, S297C+ A113D+S165E, S297C+A113D+S287N, S297C+A113D+G242L, S297C+A113D+S3 20K, S297C+A113D+N134A, S297C+A113D+N134T, S297C+A113D+N134S, S297 C+A113D+N134I, S297C+A113D+S185A, S297C+A113D+T209A, S297C+S314N+I 183V, S297C+S314N+I183N, S297C+S314N+S185A, S297C+S314N+K92A, S297 C+S314N+G242Q, S297C+T289R+S165E, S297C+T289R+G242S, S297C+T289R+G 242C, S297C+T289R+G137D, S297C+T289R+P205G, S297C+T289R+S238Q, S29 7C+T289R+N294S, S297C+T289R+S165E+S314Y, S297C+T289R+S165E+I183V,S297C+T289R+S165E+S314N, S297C+T289R+S165E+Y285A, S297C+T289R+S165E+S287H, S297C+T289R+S165E+S287M, S297C+T289R+S165E+S287K, S297C+T289R+S165E+R325A, S297C+T289R+S165E+S237G, S297C+T289R+S165E+S238A, S297C+T289R+S165E+N134F, S297C+T289R+S165E+N134E, S297C+T289R+S165E+N134L, S297C+T289R+S165E+N294S, S297C+T289R+S165E+I281N, S297C+T289R+S165E+S280T, S297C+T289R+S165E+S280L, S297C+T289R+S165E+S280K, S297C+T289R+S165E+S232N, S297C+T289R+S165E+S232Q, S297C+T289R+S165E+S232E, S297C+T289R+S165E+G242T, S297C+T289R+S165E+G242T, S297C+T289R+S165E+G137I, S297C+T289R+S165E+G137C, S297C+T289R+S165E+S177Q, S297C+T289R+S165E+S177H, S297C+T289R+S165E+S177R, S297C+T289R+S165E+S177K, S297C+T289R+S165E+S177G, S297C+T289R+S165E+T179S, S297C+T289R+S165E+N163R, S297C+T289R+S165E+N163G, S297C+T289R+S165E+S287K, S297C+T289R+S165E+S287K+G84A, S297C+T289R+S165E+S287K+G84D, S297C+T289R+S165E+S287K+G84S, S297C+T289R+S165E+S287K+G84P, S297C+T289R+S165E+S287K+L202F, S297C+T289R+S165E+S287K+L202M, S297C+T289R+S165E+S287K+L202A,S297C+T289R+S165E+S287K+L202P, S297C+T289R+S165E+S287K+K246A, S297C+T289R+S165E+S287K+K246D, S297C+T289R+S165E+S287K+K246I, S297C+T289R+S165E+S287K+K246L, S297C+T289R+S165E+S287K+K246Y, S297C+T289R+S165E+S287K+R262L, S297C+T289R+S165E+S287K+R262E, S297C+T289R+S165E+S287K+R262Q, S297C+T289R+S165E+S287K+R262Y, S297C+T289R+S165E+S287K+H302F, S297C+T289R+S165E+S287K+H302G, S297C+T289R+S165E+S287K+H302T, S297C+T289R+S165E+S287K+H140R, S297C+T289R+S165E+S287K+H140E, S297C+T289R+S165E+S287K+H140V, S297C+T289R+S165E+S287K+H140K, S297C+T289R+S165E+S287K+H140A, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143M, S297C+T289R+S165E+S287K+H143V, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143A, S297C+T289R+S165E+S287K+L202F+L293A, S297C+T289R+S165E+S287K+L202F+L293E, S297C+T289R+S165E+S287K+L202F+L293Q, S297C+T289R+S165E+S287K+L202F+L293Y, S297C+T289R+S165E+S287K+L202F+G295C, S297C+T289R+S165E+S287K+L202F+G295S, S297C+T289R+S165E+S287K+L202F+G295H, S297C+T289R+S165E+S287K+L202F+G295H+T80S,S297C+T289R+S165E+S287K+L202F+G295H+T80A, S297C+T289R+S165E+S287K+L202F+G295H+T80P, S297C+T289R+S165E+S287K+L202F+G295H+N134D, S297C+T289R+S165E+S287K+L202F+G295H+N134F, S297C+T289R+S165E+S287K+L202F+G295H+N134P, S297C+T289R+S165E+S287K+L202F+G295H+G137D, S297C+T289R+S165E+S287K+L202F+G295H+G137A, S297C+T289R+S165E+S287K+L202F+G295H+G137Y, S297C+T289R+S165E+S287K+L202F+G295H+G137C, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242K, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221W, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221Y, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221T,S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282H 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282G 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282M S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282C S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282A S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134D S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134G S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134T S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80F 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80G 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80R 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113R 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113M 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+R325 N、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92E, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S,S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302F、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A or、

[0010] S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302G.

