Double basic protease mutants and use thereof

Abstract

The application provides a double alkaline protease mutant and application thereof. The double alkaline protease mutant comprises: (a) a protein with double alkaline protease function, which is based on the amino acid sequence shown in SEQ ID NO: 1, and has deletion of amino acids at positions 1-21 and 678-777 and substitution of at least one amino acid site of K252, P255, M285, Q286, K438, I531 or I573; and (b) a protein with double alkaline protease function, which is based on the amino acid sequence shown in SEQ ID NO: 1, and has deletion of amino acids at positions 1-21 and 678-777. The mutant retains the specific cleavage function of KEX2 on paired alkaline amino acid sites, and the catalytic activity and stability in a complex environment are significantly improved, thereby providing an efficient and low-dose catalytic tool for industrial-grade protein precursor processing.

Description

Technical Field

[0001] This invention relates to the field of dual-base protease technology, and more specifically, to a dual-base protease mutant and its applications. Background Technology

[0002] KEX2 (EC 3.4.21.61), a dual-basic protease, is a calcium-dependent serine protease widely found in eukaryotes such as yeast. It specifically recognizes and cleaves the peptide bonds at the carboxyl terminus of paired basic amino acid residues (such as Lys-Arg↓, Arg-Arg↓, and Pro-Arg↓) in precursor proteins, making it a key processing enzyme in the protein secretion pathway. It has irreplaceable application value in the biopharmaceutical field, especially in the production of peptide drugs such as recombinant insulin, GLP-1 analogs, and growth hormone. KEX2 is widely used to cleave signal peptides or linker peptides in fusion proteins or precursor proteins, thereby efficiently generating biologically active mature proteins.

[0003] Currently, the KEX2 encoding gene used in industry is mainly derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Recombinant proteins are produced by Pichia pastoris (Pichia pastoris). Pichia pastoris Heterologous expression of KEX2 enzymes was obtained. However, existing commercially available KEX2 enzymes face multiple technical bottlenecks in actual production:

[0004] 1) Low catalytic efficiency leads to high enzyme consumption. Enzymatic cleavage is almost non-existent in reactions with added surfactants. The reaction time is long, usually requiring 24 hours or more. The production cost is high, which seriously restricts the large-scale and economical production of peptide drugs.

[0005] 2) Poor stability and weak environmental adaptability: In actual production processes, to improve the solubility of the target protein, reduce the size of the enzyme digestion system, and decrease the amount of solvent used, surfactants (such as SDS, Triton X-100, sodium lauryl sarcosinate, or lauroyl sarcosine), denaturants (such as urea, guanidine hydrochloride), or high ionic strength buffers are often added to the lysis or purification system. However, natural KEX2 is easily inactivated under the above conditions, significantly limiting its application in one-step enzyme digestion processes.

[0006] 3) Low expression levels and high purification costs: Although the Pichia pastoris system can achieve high-density fermentation, wild-type KEX2 is prone to misfolding, aggregation or low secretion efficiency after expression due to the presence of non-functional N-terminal and C-terminal sequences, resulting in low extracellular expression levels and poor purification yield, which further increases the cost per unit of enzyme.

[0007] In addition, although existing literature and patents (such as CN104673988A and US20150184201A1) mention the application of KEX2 in proinsulin processing, none of them disclose engineered mutants that can simultaneously achieve high specific activity and surfactant tolerance.

[0008] Therefore, there is an urgent need for a managerial design and structure-oriented KEX2 engineering modification strategy. By accurately screening key mutation sites and constructing multiple synergistic mutants, its catalytic efficiency, environmental adaptability and industrial applicability can be significantly improved, thus providing a new technical path for the low-cost and high-efficiency production of peptide drugs. Summary of the Invention

[0009] The main objective of this invention is to provide a dual-base protease mutant and its application, in order to solve the problems of low catalytic efficiency and poor surfactant tolerance of dual-base proteases in the prior art.

[0010] To achieve the above objective, according to a first aspect of the present invention, a dual-base protease mutant is provided, the dual-base protease mutant comprising:

[0011] (a) A protein that, based on the amino acid sequence shown in SEQ ID NO: 1, has the amino acids at positions 1-21 and 678-777 deleted and has at least one of the following amino acid sites replaced by: K252, P255, M285, Q286, K438, I531, M546, K114, I253, D257, L260, A279, N281, L457, P461, V489 or I573, and also has the function of a dual-base protease;

[0012] (b) A protein that has the amino acid sequence shown in SEQ ID NO: 1 missing amino acids 1-21 and 678-777 and has the function of a dual-base protease.

[0013] In (a) or (b), the position of the substituted amino acid site is defined by sequentially arranging the first amino acid of SEQ ID NO: 1 as the first position.

[0014] Furthermore, in (a) above, the substitution of the amino acid site is selected from at least one of the following sites:

[0015] K252F or K252R or K252H or K252G or K252A or K252V or K252L or K252I or K252P or K252M or K252N, P255D or P255G or P255A or P255V or P255L or P255I or P255M or P255F or P255Y or P255W or P255S or P255C or P255T or P255N or P255Q or P255E, M285L or M285G or M285A or M285V or M285I or M285P, Q286E or Q286S or Q286C or Q286T or Q286N or Q286D or Q28 6K or Q286R or Q286H or Q286G or Q286A or Q286V or Q286L or Q286I or Q286P or Q286M, K438L or K438R or K438H or K438G or K438A or K438V or K438I or K438P or K438M, I531C or I531S or I531T or I531N or I531Q or I531F or I531Y or I531W or I531G or I531A or I531V or I531L or I531P or I531M, M546L or M546G or M546A or M546V or M546I or M546P, K114H or K114G or K114A or K114V or K114L or K114I or K114P or K114M, I253G or I253A or I253V or I253L or I253P or I253M or I253S or I253C or I253T or I253N or I253Q, D257E or D257S or D257C or D257T or D257N or D257Q or D257G or D257A or D257V or D257L or D257I or D257M, L260K or L260R or L260H or L260G or L260A or L260V or L260I or L260P or L260M, A27 9G or A279V or A279L or A279I or A279P or A279M or A279S or A279C or A279T or A279N or A279Q or A279D or A279E, N281D or N281E or N281S or N281C or N281T or N281N or N281Q, L457S or L457C or L457T or L457N or L457Q or L457G or L457A or L457V or L457I or L457P or L457M, P461K or P461R or P461H or P461G or P461A or P461V or P461L or P461I or P461M,V489C or V489S or V489T or V489N or V489Q or V489G or V489A or V489L or V489I or V489P or V489M or I573S or I573C or I573T or I573N or I573Q or I573K or I573R or I573H or I573G or I573A or I573V or I573L or I573P or I573M;

[0016] In this text, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0017] Furthermore, in (a) above, the substitution of the amino acid site includes any of the following:

[0018] K114H、K114G、K114A、K114V、K114L、K114I、K114P、K114M、K252R、K252H、K252G、K252A、K252V、K252L、K252I、K252P、K252M、K252N、K252F、I253G、I253A、I253V、I253L、I253P、I253M、I253S、I253C、I253T、I253N、I253Q、P255G、P255A、P255V、P255L、P255I、P255M、P255F、P255Y、P255W、P255S、P255C、P255T、P255N、P255Q、P255D、P255E、D257E、D257S、D257C、D257T、D257N、D257Q、D257G、D257A、D257V、D257L、D257I、D257M、L260K、L260R、L260H、L260G、L260A、L260V、L260I、L260P、L260M、A279G、A279V、A279L、A279I、A279P、A279M、A279S、A279C、A279T、A279N、A279Q、A279D、A279E、N281D、N281E、N281S、N281C、N281T、N281N、N281Q、M285G、M285A、M285V、M285L、M285I、M285P、Q286S、Q286C、Q286T、Q286N、Q286D、Q286E、Q286K、Q286R、Q286H、Q286G、Q286A、Q286V、Q286L、Q286I、Q286P、Q286M、K438R、K438H、K438G、K438A、K438V、K438L、K438I、K438P、K438M、L457S、L457C、L457T、L457N、L457Q、L457G、L457A、L457V、L457I、L457P、L457M、P461K、P461R、P461H、P461G、P461A、P461V、P461L、P461I、P461M、V489C、V489A、V489S、V489T、V489N、V489Q、V489G、V489L、V489I、V489P、V489M、I531S、I531C、I531T、I531N、I531Q、I531F、I531Y、I531W、I531G、I531A、I531V、I531L、I531P、I531M、M546G、M546A、M546V、M546L、M546I、M546P、I573S、I573C、I573T、I573N、I573Q、I573K、I573R、I573H、I573G、I573A、I573V、I573L、I573P、I573M、K252F+P255D、K252F+M285L、K252F+Q286E、K252F+K438L、K252F+I531C、K252F+M546L、P255D+M285L、P255D+Q286E、P255D+K438L、P255D+I531C、P255D+M546L、M285L+Q286E、M285L+K438L、M285L+I531C、M285L+M546L、Q286E+K438L、Q286E+I531C、Q286E+M546L、K438L+I531C、K438L+M546L、I531C+M546L、K252F+P255D+M285L、K252F+P255D+Q286E、K252F+P255D+K438L、K252F+P255D+I531C、K252F+P255D+M546L、K252F+M285L+Q286E、K252F+M285L+K438L、K252F+M285L+I531C、K252F+M285L+M546L、K252F+Q286E+K438L、K252F+Q286E+I531C、K252F+Q286E+M546L、K252F+K438L+I531C、K252F+K438L+M546L、K252F+I531C+M546L、P255D+M285L+Q286E、P255D+M285L+K438L、P255D+M285L+I531C、P255D+M285L+M546L、P255D+Q286E+K438L、P255D+Q286E+I531C、P255D+Q286E+M546L、P255D+K438L+I531C、P255D+K438L+M546L、P255D+I531C+M546L、M285L+Q286E+K438L、M285L+Q286E+I531C、M285L+Q286E+M546L、M285L+K438L+I531C、M285L+K438L+M546L、M285L+I531C+M546L、Q286E+K438L+I531C、Q286E+K438L+M546L、Q286E+I531C+M546L、K438L+I531C+M546L、K252F+P255D+M285L+Q286E、K252F+P255D+M285L+K438L、K252F+P255D+M285L+I531C、K252F+P255D+M285L+M546L、K252F+P255D+Q286E+K438L、K252F+P255D+Q286E+I531C、K252F+P255D+Q286E+M546L、K252F+P255D+K438L+I531C、K252F+P255D+K438L+M546L、K252F+P255D+I531C+M546L、K252F+M285L+Q286E+K438L、K252F+M285L+Q286E+I531C、K252F+M285L+Q286E+M546L、K252F+M285L+K438L+I531C、K252F+M285L+K438L+M546L、K252F+M285L+I531C+M546L、K252F+Q286E+K438L+I531C、K252F+Q286E+K438L+M546L、K252F+Q286E+I531C+M546L、K252F+K438L+I531C+M546L、P255D+M285L+Q286E+K438L、P255D+M285L+Q286E+I531C、P255D+M285L+Q286E+M546L、P255D+M285L+K438L+I531C、P255D+M285L+K438L+M546L、P255D+M285L+I531C+M546L、P255D+Q286E+K438L+I531C、P255D+Q286E+K438L+M546L、P255D+Q286E+I531C+M546L、P255D+K438L+I531C+M546L、M285L+Q286E+K438L+I531C、M285L+Q286E+K438L+M546L、M285L+Q286E+I531C+M546L、M285L+K438L+I531C+M546L、Q286E+K438L+I531C+M546L、K252F+P255D+M285L+Q286E+K438L、K252F+P255D+M285L+Q286E+I531C、K252F+P255D+M285L+Q286E+M546L、K252F+P255D+M285L+K438L+I531C、K252F+P255D+M285L+K438L+M546L、K252F+P255D+M285L+I531C+M546L, K252F+P255D+Q286E+K438L+I531C, K252F+P255D+Q286E+K438L+M546L, K252F+ P255D+Q286E+I531C+M546L, K252F+P255D+K438L+I531C+M546L, K252F+M285L+Q286E+K438L+I531C, K252F+M285L+Q 286E+K438L+M546L, K252F+M285L+Q286E+I531C+M546L, K252F+M285L+K438L+I531C+M546L, K252F+Q286E+K438L+I5 31C+M546L, P255D+M285L+Q286E+K438L+I531C, P255D+M285L+Q286E+K438L+M546L, P255D+M285L+Q286E+I531C+M54 6L, P255D+M285L+K438L+I531C+M546L, P255D+Q286E+K438L+I531C+M546L, M285L+Q286E+K438L+I531C+M546L, K25 2F+P255D+M285L+Q286E+K438L+I531C, K252F+P255D+M285L+Q286E+K438L+M546L, K252F+P255D+M285L+Q286E+I531 C+M546L, K252F+P255D+M285L+K438L+I531C+M546L, K252F+P255D+Q286E+K438L+I531C+M546L, K252F+M285L+Q286E +K438L+I531C+M546L, P255D+M285L+Q286E+K438L+I531C+M546L or K252F+P255D+M285L+Q286E+K438L+I531C+M546L. ,

[0019] To achieve the above objectives, according to a second aspect of the present invention, a gene is provided that encodes the aforementioned dual-base protease mutant.

[0020] To achieve the above objectives, according to a third aspect of the present invention, an expression vector is provided that contains the aforementioned gene.

[0021] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, the host cell comprising the above-described gene or the above-described expression vector.

[0022] To achieve the above objectives, according to a fifth aspect of the present invention, a method for preparing a polypeptide drug is provided, the method comprising: catalyzing a polypeptide drug precursor protein with the above-mentioned dual-basic protease mutant to obtain the above-mentioned polypeptide drug; wherein the above-mentioned polypeptide drug precursor protein contains paired basic amino acid recognition sites.

