Polypeptide ligase mutant and preparation method of polypeptide

Abstract

The invention provides a polypeptide ligase mutant and a preparation method of polypeptide. The polypeptide ligase mutant comprises (a) a protein mutated on the basis of an amino acid sequence as shown in SEQ ID NO: 1, wherein mutation comprises mutation at an S297 site; or (b) protein which has more than 70% of homology with the amino acid sequence limited in (a) and has polypeptide ligase activity. The problem that in the prior art, polypeptide ligase is poor in ligation activity can be solved, and the method is suitable for the field of polypeptide synthesis.

Description

Technical Field

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

[0002] A polypeptide is a compound composed of multiple amino acids connected by peptide bonds and has certain biological activity. It is usually composed of 10 to 100 amino acid molecules, and its connection method is the same as that of proteins, with a relative molecular mass of less than 10,000. Compared with small molecule drugs and protein drugs, polypeptide drugs have higher activity and selectivity, less side effects on the human body, higher stability, and lower immunogenicity. Polypeptide drugs are widely used in medical fields such as vaccines, anti-tumor, endocrine, and cardiovascular. As of January 2023, there are about 180 polypeptide drugs on the market worldwide, with broad development prospects.

[0003] At present, solid phase synthesis (SPPS) is still the main way to easily obtain non-natural peptides. However, in the SPPS process, there are many problems such as elution impurities and difficulty in preparation and purification. In addition, the yield of SPPS-synthesized peptides decreases exponentially with chain length, and two or more preparative HPLCs are required to obtain products of sufficient purity. For medium-length peptides, the total yield of SPPS is less than 25%.

[0004] In order to synthesize longer polypeptides, the polypeptides are 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 fragments and difficulty in purification. In contrast, enzyme 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 amyloliquefaciens 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.). Afterwards, Enzypep conducted a series of mutation studies and obtained the 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] At present, although peptide ligases have been commercialized, they still face a series of challenges in practical applications. First, their catalytic activity on certain specific peptide drug fragments is limited, which limits their application range in drug synthesis. In view of these limitations, it is particularly urgent and necessary to develop new peptide ligases to achieve efficient and stable connection of various peptide drug fragments. Summary of the invention

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

[0007] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a polypeptide ligase mutant is provided, which comprises: (a) a protein mutated based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation comprises 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.