[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] According to a second aspect of the present invention, a DNA molecule is provided, which encodes the above-mentioned polypeptide ligase mutant.

[0013] According to a third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0014] According to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the aforementioned DNA molecule or the aforementioned recombinant plasmid; the host cell is not an animal or plant species.

[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] According to a fifth aspect of the present invention, a method for preparing a polypeptide is provided, which comprises utilizing the above-mentioned 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 unnatural amino acids.

[0018] In a preferred embodiment, when the number of substrate peptide chains is 2, the substrate peptide chains comprise 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:

[0019] 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: 31; 6) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 32, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 33; 7) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 34, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 35.

[0020] By using the technical solution of the present application, the polypeptide ligase mutant has higher activity and a wider substrate spectrum than the polypeptide ligase in the prior art, and can realize the connection of diversified polypeptide fragments. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings accompanying the specification of the present application are used to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0022] Figure 1 A connection diagram of a 5-peptide substrate according to Example 4 of the present application is shown.

[0023] Figure 2 A catalytic result diagram of a polypeptide ligase mutant according to Example 10 of the present application for substrates with different amino acids at the P4 position is shown. DETAILED DESCRIPTION

[0024] 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.

[0025] As mentioned in the background, prior art polypeptide ligases have low activity, resulting in limited catalytic activity for certain specific polypeptide drug fragments, a narrow substrate spectrum, and insufficient stability. Therefore, in this application, the inventors attempted to develop a novel polypeptide ligase mutant, based on which they proposed a series of protection schemes herein.

[0026] 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 S297 site; or (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0027] SEQ ID NO: 1: MEQPAKDVEKDYIVGFKSSVKTASVKKDVIKESGGKVDKQFKIINAA KATLDQDAVKELKNDPSVAYVEEDHVAHALAQTVPYGIPQIKADKVQAQGYKGANVKVGVLDTGIAASHSDLNVVGGASFVSGESYNTDGNGHGTHVAGTVAALDNSIGVLGVAPNVSLYAIKVLNSSGSGTYSAIVSGIEWATANNLDVIN MSLGGPSGSTALKQAVDKAYASGVVVVAAAGNSGTSGSSSTIGYPAKYDSVIAVGAVNSSNQRASFSSVGPELDVVAPGVSIYSTYPSNTYATLNGTSMASPHVAGAAALILSKSPALSASQVRDRLSSTATNLGDSFYYGKGLINVEAAAQ.

[0028] The activity of the peptide ligase in the prior art is low, which results in limited catalytic activity for certain specific peptide drug fragments and insufficient stability. In the early stage of this application, an enzyme library containing 200 proteases was constructed. By testing the activity of these proteases using different peptide 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 through homology modeling and AI prediction methods, and mutated the key catalytic site serine near the active center to cysteine. The ligation activity of the enzyme after single-site 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, 10 peptides were also detected. This indicates that some of these hydrolases retain some hydrolytic activity and exhibit ligation activity after undergoing single-point mutations. However, for the remaining approximately 90% of the enzymes, in addition to losing ligation activity, their hydrolytic activity is significantly reduced after undergoing single-point mutations. 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.

[0029] Through the above tests, the inventors of the present application screened out a single-point mutant of the protease from Bacillus glycinifermentans (SEQ ID NO: 1), which showed high activity when performing polypeptide ligation reactions. 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 S297 site.