[0023] Furthermore, the aforementioned polypeptide drug precursor protein includes any one or more of the following: GLP-1 precursor, GLP-1 derivative precursor, amylin precursor, or proinsulin.

[0024] Furthermore, the aforementioned GLP-1 derivative includes any one of the following: smegglutinin or liraglutinin; the aforementioned amylin is pramlinin.

[0025] Furthermore, the aforementioned dual-basic protease mutant catalyzes the aforementioned polypeptide drug precursor protein in a catalytic system.

[0026] The catalytic system includes the above-mentioned dual-base protease mutant, the above-mentioned polypeptide drug precursor protein, optional surfactant, and optional denaturant.

[0027] The surfactants mentioned above include any one or more of the following: SDS, Triton X-100, sodium fatty alcohol polyoxyethylene ether sulfate, or lauroyl sarcosine.

[0028] The denaturing agents mentioned above include any one or more of the following: urea or guanidine hydrochloride.

[0029] Furthermore, the final concentration of the above-mentioned SDS is 0.05-2%; the final concentration of the above-mentioned Triton X-100 is 0.1-2%; the final concentration of the above-mentioned sodium lauryl ether sulfate is 0.5-2%; the final concentration of the above-mentioned lauroyl sarcosine is 0.5-2%; the final concentration of the above-mentioned urea is 0.1-8.0 M; and the final concentration of the above-mentioned guanidine hydrochloride is 0.1-6.0 M.

[0030] To achieve the above objectives, according to a sixth aspect of the present invention, the application of the above-described dual-base protease mutant, the above-described gene, the above-described expression vector, the above-described host cell, or the above-described method for preparing a polypeptide drug in the preparation of a polypeptide drug is provided.

[0031] To achieve the above objectives, according to a seventh aspect of the present invention, a mutant library is provided containing a dual-base protease mutant of the first aspect of the present invention.

[0032] By applying the technical solution of this invention, a mutant of the dual basic protease KEX2 (SEQ ID NO: 1) derived from Pichia pastoris is obtained by truncating and site-directed mutagenesis, resulting in amino acid substitutions at at least one site, such as K252, P255, M285, Q286, K438, I531, M546, K114, I253, D257, L260, A279, N281, L457, P461, V489, or I573. This mutant retains the specific cleavage function of KEX2 at paired basic amino acid sites, and its catalytic activity and stability in complex environments (e.g., surfactants) are significantly improved, providing a high-efficiency, low-dosage catalytic tool for industrial-grade protein precursor processing. Attached Figure Description

[0033] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0034] Figure 1 The diagram shows an SDS-PAGE assay of KEX2 purified according to an embodiment of the present invention.

[0035] Figure 2 Mass spectrometry detection of GLP-1 and GLP-1-KR precursors according to embodiments of the present invention is illustrated.

[0036] Figure 3 The mass spectrometry detection of pramlinin peptide according to an embodiment of the present invention is shown. Detailed Implementation

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0038] Definitions:

[0039] GLP-1 (Glucagon-Like Peptide-1): refers to a mature GLP-1 polypeptide with complete biological activity. Its natural sequence usually refers to the amidated form of GLP-1(7-37) or GLP-1(7-36). It directly activates the GLP-1 receptor and has physiological activities such as lowering blood glucose, promoting insulin secretion, and delaying gastric emptying.

[0040] Precursor GLP-1: refers to a fusion protein or precursor protein containing the GLP-1 sequence and its upstream and downstream linker peptides that has not yet been cleaved by proteases. It has no biological activity on its own and must be cleaved by KEX2 to release active GLP-1.

[0041] As mentioned in the background section, the existing dual-alkaline protease KEX2 is derived from Saccharomyces cerevisiae and suffers from problems such as low expression levels, low catalytic efficiency, and poor stability in surfactants, which severely restricts its large-scale application in the production of peptide drugs such as recombinant insulin.

[0042] In this invention, the inventors attempted to construct mutants by site-directed mutagenesis and truncation of the KEX2 protein (with the amino acid sequence shown in SEQ ID NO: 1) derived from Pichia pastoris. Based on the truncated backbone by deleting residues at positions 1-21 of the N-terminus and positions 678-777 of the C-terminus, mutants with mutations at at least one of the following sites were constructed: K252, P255, M285, Q286, K438, I531, M546, K114, I253, D257, L260, A279, N281, L457, P461, V489, or I573. This significantly improved the enzyme activity of the protein in catalytic substrates (e.g., Boc-QRR-pNA) and its tolerance in industrial environments such as surfactants (e.g., SDS, Triton X-100, sodium lauryl ether sulfate, or lauroyl sarcosine) and / or denaturants (e.g., urea and guanidine hydrochloride). Therefore, the protection scheme of this invention is proposed.

[0043] In a first typical embodiment of the present invention, a dual-base protease mutant is provided. This dual-base protease mutant comprises:

[0044] (a) A protein having the amino acid sequence shown in SEQ ID NO: 1, but lacking amino acids 1-21 and 678-777, and having at least one of the following amino acid sites replaced by: K252, P255, M285, Q286, K438, I531, M546, K114, I253, D257, L260, A279, N281, L457, P461, V489, or I573, and possessing dual-base protease function; or

[0045] (b) A protein that has the amino acid sequence shown in SEQ ID NO: 1 missing amino acids 1-21 and 678-777, and possesses the function of a dual-base protease; or

[0046] (c) A protein that has more than 80% homology with the amino acid sequence defined in (a) or (b) and has the function of a dual-base protease;

[0047] In (a) or (b), the position of the substituted amino acid site is defined by sequentially arranging the first amino acid of SEQ ID NO: 1 as the first position.

[0048] The design of the above-mentioned dual-alkaline protease mutant is based on the structural and functional analysis of the wild-type KEX2 sequence from Pichia pastoris. By introducing conserved amino acid substitutions in key functional regions, the integrity of the enzyme's catalytic core structure is maintained while its substrate binding efficiency and conformational stability are enhanced.

[0049] SEQ ID NO: 1 is the original sequence, and its catalytic efficiency in its native state needs further improvement. SEQ ID NO: 3 is the truncated functional core sequence. Removing the non-essential N-terminal and C-terminal regions improves expression stability and the exposure of the catalytic center. Specifically, SEQ ID NO: 3 is the amino acid sequence obtained by deleting amino acid residues 1-21 and 678-777 from the amino acid sequence shown in SEQ ID NO: 1. Compared to SEQ ID NO: 1, the protein with the amino acid sequence shown in SEQ ID NO: 3 exhibits improved protein folding efficiency and soluble expression levels.

[0050] Amino acid substitutions at sites K252, P255, M285, Q286, K438, I531, M546, K114, I253, D257, L260, A279, N281, L457, P461, V489, or I573 are selected based on residue exchanges with similar physicochemical properties, such as hydrophobicity, charge, or steric hindrance adjustment. These mutations can improve the conformation of the substrate binding pocket, enhance calcium ion binding stability, or increase the overall flexibility of the enzyme molecule, thereby enhancing the cleavage efficiency at paired basic amino acid recognition sites without destroying the catalytic triplet structure.