[0008] Further, the mutation is selected from the S297C mutation and any one or more of the following mutations: N194 mutates to N194K; L195 mutates to L195M; S206 mutates to S206A; Y219 mutates to Y219A; V223 mutates to V223M; S258 mutates to S258E; S259 mutates to S259N; S165 mutates to S165E; S320 mutates to S320K; S185 mutates to S185A; P205 mutates to P205G; N294 mutates to N294S; Y285 mutates to Y285A; S237 mutates to S237G; I281 mutates to I281N; T179 mutates to T 179S; N163 mutated to N163R, N163G; I183 mutated to I183V or I183N; S238 mutated to S238Q or S238A; S232 mutated to S232N, S232Q or S232E; S280 mutated to S280T, S280L or S280K; G295 mutated to G295C, G295S or G295H; S287 mutated to S287H, S287M or S287K; S287 mutated to S287N, S287H, S287M or S287K; L202 mutated to L202F, L202M, L202A or L202P; R262 mutated to R26 2L, R262E, R262Q or R262Y; G84 mutated to G84A, G84S, G84Q, G84D, G84S or G84P; K246 mutated to K246A, K246D, K246I, K246L or K246Y; H302 mutated to H302F, H302G, H302T, H302A or H302G; G137 mutated to G137D, G137I, G137C, G137A or G137Y; S221 mutated to S221A, S221R, S221W, S221Y or S221T; T209 mutated to T209A, T209R, T209M, T209 9S or T209W; T289 mutated to T289R, T289S, T289M, T289K or T289A; I183 mutated to I183V, I183A, I183Q, I183M or I183V; G137 mutated to G137I, G137C, G137D, G137A or G137Y; S177 mutated to S177Q, S177H, S177R, S177K or S177G; K92 mutated to K92A, K92G, K92D, K92E, K92S or K92N; T80 mutated to T80A, T80P, T80S, T80D, T80F, T80G or T80R;Y282 mutates to Y282L, Y282G, Y282H, Y282M, Y282C or Y282A R325 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 G242A 2R; A113 mutates to A113D, A113P, A113M, A113S, A113E, A113R or A113T; R325 mutates to R325N, R325A, R325G, R325Y, R325E, R325M or R325D; N134 mutates 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,<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S314N、S297C+T289R+S165E+Y285A、S297C+T289R+ S165E+S287H、S297C+T289R+S165E+S287M、S297C+T289R+S165E+S287K、S 297C+T289R+S165E+R325A, S297C+T289R+S165E+S237G, S297C+T289R+S165E+S238A, S297C+T289R+S165E+N134F, S297C+T289R+S165E+N134E, S29 7C+T289R+S165E+N134L、S297C+T289R+S165E+N294S、S297C+T289R+S165 E+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+T 289R+S165E+G242T, S297C+T289R+S165E+G137I, S297C+T289R+S165E+G137C, S297C+T289R+S165E+S177Q, S297C+T289R+S165E+S177H, S297C+T2 89R+S165E+S177R, S297C+T289R+S165E+S177K, S297C+T289R+S165E+S177G, S297C+T289R+S165E+T179S, S297C+T289R+S165E+N163R, S297C+T289 R+S165E+N163G、S297C+T289R+S165E+S287K、S297C+T289R+S165E+S287K +G84A、S297C+T289R+S165E+S287K+G84D、S297C+T289R+S165E+S287K+G8 4S、S297C+T289R+S165E+S287K+G84P、S297C+T289R+S165E+S287K+L202F 、S297C+T289R+S165E+S287K+L202M、S297C+T289R+S165E+S287K+L202A、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+L202P, S297C+T289R+S165E+S287K+K246A, S297C+T289R+S165E+S287K+K246D, S297C+T289R+S165E+S287K+K246I, S2 97C+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+T2 89R+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+S1 65E+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+L2 02F+G295C、S297C+T289R+S165E+S287K+L202F+G295S、S297C+T289R+S165E+S287K+L202F+G295H、S297C+T289R+S165E+S287K+L202F+G295H+T80S、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+L202F+G295H+T80A、S297C+T289R+S165E+S2 87K+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+G1 37D、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+S28 7K+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+S287 K+L202F+G295H+G137A+K246A+S221R、S297C+T289R+S165E+S287K+L202F +G295H+G137A+K246A+S221W、S297C+T289R+S165E+S287K+L202F+G295H+G 137A+K246A+S221Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L<h2 style=";text-align:left;direction:ltr">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+A113DS297C+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、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134 R+T80S+A113E+T209R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+T209M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209S、S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K9 2A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N1 34R+T80S+A113E+K92D、S297C+T289R+S165E+S287K+L202F+G295H+G137A +K246A+N134R+T80S+A113E+K92E、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+K92S、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84A、S297C+T289R+S165 E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S、S297C+T2 89R+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 a protein having a homology of more than 75%, more than 80%, more than 85%, more preferably more than 95%, and even more preferably more than 99% with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0012] 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 the 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 above-mentioned DNA molecule or the above-mentioned recombinant plasmid; the host cell is not an animal or plant species.

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

[0016] According to a fifth aspect of the present invention, a method for preparing a polypeptide is provided, the method comprising using 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 non-natural amino acids.

[0018] In a preferred implementation, when the number of substrate peptide chains is 2, the substrate peptide chains include a first substrate peptide chain and a second substrate peptide chain; preferably, the combination of the first substrate peptide chain and the second substrate peptide chain is selected from any one or more of the following groups:

[0019] 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: NO:34, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO:35.

[0020] By applying the technical solution of the present invention, the above-mentioned 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 diverse polypeptide fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 A schematic diagram of the connection of 5 peptide substrates in Example 4 of the present invention is shown.

[0023] Figure 2 A schematic diagram showing the catalytic results of the polypeptide ligase mutant according to Example 10 of the present invention on substrates with different amino acids at the P4 position. DETAILED DESCRIPTION

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

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

[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 makes its catalytic activity for certain specific peptide drug fragments limited and its stability insufficient. 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 by homology modeling and AI prediction, and mutated the key catalytic site serine near the active center to cysteine, and used 5-peptide substrates (such as Ac-Ala-Asp-Ser-Lys-Leu-O-Cam-Leu-OH (SEQ ID NO: 36) and H-Ala-Leu-Arg-His-Glu-NH2 (SEQ ID NO: 37)) to test the ligation activity of the enzyme after the single-site mutation, and analyzed it by HPLC after the reaction was completed. The results showed that in about 10% of the mutants, in addition to the hydrolysis product, the product 10 peptide was also detected. This indicates that some of these hydrolases retain some hydrolytic activity and also exhibit ligation activity after single-point mutation. However, the remaining approximately 90% of the enzymes, in addition to no observed ligation activity, also have significantly reduced hydrolytic activity after single-point mutation. This indicates that the key catalytic site serine near the active center is essential for the catalysis of the protease. 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 derived from Bacillus glycinifermentans (SEQID NO: 1), which has high activity when performing a polypeptide ligation reaction, and further modified its active pocket to improve its activity and substrate selectivity, thereby obtaining a polypeptide ligase with improved amino acid residue selectivity containing at least an amino acid mutation at the S297 site.