[0030] In a preferred embodiment, the mutation is selected from the group consisting of a S297C mutation and any one or more of the following mutations: N194 to N194K; L195 to L195M; S206 to S206A; Y219 to Y219A; V223 to V223M; S258 to S258E; S259 to S259N; S165 to S165E; S320 to S320K; S185 to S185A; P205 to P205G; N294 to N294S; Y285 to Y285A; S237 to S237G; I281 to I281N; T179 to T179S; N163 to N163R, N163G; I183 to I183V or I183N; S238 to S238Q or S238A; S232 to S232N, S232Q or S232E; S280 to S280T, S280L or S280K; G295 to G295C, G295S or G295H; S287 to S287H, S287M or S287K; S287 to S287N, S287H, S287M or S287K; L202 to L202F, L202M, L202A or L202P; R262 to R262L, R262E, R262Q or R262Y; G84 to G84A, G84S, G84Q, G84D, G84S or G84P; K246 to K246A, K246D, K246I, K246L or K246Y; H302 to H302F, H302G, H302T, H302A or H302G; G137 to G137D, G137I, G137C, G137A or G137Y; S221 to S221A, S221R, S221W, S221Y or S221T; T209 to T209A, T209R, T209M, T209S or T209W; T289 to T289R, T289S, T289M, T289K or T289A; I183 to I183V, I183A, I183Q, I183M or I183V; G137 to G137I, G137C, G137D, G137A or G137Y; S177 to S177Q, S177H, S177R, S177K or S177G; K92 to K92A, K92G, K92D, K92E, K92S or K92N; T80 to T80A, T80P, T80S, T80D, T80F, T80G or T80R;Y282 mutates to Y282L, Y282G, Y282H, Y282M, Y282C or Y282AR325 mutates to R325A, R325F, R325G, R325Y, R325E or R325M; S314 mutates to S314N, S314K, S314C, S314R, S314Y or S314N; G242 mutates to G242T, G242A, G242Q, G242L, G242S, G242M or G24 2R; A113 is mutated to A113D, A113P, A113M, A113S, A113E, A113R, or A113T; R325 is mutated to R325N, R325A, R325G, R325Y, R325E, R325M, or R325D; N134 is mutated to N134F, N134D, N134G, N134T, N134R, N134P, N134E, N134L, N134S, N134I, or N134A. The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0031] In a preferred embodiment, the mutation comprises any one or more of the following amino acid mutations: S297C, S297C + K92N, S297C + T80S, S297C + G84A, S297C + L202F, S297C + K246A, S297C + R262L, S297C + H302F, S297C + N134D, S297C + N134F, S297C + G137D, S297C + N194K, S297C + L195M, S297C + S206A, S297C + Y219A, S297C + S221A, S297C + V223M, S297C + S238Q, S297C + G242Q, S297C + K246A, S297C + S258E, S297C + S259N, S297C + Y282L, S297C + S287N, S297C + T289R, S297C + T289S, S297C + T289M, S297C + T289K, S297C + T289A, S297C + A113D, S297C + A113P, S297C + A113M, S297C + A113S, S297C + S314N, S297C + S314K, S297C + S314C, S297C + S314R, S297C + S314I, S297C + S314Y, S297C + R325A, S297C + R325F, S297C + R325G, S297C + R325Y, S297C + R325E, S297C + R325M, S297C + A113D + S165E, S297C + A113D + S287N, S297C + A113D + G242L, S297C + A113D + S320K, S297C + A113D + N134A, S297C + A113D + N134T, S297C + A113D + N134S, S297C + A113D + N134I, S297C + A113D + S185A, S297C + A113D + T209A, S297C + S314N + I183V, S297C + S314N + I183N, S297C + S314N + S185A, S297C + S314N + K92A, S297C + S314N + G242Q, S297C + T289R + S165E, S297C + T289R + G242S, S297C + T289R + G242C, S297C + T289R + G137D, S297C + T289R + P205G, S297C + T289R + S238Q, S297C + T289R + N294S, S297C + T289R + S165E + S314Y,S297C+T289R+S165E+I183V、S297C+T289R+S165E+S314N、S297C+T289R+S165E+Y285A、S297C+T289R+S165E+S287H、S297C+T289R+S165E+S287M、S297C+T289R+S165E+S287K、S297C+T289R+S165E+R325A、S297C+T289R+S165E+S237G、S297C +T289R+S165E+S238A、S297C+T289R+S165E+N134F、S297C+T289R+S165E+N134E、S297C+T289R+S165E+N134L、S297C+T289R+S165E+N294S、S297C+T289R+S165E+I281N、S297C+T289R+S165E+S280T、S297C+T289R+S165E+S280L、S297C+T289R +S165E+S280K、S297C+T289R+S165E+S232N、S297C+T289R+S165E+S232Q、S297C+T289R+S165E+S232E、S297C+T289R+S165E+S232E、S297C+T289R+S165E+G242T、S297C+T289R+S165E+G137I、S297C+T289R+S165E+G137C、S297C+T289R+S165E +S177Q、S297C+T289R+S165E+S177H、S297C+T289R+S165E+S177R、S297C+T289R+S165E+S177K、S297C+T289R+S165E+S177G、S297C+T289R+S165E+T179S、S297C+T289R+S165E+N163R、S297C+T289R+S165E+N163G、S297C+T289R+S165E+S287K 、S297C+T289R+S165E+S287K+G84A、S297C+T289R+S165E+S287K+G84D、 S297C+T289R+S165E+S287K+G84S、S297C+T289R+S165E+S287K+G84P、 S297C+T289R+S165E+S287K+L202F、S297C+T289R+S165E+S287K+L202M、S297C+T289R+S165E+S287K+L202A、S297C+T289R+S165E+S287K+L202P、S297C+T289R+S165E+S287K+K246A、S297C+T289R+S165E+S287K+K246D、 S297C+T289R+S165E+S287K+K246I、S297C+T289R+S165E+S287K+K246L、S297C+T289R+S165E+S287K+K246Y、S297C+T289R+S165E+S287K+R262L、S297C+T289R+S165E+S287K+R262E、S297C+T289R+S165E+S287K+R262Q、S297C+T289R+S165E+S287K+R262Y、S29 7C+T289R+S165E+S287K+H302F、S297C+T289R+S165E+S287K+H302G、S297C+T289R+S165E+S287K+H302T、S297C+T289R+S165E+S 287K+H140R、S297C+T289R+S165E+S287K+H140E、S297C+T289R+S165E+S287K+H140V、S297C+T289R+S165E+S287K+H140K、S297C+ T289R+S165E+S287K+H140A、S297C+T289R+S165E+S287K+H143E、S297C+T289R+S165E+S287K+H143M、S297C+T289R+S165E+S287K+H143V、S297C+T289R+S165E+S287K+H143E、S297C+T289R+S165E+S287K+H143A、S297C+T289R+S165E+S287K+L202F+L293A、S29 7C+T289R+S165E+S287K+L202F+L293E、S297C+T289R+S165E+S287K+L202F+L293Q、S297C+T289R+S165E+S287K+L202F+L293Y、S 297C+T289R+S165E+S287K+L202F+G295C、S297C+T289R+S165E+S287K+L202F+G295S、S297C+T289R+S165E+S287K+L202F+G295H、S297C+T289R+S165E+S287K+L202F+G295H+T80S、S297C+T289R+S165E+S287K+L202F+G295H+T80A 、S297C+T289R+S165E+S287K+L202F+G295H+T80P 、S297C+T289R+S165E+S287K+L202F+G295H+N134D 、S297C+T289R+S165E+S287K+L202F+G295H+N134F S297C+T289R+S165E+S287K+L202F+G295H+N134P S297C+T289R+S165E+S287K+L202F+G295H+G137D S297C+T289R+S165E+S287K+L202F+G295H+G137A S297C+T289R+S165E+S287K+L202F+G295H+G137Y S297C+T289R+S165E+S287K+L202F+G295H+G137C S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242Q S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242A S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242K S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221A S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221R 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221W 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221Y 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221T 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282H, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282C, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134T, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80R,S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113D 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113R 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113M 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+R325 N、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92E, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S,S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A, or