[0051] Over 80% homology constraint ensures that the mutant maintains the integrity of the core structural domain within a sequence conservation framework, preserving enzyme function while allowing for adaptive evolution of non-conserved regions, providing a foundation of sequence diversity for constructing highly efficient industrial enzymes. This design strategy avoids the unpredictability of random mutations, improving catalytic performance while maintaining enzyme specificity through site-specific modification.

[0052] It should be noted that homology in this invention refers to "sequence identity" between two amino acid sequences, i.e., the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, it can be determined using the BLAST program in the NCBI database. For information on the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0053] The proteins described above that share 80% (e.g., 80%, 81%, 82%, 83%, 84%, 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 even 99.9%) homology with the amino acid sequences defined in (a) or (b) and possess the function of a dual-basic protease; whose active sites, active pockets, active mechanisms, protein structures, etc., are highly likely to be the same as the corresponding proteins provided in (a) or (b).

[0054] Amino acid residues can be represented by three-letter or one-letter amino acid codes according to standards known and agreed upon in the art. In this document, the abbreviations for amino acid residues are 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).

[0055] Conservative amino acid substitutions or replacements are well known in the art. For example, a conserved amino acid substitution is preferably one amino acid residue from the following group (1)-(5) replaced by another amino acid from the same group: (1) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) positively charged polar residues: His, Arg and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (5) aromatic residues: Phe, Tyr and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; and Val is substituted by Ile or Leu.

[0056] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0057] In a preferred embodiment of the present invention, in (a) above, the substitution of the amino acid site includes any of the following:

[0058] K252F or K252R or K252H or K252G or K252A or K252V or K252L or K252I or K252P or K252M or K252N, P255D or P255G or P255A or P255V or P255L or P255I or P255M or P255F or P255Y or P255W or P255S or P255C or P255T or P255N or P255Q or P255E, M285L or M285G or M285A or M285V or M285I or M285P, Q286E or Q286S or Q286C or Q286T or Q286N or Q286D or Q28 6K or Q286R or Q286H or Q286G or Q286A or Q286V or Q286L or Q286I or Q286P or Q286M, K438L or K438R or K438H or K438G or K438A or K438V or K438I or K438P or K438M, I531C or I531S or I531T or I531N or I531Q or I531F or I531Y or I531W or I531G or I531A or I531V or I531L or I531P or I531M, M546L or M546G or M546A or M546V or M546I or M546P, K114H or K114G or K114A or K114V or K114L or K114I or K114P or K114M, I253G or I253A or I253V or I253L or I253P or I253M or I253S or I253C or I253T or I253N or I253Q, D257E or D257S or D257C or D257T or D257N or D257Q or D257G or D257A or D257V or D257L or D257I or D257M, L260K or L260R or L260H or L260G or L260A or L260V or L260I or L260P or L260M, A27 9G or A279V or A279L or A279I or A279P or A279M or A279S or A279C or A279T or A279N or A279Q or A279D or A279E, N281D or N281E or N281S or N281C or N281T or N281N or N281Q, L457S or L457C or L457T or L457N or L457Q or L457G or L457A or L457V or L457I or L457P or L457M, P461K or P461R or P461H or P461G or P461A or P461V or P461L or P461I or P461M,V489C or V489S or V489T or V489N or V489Q or V489G or V489A or V489L or V489I or V489P or V489M or I573S or I573C or I573T or I573N or I573Q or I573K or I573R or I573H or I573G or I573A or I573V or I573L or I573P or I573M;

[0059] In this text, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0060] The selection of the aforementioned mutation sites was based on a comprehensive evaluation of surface charge distribution, hydrophobic core filling, and local conformational flexibility within the protein domains. These single-point mutations were experimentally verified to enhance enzyme activity and stability; their combined effect was not a simple additive one, but rather achieved through synergistic conformational adjustments to improve overall performance.

[0061] In a preferred embodiment of the present invention, in (a) above, the substitution of the amino acid site includes any of the following:

[0062] K114H、K114G、K114A、K114V、K114L、K114I、K114P、K114M、K252R、K252H、K252G、K252A、K252V、K252L、K252I、K252P、K252M、K252N、K252F、I253G、I253A、I253V、I253L、I253P、I253M、I253S、I253C、I253T、I253N、I253Q、P255G、P255A、P255V、P255L、P255I、P255M、P255F、P255Y、P255W、P255S、P255C、P255T、P255N、P255Q、P255D、P255E、D257E、D257S、D257C、D257T、D257N、D257Q、D257G、D257A、D257V、D257L、D257I、D257M、L260K、L260R、L260H、L260G、L260A、L260V、L260I、L260P、L260M、A279G、A279V、A279L、A279I、A279P、A279M、A279S、A279C、A279T、A279N、A279Q、A279D、A279E、N281D、N281E、N281S、N281C、N281T、N281N、N281Q、M285G、M285A、M285V、M285L、M285I、M285P、Q286S、Q286C、Q286T、Q286N、Q286D、Q286E、Q286K、Q286R、Q286H、Q286G、Q286A、Q286V、Q286L、Q286I、Q286P、Q286M、K438R、K438H、K438G、K438A、K438V、K438L、K438I、K438P、K438M、L457S、L457C、L457T、L457N、L457Q、L457G、L457A、L457V、L457I、L457P、L457M、P461K、P461R、P461H、P461G、P461A、P461V、P461L、P461I、P461M、V489C、V489A、V489S、V489T、V489N、V489Q、V489G、V489L、V489I、V489P、V489M、I531S、I531C、I531T、I531N、I531Q、I531F、I531Y、I531W、I531G、I531A、I531V、I531L、I531P、I531M、M546G、M546A、M546V、M546L、M546I、M546P、I573S、I573C、I573T、I573N、I573Q、I573K、I573R、I573H、I573G、I573A、I573V、I573L、I573P、I573M、K252F+P255D、K252F+M285L、K252F+Q286E、K252F+K438L、K252F+I531C、K252F+M546L、P255D+M285L、P255D+Q286E、P255D+K438L、P255D+I531C、P255D+M546L、M285L+Q286E、M285L+K438L、M285L+I531C、M285L+M546L、Q286E+K438L、Q286E+I531C、Q286E+M546L、K438L+I531C、K438L+M546L、I531C+M546L、K252F+P255D+M285L、K252F+P255D+Q286E、K252F+P255D+K438L、K252F+P255D+I531C、K252F+P255D+M546L、K252F+M285L+Q286E、K252F+M285L+K438L、K252F+M285L+I531C、K252F+M285L+M546L、K252F+Q286E+K438L、K252F+Q286E+I531C、K252F+Q286E+M546L、K252F+K438L+I531C、K252F+K438L+M546L、K252F+I531C+M546L、P255D+M285L+Q286E、P255D+M285L+K438L、P255D+M285L+I531C、P255D+M285L+M546L、P255D+Q286E+K438L、P255D+Q286E+I531C、P255D+Q286E+M546L、P255D+K438L+I531C、P255D+K438L+M546L、P255D+I531C+M546L、M285L+Q286E+K438L、M285L+Q286E+I531C、M285L+Q286E+M546L、M285L+K438L+I531C、M285L+K438L+M546L、M285L+I531C+M546L、Q286E+K438L+I531C、Q286E+K438L+M546L、Q286E+I531C+M546L、K438L+I531C+M546L、K252F+P255D+M285L+Q286E、K252F+P255D+M285L+K438L、K252F+P255D+M285L+I531C、K252F+P255D+M285L+M546L、K252F+P255D+Q286E+K438L、K252F+P255D+Q286E+I531C、K252F+P255D+Q286E+M546L、K252F+P255D+K438L+I531C、K252F+P255D+K438L+M546L、K252F+P255D+I531C+M546L、K252F+M285L+Q286E+K438L、K252F+M285L+Q286E+I531C、K252F+M285L+Q286E+M546L、K252F+M285L+K438L+I531C、K252F+M285L+K438L+M546L、K252F+M285L+I531C+M546L、K252F+Q286E+K438L+I531C、K252F+Q286E+K438L+M546L、K252F+Q286E+I531C+M546L、K252F+K438L+I531C+M546L、P255D+M285L+Q286E+K438L、P255D+M285L+Q286E+I531C、P255D+M285L+Q286E+M546L、P255D+M285L+K438L+I531C、P255D+M285L+K438L+M546L、P255D+M285L+I531C+M546L、P255D+Q286E+K438L+I531C、P255D+Q286E+K438L+M546L、P255D+Q286E+I531C+M546L、P255D+K438L+I531C+M546L、M285L+Q286E+K438L+I531C、M285L+Q286E+K438L+M546L、M285L+Q286E+I531C+M546L、M285L+K438L+I531C+M546L、Q286E+K438L+I531C+M546L、K252F+P255D+M285L+Q286E+K438L、K252F+P255D+M285L+Q286E+I531C、K252F+P255D+M285L+Q286E+M546L、K252F+P255D+M285L+K438L+I531C、K252F+P255D+M285L+K438L+M546L、K252F+P255D+M285L+I531C+M546L, K252F+P255D+Q286E+K438L+I531C, K252F+P255D+Q286E+K438L+M546L, K252F+ P255D+Q286E+I531C+M546L, K252F+P255D+K438L+I531C+M546L, K252F+M285L+Q286E+K438L+I531C, K252F+M285L+Q 286E+K438L+M546L, K252F+M285L+Q286E+I531C+M546L, K252F+M285L+K438L+I531C+M546L, K252F+Q286E+K438L+I5 31C+M546L, P255D+M285L+Q286E+K438L+I531C, P255D+M285L+Q286E+K438L+M546L, P255D+M285L+Q286E+I531C+M54 6L, P255D+M285L+K438L+I531C+M546L, P255D+Q286E+K438L+I531C+M546L, M285L+Q286E+K438L+I531C+M546L, K25 2F+P255D+M285L+Q286E+K438L+I531C, K252F+P255D+M285L+Q286E+K438L+M546L, K252F+P255D+M285L+Q286E+I531 C+M546L, K252F+P255D+M285L+K438L+I531C+M546L, K252F+P255D+Q286E+K438L+I531C+M546L, K252F+M285L+Q286E +K438L+I531C+M546L, P255D+M285L+Q286E+K438L+I531C+M546L or K252F+P255D+M285L+Q286E+K438L+I531C+M546L. ,

[0063] The aforementioned combined mutations, through multi-point synergistic effects, optimize the electrostatic environment, hydrophobic microenvironment, and conformational flexibility and solvent accessibility of the substrate binding channel at the catalytic active site without disrupting the enzyme's basic folding, thereby effectively improving substrate recognition efficiency and catalytic turnover rate. The conformational changes in these multiple mutants (e.g., K252F+P255D+Q286E+K438L+I531C+M546L) also ensure their stable activity in complex reaction systems (e.g., surfactants).

[0064] In a second typical embodiment of the present invention, a gene is provided. This gene encodes the aforementioned dual-base protease mutant.

[0065] The aforementioned gene sequence has undergone codon optimization to adapt to the host expression system, enabling efficient transcription and translation in host cells. Its coding region contains a mutated KEX2 catalytic domain, retaining the conserved catalytic triplet (His-Asp-Ser) and substrate recognition motif of calcium-dependent serine proteases, while eliminating non-essential terminal sequences to improve secretion efficiency. This gene can be independently expressed as a functional protein, and its sequence design meets the post-translational modification requirements of host cells (e.g., eukaryotes), including the retention or removal of glycosylation sites to ensure product activity and purification feasibility.

[0066] In three typical embodiments of the present invention, an expression vector is provided. This expression vector contains the aforementioned gene.

[0067] This expression vector is a functional DNA molecule that can stably replicate in host cells and drive the efficient expression of exogenous genes. It contains regulatory elements such as promoters, terminators, and selection markers to support the transcription and translation of the aforementioned genes.

[0068] In a preferred embodiment of the present invention, the expression vector includes any one or more of the following: pPIC9K or pPICZa.

[0069] The aforementioned Pichia pastoris expression vector, utilizing its potent and methanol-induced AOX1 promoter system, achieved efficient and regulated expression of the codon-optimized and truncated KEX2 mutant gene in the Pichia pastoris host. This expression strategy not only significantly improved the secretion and expression stability of recombinant proteins but also effectively avoided inclusion body formation or toxicity problems that may result from intracellular expression, thereby greatly increasing the actual yield of the KEX2 mutant. This lays a solid material foundation for reducing enzyme preparation costs and improving the enzymatic conversion efficiency of peptide drug precursors (such as GLP-1 precursors) in subsequent large-scale industrial production.

[0070] In a fourth typical embodiment of the present invention, a host cell is provided. This host cell includes the aforementioned gene or the aforementioned expression vector.

[0071] In a preferred embodiment of the present invention, the host cell includes any one or more of the following: Pichia pastoris GS115, Pichia pastoris X33, or Pichia pastoris SMD1168. These strains are all common strains in the prior art and are commercially available.

[0072] The host cells possess a complete secretory system and protein folding cofactors, enabling stable integration and sustained expression of the aforementioned genes, thus facilitating the intracellular synthesis and secretion of mutant proteins. The cells' genetic background allows for efficient homologous recombination integration, ensuring the stable presence of single or multiple copies of the foreign gene in the genome and preventing plasmid loss. Under induction conditions, these host cells can achieve efficient accumulation of mutant proteins, and high-purity active enzymes can be obtained from the culture supernatant, providing a reliable source for subsequent purification and application.

[0073] In five typical embodiments of the present invention, a method for preparing a polypeptide is provided, the method comprising: using the above-mentioned dual-basic protease mutant to catalyze a polypeptide drug precursor protein to obtain the above-mentioned polypeptide drug; wherein the above-mentioned polypeptide drug precursor protein contains paired basic amino acid recognition sites.