[0030] In a preferred embodiment, the mutation is selected from the group consisting of S297C mutation and any one or more of the following mutations: N194 mutation to N194K; L195 mutation to L195M; S206 mutation to S206A; Y219 mutation to Y219A; V223 mutation to V223M; S258 mutation to S258E; S259 mutation to S259N; S165 mutation to S165E; S320 mutation to S320K; S185 mutation to S185A; P205 mutation to P205G; N294 mutation to N294S; Y285 mutation to Y285A; S237 mutation to S237G; I281 mutation to I281N; T179 Mutation to T179S; N163 mutation to N163R, N163G; I183 mutation to I183V or I183N; S238 mutation to S238Q or S238A; S232 mutation to S232N, S232Q or S232E; S280 mutation to S280T, S280L or S280K; G295 mutation to G295C, G295S or G295H; S287 mutation to S287H, S287M or S287K; S287 mutation to S287N, S287H, S287M or S287K; L202 mutation to L202F, L202M, L202A or L202P; R262 mutation to R262L, R262E, R262Q or R262Y; G84 mutated to G84A, G84S, G84Q, G84D, G84S or G84P; K246 mutated to K246A, K246D, K246I, K246L or K246Y; H302 mutated to H302F, H302G, H302T, H302A or H302G; G137 mutated to G137D, G137I, G137C, G137A or G137Y; S221 mutated to S221A, S221R, S221W, S221Y or S221T; T209 mutated to T209A, T209R, T209M, T2 09S or T209W; T289 mutated to T289R, T289S, T289M, T289K or T289A; I183 mutated to I183V, I183A, I183Q, I183M or I183V; G137 mutated to G137I, G137C, G137D, G137A or G137Y; S177 mutated to S177Q, S177H, S177R, S177K or S177G; K92 mutated to K92A, K92G, K92D, K92E, K92S or K92N; T80 mutated to T80A, T80P, T80S, T80D, T80F, T80G or T80R;Y282 mutates to Y282L, Y282G, Y282H, Y282M, Y282C or Y282A R325 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 G242A 2R; A113 mutates to A113D, A113P, A113M, A113S, A113E, A113R or A113T; R325 mutates to R325N, R325A, R325G, R325Y, R325E, R325M or R325D; N134 mutates 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 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+G2 42Q, S297C+K246A, S297C+S258E, S297C+S259N, S297C+Y282L, S297C+S28 7N, S297C+T289R, S297C+T289S, S297C+T289M, S297C+T289K, S297C+T289 A. S297C+A113D, S297C+A113P, S297C+A113M, S297C+A113S, S297C+S314N , S297C+S314K, S297C+S314C, S297C+S314R, S297C+S314I, S297C+S314Y, S 297C+R325A, S297C+R325F, S297C+R325G, S297C+R325Y, S297C+R325E, S2 97C+R325M, S297C+A113D+S165E, S297C+A113D+S287N, S297C+A113D+G24 2L, S297C+A113D+S320K, S297C+A113D+N134A, S297C+A113D+N134T, S297 C+A113D+N134S, S297C+A113D+N134I, S297C+A113D+S185A, S297C+A113D+ T209A, S297C+S314N+I183V, S297C+S314N+I183N, S297C+S314N+S185A, S 297C+S314N+K92A, S297C+S314N+G242Q, S297C+T289R+S165E, S297C+T28 9R+G242S, S297C+T289R+G242C, S297C+T289R+G137D, S297C+T289R+P205 G. S297C+T289R+S238Q, S297C+T289R+N294S, S297C+T289R+S165E+S314Y,<h2 style=";text-align:left;direction:ltr">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+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、<h2 style=";text-align:left;direction:ltr">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、<h2 style=";text-align:left;direction:ltr">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<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282H、S297C+T28 9R+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+N 134T、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、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183Q、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134 R+T80S+A113E+I183M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209R、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209M、S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209S、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T2 09W、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N 134R+T80S+A113E+K92G、S297C+T289R+S165E+S287K+L202F+G295H+G137A +K246A+N134R+T80S+A113E+K92D、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+K92E、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92S、S297C+T289R+S165 E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84A、S297C+T2 89R+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+H302G.

[0033] The connection efficiency of peptide ligase is related to the six substrate recognition pockets (S4-S1, S1' and S2') in the active center of the enzyme. S4-S1 corresponds to the amino acids that recognize the acyl donor P4-P3-P2-P1, and S1' and S2' correspond to the amino acids that recognize the acyl acceptor P1'-P2'. When the peptide ligase Omnilgase-1 in the prior art catalyzes the connection of peptide substrates, if there are amino acids that affect the catalytic efficiency (such as histidine, glutamic acid, lysine, aspartic acid and some non-natural amino acids, etc.) 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.