[0032] S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A, or

[0033] The ligation efficiency of the polypeptide ligase is related to the six substrate recognition pockets (S4-S1, S1' and S2') of the enzyme active center, S4-S1 corresponds to the recognition of the amino acids of the acyl donor P4-P3-P2-P1, and S1' and S2' correspond to the recognition of the amino acids of the acyl acceptor P1'-P2'. The polypeptide ligase Omnilgase-1 in the prior art has reduced catalytic activity when the positions of P4 and P1 of the polypeptide substrate have amino acids that affect the catalytic efficiency (such as histidine, glutamic acid, lysine, aspartic acid and some unnatural amino acids, etc.) when catalyzing the ligation of polypeptide substrates. This limits its application in the catalytic synthesis of polypeptide drugs.

[0034] The protease derived from Bacillus glycinifermentans (SEQ ID NO: 1) is modified in the present application, and the mutant of the above-mentioned mutation combination shows better acceptance to the amino acids and unnatural amino acids at the position of P4 of the polypeptide substrate, and has higher catalytic efficiency than Omnilgase-1. Moreover, when the polypeptide ligase mutant obtained in the present application is applied in the ligation reaction of semaglutide and liraglutide, the product purity and yield can both maintain a high level, which indicates that the polypeptide ligase mutant of the present application has high activity, wide substrate spectrum, high selectivity to substrate amino acids, and the polypeptide ligase of the present application has higher stability than Omnilgase-1, and can be applied in the synthesis of polypeptide drugs.

[0035] In a preferred embodiment, the polypeptide ligase mutant comprises a protein having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and further preferably 99% or more homology to the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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).

[0040] 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:

[0041] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0042] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0043] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0044] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0045] 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.

[0046] 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.

[0047] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned polypeptide ligase mutant.

[0048] 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.

[0049] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0050] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the aforementioned DNA molecule or the aforementioned recombinant plasmid; the host cell is not an animal or plant species.

[0051] 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.

[0052] 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.

[0053] In a fifth typical embodiment of the present application, a method for preparing a polypeptide is provided, which comprises utilizing the above-mentioned polypeptide ligase mutant to catalyze the binding of substrate peptide chains to prepare the polypeptide.

[0054] In a preferred embodiment, the number of substrate peptide chains is 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.

[0055] 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:

[0056] 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: 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: The amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 34; preferably, the C-terminus of the first substrate peptide chain contains an acyl group.

[0057] 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.

[0058] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0059] Unless otherwise specified, all reagents in the examples of this application are conventional commercially available products.

[0060] Example 1

[0061] Expression and purification of peptide ligase mutants in Bacillus subtilis:

[0062] The mutant S297C gene (N-terminal or C-terminal His tag) from Bacillus glycinifermentans (SEQ ID NO: 1) was cloned into the Escherichia coli-Bacillus subtilis shuttle expression vector pBE-S between the MLuI and BamHI 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.