[0074] The above method utilizes the mutant KEX2 to specifically cleave polypeptide precursors containing Lys-Arg, Arg-Arg, or Pro-Arg sequences. Its catalytic efficiency is higher than that of wild-type enzymes, and complete hydrolysis can be achieved with shorter reaction times or lower enzyme dosages. This method does not rely on extreme pH or high temperature conditions and is suitable for mild in vitro enzymatic reaction systems, especially for processing heat-sensitive or easily aggregated polypeptide precursors such as GLP-1 or proinsulin, avoiding side reactions.

[0075] In a preferred embodiment of the present invention, the aforementioned polypeptide precursor protein includes any one or more of the following: GLP-1 precursor, GLP-1 derivative precursor, amylin precursor, or proinsulin. In a preferred embodiment of the present invention, the aforementioned GLP-1 derivative includes any one of the following: smegglutide or liraglutide; the aforementioned amylin is pramlinide.

[0076] The aforementioned peptide precursor protein is a recombinantly expressed fusion protein containing tandemly repeated enzyme recognition sites for the mass production of the target active peptide. The mutant KEX2 can effectively recognize and cleave multiple sites, improving the yield per batch. This strategy avoids the costs and purification complexities of chemical synthesis or solid-phase peptide synthesis, making it suitable for the high-throughput, large-scale preparation of peptide drugs.

[0077] In a preferred embodiment of the present invention, the above-mentioned dual-base protease mutant catalyzes the above-mentioned polypeptide drug precursor protein in a catalytic system; wherein the catalytic system includes the above-mentioned dual-base protease mutant, the above-mentioned polypeptide drug precursor protein, an optional surfactant, and an optional denaturant; wherein the above-mentioned surfactant includes any one or more of the following: SDS, Triton X-100, sodium lauryl ethoxylate sulfate, or lauroyl sarcosine; the above-mentioned denaturant includes any one or more of the following: urea or guanidine hydrochloride.

[0078] The dual-alkaline protease KEX2 mutant provided in this application significantly improves the catalytic stability and activity of the enzyme in the presence of different surfactants (such as SDS, Triton X-100, sodium lauryl ether sulfate, or lauroyl sarcosine) and denaturants (such as urea and guanidine hydrochloride). This overcomes the problems of poor adaptability and low catalytic efficiency of wild-type KEX2 in industrial production, thereby greatly reducing the amount of enzyme used in the production of peptide drugs (such as GLP-1), effectively reducing production costs and improving the conversion rate and purity of peptide modification.

[0079] In a preferred embodiment of the present invention, the final concentration of the SDS is 0.05-2%; the final concentration of the Triton X-100 is 0.1-2%; the final concentration of the sodium fatty alcohol polyoxyethylene ether sulfate is 0.5-2%; the final concentration of the lauroyl sarcosine is 0.5-2%; the final concentration of the urea is 0.1-8.0 M; and the final concentration of the guanidine hydrochloride is 0.1-6.0 M.

[0080] The aforementioned concentration range covers the cell lysis and protein dissolution conditions commonly used in industrial production, ensuring that the mutant KEX2 retains its functionality in high ionic strength or detergent environments. This technical solution, by limiting the specific concentration ranges of surfactants (SDS, Triton X-100, sodium lauryl ether sulfate, lauroyl sarcosine) and denaturants (urea, guanidine hydrochloride), ensures that the dual-basic protease KEX2 mutant maintains high catalytic activity in systems containing these components that typically lead to enzyme inactivation or inhibition. The principle lies in the enhanced structural stability of the mutant, which resists the interference of surfactants on the hydrophobic core of the enzyme molecule and the disruption of hydrogen bond network stability by denaturants. This enables efficient and stable catalysis of specific cleavage of peptide precursor proteins in complex reaction media (such as high-concentration detergent extracts or folding environments containing denaturants), significantly improving enzyme tolerance and process compatibility in industrial applications.

[0081] In a sixth typical embodiment of the present invention, the application of the above-described dual-base protease mutant, the above-described gene, the above-described expression vector, the above-described host cell, or the above-described method for preparing a polypeptide drug is provided in the preparation of a polypeptide drug.

[0082] The dual-alkaline protease KEX2 mutant provided by this invention significantly enhances the enzyme's catalytic activity and structural stability, which helps to achieve industrial breakthroughs in the efficient, low-cost, and high-purity preparation of recombinant peptide drugs (such as GLP-1), significantly improves product yield and process economy, and provides a stable, efficient, and interference-resistant novel tool enzyme platform for the large-scale production of peptide drugs.

[0083] In a seventh typical embodiment of the present invention, a mutant library is provided containing the dual-base protease mutant of the first typical embodiment of the present invention.

[0084] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0085] Example 1: Construction of a genetically engineered yeast strain expressing KEX2

[0086] The inventors screened proteases from multiple species in the early stages, and finally screened out the Pichia pastoris strain used in this application. Pichia pastoris The expression level of the enzyme KEX2 is the highest among the enzymes, therefore, this application chooses to optimize the enzyme through mutation.

[0087] The inventors first studied the yeast derived from Pichia pastoris ( Pichia pastoris The protease KEX2 (amino acid sequence shown in SEQ ID NO: 1) was expressed. Based on the strategy of expressing recombinant proteins in yeast, the target gene (objective gene) was inserted into the pPIC9K vector (existing technology) using molecular cloning technology. The codon-optimized nucleotide sequence of KEX2 (as shown in SEQ ID NO: 2) was synthesized by Genewiz. An enzyme was introduced at the 5' end of the above nucleotide sequence. EcoR An enzyme cleavage site is introduced at the 3' end of the above nucleotide sequence. Not The I restriction site was constructed in the pPIC9K cloning vector.

[0088] The sequence of SEQ ID NO: 1 is as follows:

[0089] MYLPALRLACWILIGLRSTEALETSEREIFALKLDKSWLPTFLETFQDKFRYERQINGLDDYHVFSHSKNEEFQLENFKVKTLLTRDNANLHSELISHNVDEVHMLRPSHYLHKRAPVVMDKSEELREQIAKDFDIDDPLFAKQWHLFNPRYPGHDVNVSQVWYDGITGKGVVTAIVDDGLDMDSKDLKESFCEEGSWDFNANTRLPKPRLRDDHHGTRCAAEIAAKKGNKYCGVGVAYDSKVSGIRILSDKITPEDEALSLIYGLDVNDIYSCSWGPADNGITMQGPSSLVKEAMLKGVQDGRKGKGALYVFASGNGASSGDNCNFDGYTNSIYSITVGAIDIKGLHPPYAEACSAVMTVTYSSGSGEHIHTTDINDKCSDTHGGTSAAAPLAAGLYSLVYQANPDLTWRDIQWLTVLTAVPVNEQEPGWQKTAIGKMYSHKYGYGKIDAYALVNLARSPDFPYLKPQSWIYGTEVHESLNTSEANGVLTSKYELTQEAKDLMNFEKIEHVTVTVDIKAAERGKVLVELISPSGVVSELAPYRRMDKDKEGFPNWTFMSVAHWGEDGLGEWILKITNKEGNSVVLNSWQIKFFGESQDPEKAEKFSLTKKYDEILVNPPSSSTSTTVDTSSTEATFSSSSVSEASATETDVKETSTTGDEDFENEEGSYKHSNSSHITEYLAFLLGLGFLICIIFLFTNRNKLERRQRRNRRDEYEFDLIPADDDFDTEEDQEANSQFTLDSDAELMFEDTSQREASPHEYQDSLGSNEHPKRAALHHHHHH.