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

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

[0036] The above amino acid mutations are all experimentally explored in the examples of this application, and compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, they all have the activity of catalyzing the binding of the substrate peptide chain to obtain the target polypeptide. The above mutation sites are all mutations made around the amino acid active site, and such mutations can improve the binding ability and / or catalytic ability of the mutant with the substrate. For mutations far away from the active site, the catalytic ability of the enzyme is less affected, so it is possible to obtain proteins with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or more homology with the above amino acid sequence and the same catalytic activity.

[0037] The homology in this specification refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0038] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those provided by the sequence (a), are homologous proteins obtained by amino acid mutation.

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

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

[0042] The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

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

[0045] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0046] The "AlphaFold2-Multimer" used in this application is a public artificial intelligence model that can predict the conformation of protein complexes. The prediction of protein three-dimensional structure can be very close to the level observed by cryo-electron microscopes and other equipment in real experiments. It can obtain a more realistic protein structure, thereby guiding the exploration of protein structure and protein activity.

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

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

[0053] In a fifth typical embodiment of the present application, a method for preparing a polypeptide is provided, which comprises using 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 includes 2 to 3; preferably, the number of substrate peptide chains is 2; preferably, the substrate peptide chain contains 5 to 30 amino acids. Preferably, the amino acids in the substrate peptide chain contain non-natural amino acids.

[0055] In a preferred implementation, when the number of substrate peptide chains is 2, the substrate peptide chains include a first substrate peptide chain and a second substrate peptide chain; preferably, the combination of the first substrate peptide chain and the second substrate peptide chain is selected from any one or more of the following groups:

[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, 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.

[0057] The first substrate peptide chain refers to the upper substrate in the polypeptide ligation reaction, and the second substrate peptide chain refers to the lower substrate in the polypeptide ligation reaction. In the ligation reaction, since the C-terminus of the first substrate peptide chain contains a protective ester acyl group, it can also be called an acyl donor, and the second substrate peptide chain can be called an acyl acceptor.

[0058] The beneficial effects of the present application will be further explained in detail below in conjunction with 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) derived from Bacillus glycinifermentans (SEQ ID NO: 1) was cloned into the restriction enzyme sites MLuI and BamH I of the Escherichia coli-Bacillus subtilis shuttle expression vector pBE-S. After obtaining the recombinant expression plasmid, it was transformed into the Bacillus subtilis WB600 strain by the Spizizen method (Spizizen J. Transformation of biochemically deficient strain of B. subtilis by deoxyribonucleate. Proceeding of National Academy of Science USA, 1958, 44: 1072-1078.), and the transformants were selected on LB plates containing 50 μg / mL kanamycin sulfate.

[0063] Positive transformants were picked into 5 mL LB liquid medium containing 50 μg / mL kanamycin sulfate, cultured at 37°C 200 rpm for 16 h, and then transferred to 500 mL TB medium (Terrific broth medium, 12 g / L tryptone, 24 g / L yeast powder, 0.4% glycerol) at a 1% (v / v) inoculation amount, and cultured at 37°C 200 rpm for 48 h. After the fermentation was completed, the fermentation supernatant was collected by centrifugation at 4°C and 8000 rpm for 10 min, and the precipitate was discarded.

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

[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 restriction enzyme cutting site Nco I-BamH I of the expression vector pET28a (+) and transformed into the competent Escherichia coli BL21 (DE3). The strain was cultured on an LB plate containing 50 μg / mL of kanamycin sulfate at 37°C for 16 hours, and then picked and inoculated into LB liquid medium containing 50 μg / mL of kanamycin sulfate. The strain was cultured at 37°C until the OD 600 When the concentration was about 0.8, 0.1 mM IPTG was added, and the culture was continued at 18°C ​​for 17 h before collecting the bacterial sludge.

[0068] Weigh the bacterial mud, add lysis solution (50mM Tris-HCl, 500mM NaCl, 0.1% Triton, pH 8.0), use an ultrasonic disruptor to lyse the bacteria, centrifuge the lysate of the bacteria (12000rpm for 20 minutes), and purify the supernatant. The specific process is: the sample is loaded at a flow rate of 2mL / min, and then rinsed with buffer A (25mM Tricine, pH7.5, 0.5MNaCl, 20mM imidazole) until the unbound protein is completely eluted, followed by eluting the impurity protein with a linear gradient imidazole for 5 column volumes (imidazole concentration is increased from 20mM to 50mM), and then eluting the target protein at 200mM. The affinity-purified protein is further centrifuged and replaced with an ultrafiltration tube to remove imidazole and salt, and placed at -20°C for standby use. The protein concentration is determined by the Bradford method, and the purity is analyzed by 12% separation gel SDS-PAGE. Finally, an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing about 5 mg / mL (purity greater than 90%) of the obtained enzyme was obtained and used for polypeptide fragment connection.