[0063] 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.

[0064] 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.

[0065] Example 2

[0066] Expression and purification of peptide ligase mutants in E. coli:

[0067] The mutant S297C gene (N-terminal or C-terminal His tag) from Bacillus glycinifermentans (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.

[0068] 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.

[0069] Example 3

[0070] Expression and purification of peptide ligase mutants in yeast:

[0071] The mutant S297C gene (N-terminal or C-terminal His tag) from Bacillus glycinifermentans (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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Other polypeptide ligase mutants of the present application can be expressed and purified using any of the methods in Examples 1 to 3 to obtain the enzyme solution of the present application, which can be used to catalyze the ligation of polypeptide substrates.

[0076] Example 4

[0077] In this example, a protease derived from Bacillus glycinifermentans (SEQ ID NO: 1) was modified by mutating the key catalytic site serine near the active center to cysteine. A ligation reaction was performed between the mutant S297C and Omnilgase-1 using a pentapeptide (with the unnatural amino acid Aib (α-aminoisobutyric acid) at the P4 position of the acyl donor) as a substrate. The reaction system was as follows:

[0078] A 1 mL reaction system contained: 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), 0.5 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 4 h 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 min, the supernatant aqueous phase was collected and analyzed for conversion by HPLC. The results are shown in Table 1.

[0079] Table 1

[0080]

[0081] Note: In Table 1, * indicates a conversion rate less than 0.1%, ** indicates a conversion rate greater than or equal to 0.1% and less than 10% (excluding 10%), **** indicates a conversion rate greater than or equal to 30% and less than 50% (excluding 50%), ***** indicates a conversion rate greater than or equal to 50% and less than 70% (excluding 70%), and ****** indicates a conversion rate greater than or equal to 70% and less than 90% (excluding 90%). Acyl donor refers to the substrate containing an acyl ester 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.

[0082] 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 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.

[0083] In this example, both mutant S297C and Omniligase-1 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 P4 position, S297C exhibited approximately several hundred times the catalytic activity of Omniligase-1. This suggests that the Bacillus glycinifermentans mutant has catalytic potential in ligation reactions in which the unnatural amino acid Aib is present at the P4 position.

[0084] Example 5

[0085] Enzyme evolution was continued on mutant S297C from Bacillus glycinifermentans. Ac-Asp-Aib-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 2) with an unnatural amino acid at position P4 was used as the acyl donor, and H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4) was used as the acyl acceptor. The resulting peptide ligase mutants were screened. The reaction system was as follows:

[0086] A 1 mL reaction system contains: 2.5 mM acyl donor, 3.75 mM acyl acceptor, enzyme reduced to 0.3 mg / mL, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction is incubated at 25°C for 4 hours. The reaction is then terminated by adding one volume of anhydrous ethanol. After thorough vortexing, the reaction is centrifuged at 8000 rpm for 1 minute. The supernatant aqueous phase is then analyzed for conversion by HPLC.

[0087] After primary and secondary screening, the following mutants showing improved activity were obtained, and the specific results are shown in Table 2.

[0088] Table 2

[0089]

[0090]

[0091] Note: In Table 2, ** 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%), and **** represents a conversion rate greater than or equal to 30% and less than 50%.

[0092] In this example, the single point mutation S297C was used as the parent to construct a combination of mutations, and the P4 position was used as the substrate of Aib for screening and activity verification, thereby obtaining a series of polypeptide ligase mutants with enhanced activity.

[0093] Example 6

[0094] Evolution was continued based on the mutants of Example 5, and the combined mutations were tested under the same reaction conditions as in Example 5. The results are shown in Table 3.

[0095] Table 3

[0096]

[0097]

[0098] Note: In Table 3, * 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 10% and less than 30% (excluding 30%), **** 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%), ****** represents a conversion rate of greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate of greater than or equal to 90%.

[0099] In this example, beneficial mutations were combined to further obtain a series of polypeptide ligase mutants with enhanced activity.

[0100] Example 7

[0101] Mutations were continued based on the mutants of Example 6, and the mutants were tested for activity according to the following reaction system.

[0102] A 1 mL reaction system contained: 5 mM acyl donor (SEQ ID NO: 2), 7.5 mM acyl acceptor (SEQ ID NO: 4), and the enzyme concentration was further reduced to 0.1 mg / mL. The reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP) was used. The reaction was incubated at 25°C for 4 hours. The reaction was then terminated by adding one volume of anhydrous ethanol. After thorough mixing, the reaction was centrifuged at 8000 rpm for 1 minute. The supernatant aqueous phase was collected and analyzed for conversion by HPLC.

[0103] After primary and secondary screening, the following mutants were found to have improved activity. The specific results are shown in Table 4.