[0090] Wherein, the sequence of SEQ ID NO: 2 is:

[0091]

[0092] Post-construction plasmid use Sac Linearization was achieved by enzyme digestion with 2-5 μg of the plasmid. Electrophoresis was used to check for complete digestion. After confirming complete digestion, the linearized DNA fragment was recovered. Approximately 1 μg of the recovered fragment was transferred and mixed into yeast GS115 competent cells (existing technology). The cells were incubated on ice for 3-5 min, and then the mixture was transferred again to a pre-cooled electroporation cuvette. Yeast was selected for the electroporation program. Immediately after electroporation, 1 ml of antibiotic-free YPD medium (1% yeast extract, 2% tryptone, 2% glucose) was added to the cuvette, and the cells were incubated at 30°C and 160 rpm for 1 h. After incubation, the solution was plated onto YPD solid plates containing 200 μg / mL G418 resistance and incubated at 30°C for 3-5 days.

[0093] The grown single clones were identified by bacterial PCR to confirm gene integration. Three correct clones of each strain were selected and activated in YPD medium, incubated overnight at 30°C and 220 rpm. Cells were then inoculated at a 2% inoculum into 50 mL BMGY medium (1% yeast extract, 2% peptone, 10% KPB pH 6.0, 1.34% YNB, 1% glycerol, 1000× biotin) in shake flasks and cultured at 30°C and 220 rpm for 12 h with shaking. The cells were then resuspended and transferred to 50 mL BMMY medium (1% yeast extract, 2% trypone, 10% KPB pH 6.0, 1.34% YNB, 1% methanol, 1000× biotin). Methanol was added every 24 h to a final concentration of 0.5%. Induction was performed at 30°C for 72 h. After induction, the protein expression supernatant was collected by centrifugation. Protein expression results were analyzed by 12% separating gel SDS-PAGE.

[0094] Example 2: KEX2 Purification

[0095] KEX2 was produced using a recombinant method. The supernatant of the recombinant protein expression was filtered through a 0.45 μm filter to obtain a crude enzyme solution. The pH of the crude enzyme solution was adjusted to 8.0, and the supernatant was obtained by centrifugation.

[0096] KEX2 purification was performed using an AKTA system equipped with a Ni column (Ni FF, 5 mL). The crude protease sample was loaded at a flow rate of 5 mL / min. The binding buffer was 50 mM Tris, 0.2 M NaCl, pH 8.0. After loading, the sample was washed with binding buffer until all unbound protein was eluted. Then, a gradient elution was performed using 50 mM Tris, 0.2 M NaCl, 1 M imidazole, pH 8.0, at a gradient of approximately 300 mM imidazole to obtain the target protein. SDS-PAGE analysis showed that the target protein achieved a purity of over 90%. Figure 1After purification, the sample was treated with 25 mM Tris, pH 8.0 storage buffer, encapsulated in a 10 KD membrane, desalted, and stored at -20 °C.

[0097] Example 3: Enzyme activity detection of KEX2

[0098] The enzyme activity of KEX2 (amino acid sequence shown in SEQ ID NO: 1) was evaluated. In a reaction system of 25℃, 50 mM Tris-HCl, 2 mM CaCl2, and pH 8.0, the amount of enzyme that catalyzes the release of 1 μmol of 4-nitroaniline from the substrate Boc-QRR-pNA (Chinese name: tert-butyloxycarbonyl-glutamine-arginine-arginine-p-nitroaniline) is defined as one enzyme activity unit. This substance exhibits specific absorption at 405 nm, and the product can be detected using a microplate reader for enzyme activity verification and mutant screening.

[0099] Example 4 Screening for mutation points in enzyme modification

[0100] A mutant expression strain of KEX2 (amino acid sequence as shown in SEQ ID NO: 1) was constructed. Based on the above amino acid sequence, to ensure correct expression of the target protein in the yeast strain, amino acids 1-21 (the protein's own signal peptide; since the yeast expression vector pPIC9K carries a more efficient afactor signal peptide, this region was deleted to avoid the influence of the two signal peptides on protein expression) and amino acids 678-777 (the transmembrane region; during protein expression, this region anchors the protein to the Golgi apparatus, causing protein degradation or retention within the cell, preventing proper secretion and expression) were deleted to obtain the mutant. The enzyme activity of wild-type KEX2 (unmodified sequence, amino acid sequence as shown in SEQ ID NO: 1) and the mutant (amino acid sequence as shown in SEQ ID NO: 3) was detected (purified enzymes were used for enzyme activity determination in Examples 4-8 of this application), and the results are shown in Table 1.

[0101] Table 1

[0102]

[0103] Note: In Table 1, This indicates an enzyme activity of less than 5 U / mg. This indicates an enzyme activity greater than or equal to 10 U / mg and less than 15 U / mg (excluding 15 U / mg).

[0104] In this embodiment, by truncating the KEX2 sequence, it was found that the mutant enzyme activity was significantly higher than that of the unmodified sequence, and the protein yield was significantly increased.

[0105] The sequence of SEQ ID NO:3 is as follows:

[0106] .

[0107] Example 5: Screening for single-point mutations in enzymes

[0108] Based on the mutant of Example 4, KEX2 was further mutated to construct mutant expression strains. Through primary and secondary screening of enzyme activity against substrates, the following mutants with enhanced activity were obtained, and the results are shown in Table 2.

[0109] Table 2

[0110]

[0111]

[0112]

[0113]

[0114] Note: In Table 2, This indicates an enzyme activity of less than 5 U / mg. This indicates an enzyme activity greater than or equal to 10 U / mg and less than 15 U / mg (excluding 15 U / mg). This represents an enzyme activity greater than or equal to 15 U / mg and less than 20 U / mg (excluding 20 U / mg).

[0115] Example 6 Screening for combined mutation points of enzymes

[0116] Based on the mutant of Example 5, KEX2 was further mutated to construct a combined mutant expression strain. Through primary and secondary screening of enzyme activity against substrates, the following mutants with enhanced activity were obtained, and the results are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120]

[0121] Note: In Table 3, This indicates an enzyme activity greater than or equal to 10 U / mg and less than 15 U / mg (excluding 15 U / mg). This indicates an enzyme activity greater than or equal to 15 U / mg and less than 20 U / mg (excluding 20 U / mg). This indicates an enzyme activity greater than or equal to 20 U / mg and less than 25 U / mg (excluding 25 U / mg). This indicates an enzyme activity greater than or equal to 25 U / mg.