[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 restriction enzyme sites EcoR I and Not I of the Pichia pastoris expression vector pPICZ A. After obtaining the recombinant expression plasmid, the recombinant expression plasmid was linearized at 37°C with Sac I restriction endonuclease. After agarose electrophoresis verified that the enzyme digestion was complete, the linearized plasmid was recovered. The linearized plasmid was transformed into the Pichia pastoris X33 strain by electroporation, spread on a low-salt YPD plate containing 100 mg / mL bleomycin, and cultured at 30°C for 3 days. The transformant was picked into 10 mL BMGY liquid medium (10 g / L yeast powder, 20 g / L tryptone, 100 mM potassium phosphate pH 6.0, 13.4 g / L YNB, 4×10 -4 g / L biotin, 10 g / L glycerol), cultured at 30°C 200 rpm for 18 h, then transferred to 100 mL BMGY liquid culture medium with a 1% (v / v) inoculum, and cultured further at 30°C 200 rpm for 36 h.

[0072] At room temperature, the bacterial cells were collected by centrifugation at 1500-3000g and BMMY liquid medium (10 g / L yeast powder, 20 g / L tryptone, 100 mM potassium phosphate pH 6.0, 13.4 g / L YNB, 4×10 -4 g / L biotin, 5g / L methanol) to OD 600 ≈1.0, cultured at 30°C, 200 rpm, and added with methanol at a final concentration of 5 g / L every 24 h to induce expression.

[0073] After 5 days of fermentation, the supernatant of the fermentation broth was collected by centrifugation at 4°C and 8000rpm for 10min, the precipitate was discarded, and the supernatant of the fermentation broth was used for subsequent purification. The specific process is: the sample is loaded at a flow rate of 2mL / min, and then rinsed with buffer A (20Mm KPB, pH7.5, 0.5M NaCl, 20mM imidazole) until the unbound protein is completely eluted, followed by a linear gradient imidazole elution of 5 column volumes (imidazole concentration increased from 20mM to 100mM), and then the target protein is eluted at 200mM. The affinity-purified protein is further centrifuged and replaced with an ultrafiltration tube to remove imidazole and salt, and stored at -20°C for use.

[0074] The protein concentration was determined by Bradford method and the purity was analyzed by 12% separation gel SDS-PAGE. Finally, an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing 2 mg / mL (purity greater than 90%) of the obtained enzyme was obtained for polypeptide fragment connection.

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

[0076] Example 4

[0077] In this example, the protease from Bacillus glycinifermentans (SEQ ID NO: 1) was modified, the key catalytic site serine near the active center was mutated to cysteine, and the mutant S297C and Omnilgase-1 were subjected to a ligation reaction to test the activity using a pentapeptide (acyl donor P4 position is non-natural amino acid Aib, α-aminoisobutyric acid) as a substrate. The reaction system is as follows:

[0078] 1mL reaction system includes: 2.5mM acyl donor Ac-Asp-Aib-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 2) or Ac-Asp-Phe-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 3), 3.75mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), 0.5mg / mL enzyme, reaction buffer (0.1M Tricine, pH8.0, 0.8mg / mL TCEP). The reaction time is 4h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, the supernatant water phase is obtained by centrifugation at 8000rpm for 1min, and the conversion rate is detected by HPLC. The specific results are shown in Table 1.

[0079] Table 1

[0080]

[0081] Note: In Table 1, * represents a conversion rate of less than 0.1%, ** represents a conversion rate of greater than or equal to 0.1% and less than 10% (excluding 10%), **** represents a conversion rate of greater than or equal to 30% and less than 50% (excluding 50%), ***** represents a conversion rate of greater than or equal to 50% and less than 70% (excluding 70%), and ****** represents a conversion rate of greater than or equal to 70% and less than 90% (excluding 90%). Acyl donor refers to the substrate with an ester acyl group at the C-terminus in the first half of the ligation reaction; acyl acceptor refers to the substrate in the second half of the ligation reaction.

[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 at the C-terminus of the polypeptide substrate, and the "Leu-OH" group is a group connected after the "O-Cam" group. The "Leu" connected after "O-Cam" is an amino acid residue that is known to those skilled in the art and can improve the ligation efficiency in the ligation reaction. The specific structure of "O-Cam-Leu-OH" is as follows Figure 1 As shown in , the connection method and connection purpose of the same groups in the sequence of the remaining substrate peptide chains of the present application are the same as described here.

[0083] In this example, both mutant S297C and Omniligase-1 can catalyze the ligation reaction without non-natural amino acids in the acyl donor and show a high conversion rate. However, in the reaction where the acyl donor P4 position is the non-natural amino acid Aib, the catalytic activity of S297C is about hundreds of times that of Omniligase-1. This indicates that the mutant derived from Bacillus glycinifermentans has catalytic potential in the ligation reaction with the non-natural amino acid Aib at the P4 position.