[0104] Table 4

[0105]

[0106]

[0107] Note: In Table 4, * 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 10% and less than 30% (excluding 30%), **** 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%), ****** represents a conversion rate of greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate of greater than or equal to 90%.

[0108] In this example, the peptide ligase mutant S297C+T289R+S165E+S287K with the best catalytic effect obtained in Example 6 was used as the parent for further evolution, and the enzyme amount was reduced to 0.1 mg / mL for screening, and a series of peptide ligase mutants with improved activity were further obtained.

[0109] Example 8

[0110] Evolution was continued based on the mutants of Example 7, and the mutants were tested for activity according to the following reaction system.

[0111] A 1 mL reaction system contained: 5 mM acyl donor (SEQ ID NO: 2), 7.5 mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), and the enzyme concentration was further reduced to 0.05 mg / mL. The reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP) was used. 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.

[0112] After primary and secondary screening, the following mutants were found to have improved activity. The specific results are shown in Table 5.

[0113] Table 5

[0114]

[0115]

[0116] Note: In Table 5, * 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 10% and less than 30% (excluding 30%), **** 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%), ****** represents a conversion rate of greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate of greater than or equal to 90%.

[0117] In this example, the mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A with the best catalytic effect obtained in Example 7 was used as the parent for further evolution, and the enzyme amount was reduced to 0.05 mg / mL for screening to further obtain polypeptide ligase mutants with improved activity.

[0118] Example 9

[0119] Enzyme stability test:

[0120] Stability tests were performed on some of the mutants evolved in Examples 4 to 8 (screened using the P4 site as a substrate for Aib) to identify 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.

[0121] 1 mL reaction system includes: 2.5 mM acyl donor (SEQ ID NO: 2), 3.75 mM acyl acceptor (SEQ ID NO: 4), enzyme amount 0.1 mg / mL (treated at 70°C for one hour), reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). Reaction time is 2h at 25°C, then add one volume of absolute ethanol to terminate the reaction, shake well after mixing, centrifuge at 8000 rpm for 1 min to obtain the supernatant water phase, and send to HPLC for detection of conversion rate. The stability test results of part of the mutants are shown in Table 6:

[0122] Table 6

[0123]

[0124]

[0125] Note: The residual activity in Table 6 refers to the conversion rate of the enzyme solution after being treated at 70°C for 1h divided by the conversion rate of the enzyme solution directly reacted without high temperature treatment multiplied by 100%. Among them, * represents residual activity greater than or equal to 10% and less than 30% (not including 30%), ** represents residual activity greater than or equal to 30% and less than 50% (not including 50%), *** represents residual activity greater than or equal to 50% and less than 70% (not including 70%), **** represents residual activity greater than or equal to 70% and less than 90%.

[0126] Example 10

[0127] Substrate spectrum test:

[0128] The catalytic ability of polypeptide ligase mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q for 5-peptide acyl donor with different amino acids at P4 position was tested. The acyl donor is 20 different 5-peptides, the sequence is Ac-Asp-Xxx-Tyr-Ser-Leu-O-Cam-Leu-OH (Xxx refers to 20 different amino acids, including L, Y, C, V, I, M, F (SEQ ID NO: 3), N, S, W, R, A, H, P, A, T, D, G, E and K, corresponding to SEQ ID NO: 5-23) The acyl acceptor is H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4). The reaction system is as follows:

[0129] 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.

[0130] The catalytic results of the above peptide ligase mutants for different amino acids at the P4 position are as follows Figure 2 shown.

[0131] In this example, enzyme evolution screening was performed using a pentapeptide containing the unnatural amino acid Aib at the P4 position as the target substrate, and the resulting polypeptide ligase mutants exhibited a wider substrate adaptability at the P4 position.

[0132] Example 11

[0133] Using the peptide ligase mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q as a catalyst, the ligation reaction of substrate combinations of different lengths was tested (as shown in Table 7), 8+8, 12+12, 15+15, 20+18 (the numbers represent the lengths of the acyl donor and acyl acceptor, respectively). The reaction system is as follows:

[0134] 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 7.

[0135] Table 7

[0136]

[0137]

[0138] Note: In Table 7, S / H refers to the ratio of the amount of enzyme-synthesized product to the amount of enzyme-hydrolyzed substrate.

[0139] The mutants obtained in this example can be used for ligation reactions of polypeptide substrates of different lengths, and all can exhibit good ligation activity and S / H ratio.

[0140] Example 12

[0141] The semaglutide peptide was enzymatically synthesized using 17-mer+14-mer (acyl donor+acyl acceptor) fragment:

[0142] 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), 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), 0.01 mg / mL mutant based on SEQ ID NO: 1 mutant (S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q) enzyme solution, 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 2 h. HPLC detection showed that the donor substrate reaction was complete, the conversion rate was 91% (no product isomer was detected), and the S / H was 42. Then the system was adjusted to pH 1-2, and the protein was denatured. The denatured system was centrifuged at 8000 rpm for 10 min to obtain the supernatant aqueous phase, which was then purified by HPLC, and the product semaglutide was obtained after lyophilization.

[0143] The product purity was >98% by HPLC and Q-NMR detection, and the yield was 74%.

[0144] Example 11

[0145] The liraglutide peptide was enzymatically synthesized using 17-mer+14-mer (acyl donor+acyl acceptor) fragment:

[0146] 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) and 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, and 0.01 mg / mL of SEQ ID NO: A mutant enzyme solution containing mutations based on NO: 1 (S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q) was added to a total volume of 50 mL with buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The pH was adjusted to 7.8-8.2, and the reaction was stirred at 25°C for 3 hours. HPLC analysis confirmed complete reaction of the donor substrate, with a conversion rate of 94% (no product isomers were detected) and an S / H ratio of 47. The system was then acidified to pH 1-2 to denature the protein. The denatured system was centrifuged at 8000 rpm for 10 minutes to obtain the supernatant aqueous phase, which was then purified by HPLC and lyophilized to obtain the product, liraglutide.

[0147] HPLC and Q-NMR detection showed that the product purity was >98% and the yield was 76%.

[0148] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: (1) The present application obtains a ligase with high polypeptide ligation activity through enzyme evolution, and shows good catalytic potential for the reaction of the non-natural amino acid Aib at the P4 position. (2) Using the 5-peptide with Aib at the P4 position as a 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 mutants obtained in the present application can be used for the efficient synthesis of various polypeptide drugs. (5) Compared with traditional chemical methods, the enzymatic method of the present application does not require complex processes and steps, and the operation is simpler and milder. Protein racemization will not occur during the reaction process, no isomers will be generated, the system generates fewer impurities, purification is simple, the purity is high, and it is more suitable for industrial scale-up production.