[0084] Example 5

[0085] The mutant S297C from Bacillus glycinifermentans was further subjected to enzyme evolution, with Ac-Asp-Aib-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 2) with a non-natural amino acid at the P4 position as the acyl donor and H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4) as the acyl acceptor, and the evolved polypeptide ligase mutants were screened. The reaction system is as follows:

[0086] 1mL reaction system includes: 2.5mM acyl donor, 3.75mM acyl acceptor, enzyme amount reduced to 0.3mg / mL, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). Reaction time is 4h at 25℃, then add one volume of anhydrous ethanol to terminate the reaction, shake and mix thoroughly, centrifuge at 8000rpm for 1min, obtain the supernatant water phase, and send to HPLC to detect the conversion rate.

[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 combined mutation, 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 improved activity.

[0093] Example 6

[0094] The evolution was continued on the basis of the mutants in 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 less than 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%), ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate greater than or equal to 90%.

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

[0100] Example 7

[0101] Mutation was continued on the basis of the mutant in Example 6, and the mutant was tested for activity according to the following reaction system.

[0102] 1mL reaction system includes: 5mM acyl donor (SEQ ID NO: 2), 7.5mM acyl acceptor (SEQ ID NO: 4), enzyme amount is further reduced to 0.1mg / mL, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 4h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation is performed at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC for detection of conversion rate.

[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 less than 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%), ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate greater than or equal to 90%.

[0108] In this example, the polypeptide 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 polypeptide ligase mutants with improved activity were further obtained.

[0109] Example 8

[0110] The evolution was continued on the basis of the mutant in Example 7, and the mutant was tested for activity with reference to the following reaction system.

[0111] 1mL reaction system includes: 5mM acyl donor (SEQ ID NO: 2), 7.5mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), enzyme amount is further reduced to 0.05mg / mL, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation is performed at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC for detection of conversion rate.

[0112] After primary and secondary screening, the following mutants were obtained that showed improved activity. The specific results are shown in Table 5:

[0113] Table 5

[0114]

[0115]

[0116] Note: In Table 5, * represents a conversion rate less than 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%), ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate 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] The stability test was performed on some mutants evolved in Examples 4 to 8 (screened with the P4 site as the substrate of Aib) to screen mutants with improved stability. The mutants were prepared into enzyme solution according to the method described in Examples 1, 2 or 3, and the enzyme solution was treated at 70° C. for one hour, and then the mutants were tested for activity according to the following reaction system.

[0121] 1mL reaction system includes: 2.5mM acyl donor (SEQ ID NO: 2), 3.75mM acyl acceptor (SEQ ID NO: 4), enzyme amount 0.1mg / mL (treated at 70℃ for one hour), reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation at 8000rpm for 1min is performed to obtain the supernatant water phase, which is sent to HPLC to detect the conversion rate. The stability test results of some 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 treated at 70°C for 1 hour divided by the conversion rate of the enzyme solution directly reacted without high temperature treatment multiplied by 100%. Among them, * represents the residual activity greater than or equal to 10% and less than 30% (excluding 30%), ** represents the residual activity greater than or equal to 30% and less than 50% (excluding 50%), *** represents the residual activity greater than or equal to 50% and less than 70% (excluding 70%), and **** represents the residual activity greater than or equal to 70% and less than 90%.

[0126] Example 10

[0127] Substrate spectrum test:

[0128] The peptide ligase mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q was used as a catalyst to test the catalytic ability of 5-peptide acyl donors with different amino acids at the P4 position. The acyl donors were 20 different 5-peptides with the sequence of 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) and the acyl acceptor was H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4). The reaction system is as follows:

[0129] 1mL reaction system includes: 5mM acyl donor, 7.5mM acyl acceptor, 0.05mg / mL enzyme, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifuge at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC to detect the conversion rate.

[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, by performing enzyme evolution screening using a pentapeptide with the non-natural amino acid Aib at the P4 position as the target substrate, the obtained polypeptide ligase mutant exhibited a wider substrate adaptability at the P4 position.

[0132] Embodiment 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 the acyl acceptor, respectively), and the reaction system is as follows:

[0134] 1mL reaction system includes: 2.5mM acyl donor, 3.75mM acyl acceptor, enzyme amount 0.1mg / mL, reaction buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP). The reaction time is 2h at 25℃, and then one volume of anhydrous ethanol is added to terminate the reaction. After sufficient shaking and mixing, centrifugation is performed at 8000rpm for 1min to obtain the supernatant water phase, which is sent to HPLC to detect the conversion rate. The reaction results are shown in Table 7.