[0149] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

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: S297C, S297C+K92N, S297C+T80S, S297C+G84A, S297C+L202F, S297C+R262L, S297C+H302F, S297C+N134D, S297C+N134F, S297C+G137D, S297C+N194K, S297C+L195M, S297C+S206A, S297C+Y219A, S297C+S221A, S297C+V223M, S297C+S238Q, S297C+G242Q, S297C+K2 46A, S297C+S258E, S297C+S259N, S297C+Y282L, S297C+S287N, S297C+T2 89R, S297C+T289S, S297C+T289M, S297C+T289K, S297C+T289A, S297C+A1 13D, S297C+A113P, S297C+A113M, S297C+A113S, S297C+S314N, S297C+S3 14K, S297C+S314C, S297C+S314R, S297C+S314I, S297C+S314Y, S297C+R32 5A, S297C+R325F, S297C+R325G, S297C+R325Y, S297C+R325E, S297C+R32 5M, S297C+A113D+S165E, S297C+A113D+S287N, S297C+A113D+G242L, S29 7C+A113D+S320K, S297C+A113D+N134A, S297C+A113D+N134T, S297C+A11 3D+N134S, S297C+A113D+N134I, S297C+A113D+S185A, S297C+A113D+T209 A. S297C+S314N+I183V, S297C+S314N+I183N, S297C+S314N+S185A, S297 C+S314N+K92A, S297C+S314N+G242Q, S297C+T289R+S165E, S297C+T289R +G242S, S297C+T289R+G242C, S297C+T289R+G137D, S297C+T289R+P205G , S297C+T289R+S238Q, S297C+T289R+N294S, S297C+T289R+S165E+S314Y,S297C+T289R+S165E+I183V、S297C+T289R+S165E+S314N、S297C+T289R+S165E+Y285A、S297C+T289R+S165E+S287H、S297C+T289R+S165E+S287M、S2 97C+T289R+S165E+R325A、S297C+T289R+S165E+S237G、S297C+T289R+S165E+S238A、S297C+T289R+S165E+N134F、S297C+T289R+S165E+N134E、S297C +T289R+S165E+N134L、S297C+T289R+S165E+N294S、S297C+T289R+S165E+ I281N、S297C+T289R+S165E+S280T、S297C+T289R+S165E+S280L、S297C+T 289R+S165E+S280K、S297C+T289R+S165E+S232N、S297C+T289R+S165E+S232Q、S297C+T289R+S165E+S232E、S297C+T289R+S165E+G242T、S297C+T289 R+S165E+G137I、S297C+T289R+S165E+G137C、S297C+T289R+S165E+S177Q、S297C+T289R+S165E+S177H、S297C+T289R+S165E+S177R、S297C+T289R+ S165E+S177K、S297C+T289R+S165E+S177G、S297C+T289R+S165E+T179S、S297C+T289R+S165E+N163R、S297C+T289R+S165E+N163G、S297C+T289R+S16 5E+S287K、S297C+T289R+S165E+S287K+G84A、S297C+T289R+S165E+S287K +G84D、S297C+T289R+S165E+S287K+G84S、S297C+T289R+S165E+S287K+G8 4P、S297C+T289R+S165E+S287K+L202F、S297C+T289R+S165E+S287K+L202M、S297C+T289R+S165E+S287K+L202A、S297C+T289R+S165E+S287K+L202P、S297C+T289R+S165E+S287K+K246A、S297C+T289R+S165E+S287K+K246D、S297C+T289R+S165E+S287K+K246I、S297C+T289R+S165E+S287K+K246L、 S297C+T289R+S165E+S287K+K246Y、S297C+T289R+S165E+S287K+R262L、 S297C+T289R+S165E+S287K+R262E、S297C+T289R+S165E+S287K+R262Q、S 297C+T289R+S165E+S287K+R262Y、S297C+T289R+S165E+S287K+H302F、S 297C+T289R+S165E+S287K+H302G、S297C+T289R+S165E+S287K+H302T、S 297C+T289R+S165E+S287K+H140R、S297C+T289R+S165E+S287K+H140E、S297C+T289R+S165E+S287K+H140V、S297C+T289R+S165E+S287K+H140K、S2 97C+T289R+S165E+S287K+H140A、S297C+T289R+S165E+S287K+H143E、S297C+T289R+S165E+S287K+H143M、S297C+T289R+S165E+S287K+H143V、S2 97C+T289R+S165E+S287K+H143A、S297C+T289R+S165E+S287K+L202F+L293A、S297C+T289R+S165E+S287K+L202F+L293E、S297C+T289R+S165E+S28 7K+L202F+L293Q、S297C+T289R+S165E+S287K+L202F+L293Y、S297C+T289R+S165E+S287K+L202F+G295C、S297C+T289R+S165E+S287K+L202F+G29 5S、S297C+T289R+S165E+S287K+L202F+G295H、S297C+T289R+S165E+S287K+L202F+G295H+T80S、S297C+T289R+S165E+S287K+L202F+G295H+T80A、S297C+T289R+S165E+S287K+L202F+G295H+T80P、S297C+T289R+S165E+S287K+L202F+G295H+N134D、S297C+T289R+S165E+S287K+L202F+G295H+ N134F、S297C+T289R+S165E+S287K+L202F+G295H+N134P、S297C+T289R+S165E+S287K+L202F+G295H+G137D、S297C+T289R+S165E+S287K+L202F +G295H+G137A、S297C+T289R+S165E+S287K+L202F+G295H+G137Y、S297C+T289R+S165E+S287K+L202F+G295H+G137C、S297C+T289R+S165E+S287 K+L202F+G295H+G137A+G242Q、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+G242A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+G24 2K、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M、S297C+T289R+S165E+S2 87K+L202F+G295H+G137A+K246A+S221A、S297C+T289R+S165E+S287K+L 202F+G295H+G137A+K246A+S221R、S297C+T289R+S165E+S287K+L202F+G 295H+G137A+K246A+S221W、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+S221T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282H、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282M、S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+Y282C、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282A、S297C+T289R+S165E+S287K+L202 F+G295H+G137A+K246A+N134D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R、S2 97C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D、S297C+ T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80F、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80G、S297C+T289 R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80R、S297C+T289R+ S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113D、S297C+T2 89R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80 S+A113T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R +T80S+A113E+S314A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K2 46A+N134R+T80S+A113E+S314G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314D、S297C+T289R+S165E+S287K+L 202F+G295H+G137A+K246A+N134R+T80S+A113E+S314Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242A、S297C+ T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80 S+A113E+G242R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A +N134R+T80S+A113E+I183A、S297C+T289R+S165E+S287K+L202F+G295H+G1 37A+K246A+N134R+T80S+A113E+I183Q、S297C+T289R+S165E+S287K+L202 F+G295H+G137A+K246A+N134R+T80S+A113E+I183M、S297C+T289R+S165E+S 287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A1 13E+T209M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134 R+T80S+A113E+T209S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209W, S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+K92A, S297C+T289R+S165E+S287K+ L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92G, S297C+T289R+S165 E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92D, S297C+T2 89R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92E, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A1 13E+K92S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R +T80S+A113E+G84A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K2 46A+N134R+T80S+A113E+G84S, S297C+T289R+S165E+S287K+L202F+G295H+ G137A+K246A+N134R+T80S+A113E+G84Q, S297C+T289R+S165E+S287K+L20 2F+G295H+G137A+K246A+N134R+T80S+A113E+H302F, S297C+T289R+S165E+ S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A or S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302G.

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: using 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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