[0135] Table 7

[0136]

[0137]

[0138] Note: S / H in Table 7 refers to the ratio of the amount of enzyme synthesis 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 can all exhibit good ligation activity and S / H ratio.

[0140] Example 12

[0141] Enzymatic synthesis of semaglutide using 17-mer + 14-mer (acyl donor + acyl acceptor) fragments:

[0142] In a 200 mL four-necked bottle, 5 mM acyl donor His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Gly-Gln-O-Cam-Leu-OH (SEQ ID NO: 32), 7.5 mM acyl acceptor H-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-NH2 (SEQ ID NO: 33) were added, and 0.01 mg / mL of SEQ ID NO: NO: 1-based mutant (S297C + T289R + S165E + S287K + L202F + G295H + G137A + K246A + N134R + T80S + A113E + G84Q) enzyme solution, add buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP) to a total volume of 50mL, adjust pH = 7.8-8.2, stir and react at 25°C for 2h, HPLC detection of donor substrate reaction is complete, conversion rate 91% (no product isomers detected), S / H is 42. Then adjust the system to pH = 1-2, and denature the protein. Centrifuge the denatured system at 8000rpm for 10min to obtain the supernatant aqueous phase, which is then purified by HPLC and freeze-dried to obtain the product semaglutide.

[0143] Detection by HPLC and Q-NMR showed that the purity of the product was >98% and the yield was 74%.

[0144] Embodiment 11

[0145] Enzymatic synthesis of liraglutide using a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0146] In a 200 mL four-necked bottle, 5 mM acyl donor His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-O-Cam-Leu-OH (SEQ ID NO: 34), 7.5 mM acyl acceptor H-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-NH2 (SEQ ID NO: 35) were added, and 0.01 mg / mL of SEQ ID NO: The mutant (S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q) enzyme solution based on NO: 1 was added with buffer (0.1M Tricine, pH 8.0, 0.8mg / mL TCEP) to a total volume of 50mL, and the pH was adjusted to 7.8-8.2. The reaction was stirred at 25°C for 3h. The donor substrate reaction was complete by HPLC detection, the conversion rate was 94% (no product isomers were detected), and the S / H was 47. Then the system was acidified to pH=1-2 to denature the protein. The denatured system was centrifuged at 8000rpm for 10min to obtain the supernatant aqueous phase, which was then purified by HPLC and freeze-dried 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 exhibits good catalytic potential for the reaction of the non-natural amino acid Aib at the P4 position. (2) Taking the 5-peptide with Aib at the P4 position as the substrate, further enzyme evolution is carried out to obtain mutants with further improved activity. These mutants also show higher stability and catalytic ability for a wider substrate spectrum. (3) The obtained polypeptide ligase mutants can efficiently catalyze the synthesis of polypeptide substrates of different lengths. (4) The mutants obtained in the present application can be used for the efficient synthesis of a variety of polypeptide drugs. (5) Compared with the traditional chemical method, the enzymatic method of the present application does not require complicated processes and steps, and the operation is simpler and gentler. The reaction process will not cause protein racemization, no isomers will be generated, the system generates fewer impurities, the purification is simple, the purity is higher, and it is more suitable for industrial scale-up production.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide ligase mutant, characterized in that: The polypeptide ligase mutants include: (a) a protein having a mutation based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation comprises a mutation at the S297 site; or (b) A protein having 70% or more homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

2. The polypeptide ligase mutant according to claim 1, characterized in that The mutation is selected from the group consisting of S297C mutation and any one or more of the following mutations: N194 mutated to N194K; L195 mutated to L195M; S206 mutated to S206A; Y219 mutated to Y219A; V223 mutated to V223M; S258 mutated to S258E; S259 mutated to S259N; S165 mutated to S165E; S320 mutated to S320K; S185 mutated to S185A; P205 mutated to P205G; N294 mutated to N294S; Y285 mutated to Y285A; S237 mutated to S237G; I281 mutated to I281N; T179 mutated to T179S; N163 mutated to N163R, N163G; I183 mutated to I183V or I183N; S238 mutated to S238Q or S238A; S232 mutation to S232N, S232Q, or S232E; S280 mutation to S280T, S280L, or S280K; G295 mutated to G295C, G295S, or G295H; S287 mutation to S287H, S287M, or S287K; S287 mutation to S287N, S287H, S287M, or S287K; L202 mutation is L202F, L202M, L202A or L202P; R262 mutation to R262L, R262E, R262Q or R262Y; G84 mutation to G84A, G84S, G84Q, G84D, G84S, or G84P; K246 mutation is K246A, K246D, K246I, K246L, or K246Y; H302 mutated to H302F, H302G, H302T, H302A, or H302G; G137 mutation to G137D, G137I, G137C, G137A, or G137Y; S221 mutation to S221A, S221R, S221W, S221Y, or S221T; T209 mutation is T209A, T209R, T209M, T209S, or T209W; T289 mutation is T289R, T289S, T289M, T289K, or T289A; I183 mutation is I183V, I183A, I183Q, I183M or I183V; G137 mutation to G137I, G137C, G137D, G137A, or G137Y; S177 mutation to S177Q, S177H, S177R, S177K, or S177G; K92 mutation is K92A, K92G, K92D, K92E, K92S, or K92N; T80 mutation is T80A, T80P, T80S, T80D, T80F, T80G, or T80R; Y282 mutation is Y282L, Y28G, Y282H, Y282M, Y282C or Y282A R325 mutation to R325A, R325F, R325G, R325Y, R325E or R325M; S314 mutation to S314N, S314K, S314C, S314R, S314Y, or S314N; G242 mutation is G242T, G242A, G242Q, G242L, G242S, G242M or G242R; A113 mutation is 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.

3. The polypeptide ligase mutant according to claim 1, characterized in that The mutation includes any one or more of the following amino acid mutations: <h2 style=";text-align:left;direction:ltr">S297C、S297C+K92N、S297C+T80S、S297C+G84A、S297C+L202F、S297C+K246A 、S297C+R262L、S297C+H302F、S297C+N134D、S297C+N134F、S297C+G137D、S 297C+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, S297 C+R325Y, S297C+R325E, S297C+R325M, S297C+A113D+S165E, S297C+A113D+S287N, S297C+A113D+G242L, S297C+A113D+S320K, S297C+A113D+N134A, S2 97C+A113D+N134T、S297C+A113D+N134S、S297C+A113D+N134I、S297C+A113 D+S185A、S297C+A113D+T209A、S297C+S314N+I183V、S297C+S314N+I183N、S 297C+S314N+S185A、S297C+S314N+K92A、S297C+S314N+G242Q、S297C+T289 R+S165E、S297C+T289R+G242S、S297C+T289R+G242C、S297C+T289R+G137D、S 297C+T289R+P205G、S297C+T289R+S238Q、S297C+T289R+N294S、S297C+T289R+S165E+S314Y、S297C+T289R+S165E+I183V、S297C+T289R+S165E+S314N、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+Y285A、S297C+T289R+S165E+S287H、S297C+T289R+S165E+S287M、S297C+T289R+S165E+S287K、S297C+T289R+S165E+R325A、S2 97C+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+T 289R+S165E+S232N, S297C+T289R+S165E+S232Q, S297C+T289R+S165E+S232E, S297C+T289R+S165E+G242T, 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、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+K246A、S297C+T289R+S165E+S287K+K246D、S297C+T289R+S165E+S287K+K246I、S297C+T289R+S165E+S287K+K246L、S297 C+T289R+S165E+S287K+K246Y、S297C+T289R+S165E+S287K+R262L、S297C+T289R+S165E+S287K+R262E、S297C+T289R+S165E+S287K+R262Q、S297C+T2 89R+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+S1 65E+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+S28 7K+H143E, S297C+T289R+S165E+S287K+H143A, S297C+T289R+S165E+S287K+L202F+L293A, S297C+T289R+S165E+S287K+L202F+L293E, S297C+T289R+S 165E+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、<h2 style=";text-align:left;direction:ltr">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+G137 A+G242Q、S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242A、S297C+ T289R+S165E+S287K+L202F+G295H+G137A+G242K、S297C+T289R+S165E+S28 7K+L202F+G295H+G137A+K246A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A、S2 97C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221A、S297C+T289 R+S165E+S287K+L202F+G295H+G137A+K246A+S221R、S297C+T289R+S165E+S 287K+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、<h2 style=";text-align:left;direction:ltr">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+L 202F+G295H+G137A+K246A+N134F、S297C+T289R+S165E+S287K+L202F+G 295H+G137A+K246A+N134D、S297C+T289R+S165E+S287K+L202F+G295H+G 137A+K246A+N134F、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N1 34T, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S, S29 7C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D、S297 C+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+T28 9R+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、<h2 style=";text-align:left;direction:ltr">S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134 R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209M、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+T209S、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W、S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92 G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+ A113E+K92D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N13 4R+T80S+A113E+K92E、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92S、S297C+T289R+S165E+S287K+L202F+G295 H+G137A+K246A+N134R+T80S+A113E+G84A、S297C+T289R+S165E+S287K+L 202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S、S297C+T289R+S165E +S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q、S297C+T28 9R+S165E+S287K+L202F+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.

4. The polypeptide ligase mutant according to any one of claims 1 to 3, characterized in that The polypeptide ligase mutant includes a protein having a homology of more than 75%, more than 80%, more than 85%, more preferably more than 95%, and further preferably more than 99% with the amino acid sequence defined in (a) and having polypeptide ligase activity.

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

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

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

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

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

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

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