[0122] Example 7 Evaluation of KEX2 mutants in different surfactants

[0123] For the mutants with enhanced enzyme activity obtained in Examples 5 and 6, enzyme activity was tested using different surfactants or denaturants. The enzyme activity was measured by adding 0.1% SDS, 1% Triton X-100, 1% sodium fatty alcohol polyoxyethylene ether sulfate, 0.5% lauroyl sarcosine, 0.5 M urea, or 0.2 M guanidine hydrochloride to the protease preservation solution, respectively, and then detecting the enzyme activity using the method described in Example 3. The results are shown in Table 4.

[0124] Table 4

[0125]

[0126]

[0127] Note: In Table 4, This indicates an enzyme activity of less than 5 U / mg. This indicates an enzyme activity greater than or equal to 10 U / mg and less than 15 U / mg (excluding 15 U / mg). This indicates an enzyme activity greater than or equal to 15 U / mg and less than 20 U / mg (excluding 20 U / mg). This indicates an enzyme activity greater than or equal to 20 U / mg and less than 25 U / mg (excluding 25 U / mg). This indicates an enzyme activity greater than or equal to 25 U / mg.

[0128] Example 8: Application of KEX2-digested peptides

[0129] The GLP-1 precursor was prepared by enzymatic digestion using the mutants with enhanced enzyme activity obtained in Examples 5 and 6:

[0130] The amino acid sequence (SEQ ID NO: 5) of the constructed multi-copy polypeptide GLP-1 precursor is as follows:

[0131] EGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREQGGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0132] According to the GLP-1 precursor preparation method in patent CN 118440968 B, a multi-copy polypeptide expression plasmid was constructed, and the tandem linker region was selected as KREQGGKR (SEQ ID NO:4). The strain expressed protein was extracted using 50 mM Tris buffer containing 0.5% SDS and 1% Triton X-100 at pH 8.0 to obtain the fusion multi-copy protein. The precursor GLP-1 and GLP-1-KR terminal monomers were formed by digestion with 0.2 wt% of the mutant KEX2-del 1-21-del-678-777+P255D+M285L+Q286E+K438L+I531C for 16 h. Under the same conditions, KEX2-del 1-21-del-678-777 with 1 wt% enzyme added could be digested to form the same amount of precursor GLP-1 and GLP-1-KR terminal monomers.

[0133] Among them, "precursor GLP-1" refers to GLP-1 protein that is not fully mature and still has a leader peptide or linker peptide; "GLP-1-KR terminal monomer" refers to a single GLP-1 molecule obtained after enzyme digestion, whose C-terminus retains the Lys-Arg(KR) dipeptide (i.e. the residual part of the cleavage site), which is a typical "terminal residue" pattern after KEX2 cleavage.

[0134] The product was identified using LC-MS, with the following procedures: The sample was first separated using an HPLC column: Agilent ZORBAX Edipse Plus C18, 4.6. 100 mm, 3.5 μm, mobile phase A: 0.1% trifluoroacetic acid, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution, gradient elution: 0 min 10% B, 9 min 95% B, 12 min 100% B, 12.1 min 10% B, 15 min 10% B, column temperature 50℃, UV detector 210 nm, flow rate 0.3 ml / min.

[0135] The fractions separated by HPLC were analyzed using a Q Exactive HF quadrupole Orbitrap mass spectrometer with a dual AJS ESI electrospray ionization source in positive ion mode. The sheath gas flow rate was 35 arb, the auxiliary gas flow rate was 8 arb, the spray voltage was 3800 V, the ion transfer tube temperature was 320 °C, and the scan range was 200–3000 m / z. The mass spectrometry data were processed using BioPharma Finder software. The results are as follows: Figure 2 As shown. The detected product accounted for 90%, and the sample was lyophilized and stored. Figure 2 The product corresponding to the mutant KEX2-del 1-21-del-678-777+P255D+M285L+Q286E+K438L+I531C is shown.

[0136] Example 9

[0137] Similarly, using the enzyme-enhanced mutants obtained in Examples 5 and 6, the polypeptide pramlin peptide was prepared by enzymatic digestion:

[0138] The amino acid sequence (SEQ ID NO: 6) of the constructed multicopy polypeptide pramlineptide is as follows:

[0139] KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTYKREQGGKRKCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY.

[0140] According to the peptide preparation scheme of patent CN 118440968 B, a multi-copy peptide expression plasmid was constructed, and the tandem linker region was also selected as KREQGGKR (SEQ ID NO:4). The strain expressed protein was extracted using 50 mM Tris buffer containing 0.5% SDS and 1% Triton X-100 at pH 8.0 to obtain the fusion multi-copy protein. In the experiment, 0.2 wt% of the mutant KEX2-del 1-21-del-678-777+P255D+M285L+Q286E+K438L+I531C and 1 wt% carboxypeptidase B were used for double enzyme digestion for 16 h to prepare pramlin peptide. Under the same conditions, double enzyme digestion with 1 wt% KEX2-del 1-21-del-678-777 and 1 wt% carboxypeptidase B could form an equal amount of pramlin peptide. The enzyme digestion products were separated by HPLC, and the obtained products were analyzed by mass spectrometry. The results are as follows: Figure 3 As shown (the specific detection method is the same as in Example 8), through Figure 3 The corresponding intensity of the mass spectrometry molecular weight was calculated, indicating that the proportion of pramlin peptide product was over 99%. Figure 3 The product corresponding to the mutant KEX2-del 1-21-del-678-777+P255D+M285L+Q286E+K438L+I531C is shown.

[0141] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0142] The KEX2 mutant of the dual-basic protease provided by this invention significantly enhances the enzyme's specific activity in catalyzing the cleavage of paired basic amino acid sites and its tolerance to surfactants. This mutant achieves a breakthrough enhancement in enzymatic reaction efficiency without altering its natural substrate recognition characteristics. It provides a novel, highly active, and highly stable biocatalyst for reducing enzyme usage in industrial production and improving the efficiency of peptide precursor processing, possessing significant industrial application value and economic significance.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dual-basic protease mutant, characterized in that, The amino acid sequence of the dual-alkaline protease mutant is based on the amino acid sequence shown in SEQ ID NO: 1, with the deletion of amino acids 1-21 and 678-777 and the substitution of any one of the following amino acid sites: K252F、K252F+P255D、K252F+M285L、K252F+Q286E、K252F+K438L、K252F+I531C、K252F+M546L、K252F+P255D+M285L、K252F+P255D+Q286E、K252F+P255D+K438L、K252F+P255D+I531C、K252F+P255D+M546L、K252F+M285L+Q286E、K252F+M285L+K438L、K252F+M285L+I531C、K252F+M285L+M546L、K252F+Q286E+K438L、K252F+Q286E+I531C、K252F+Q286E+M546L、K252F+K438L+I531C、K252F+K438L+M546L、K252F+I531C+M546L、K252F+P255D+M285L+Q286E、K252F+P255D+M285L+K438L、K252F+P255D+M285L+I531C、K252F+P255D+M285L+M546L、K252F+P255D+Q286E+K438L、K252F+P255D+Q286E+I531C、K252F+P255D+Q286E+M546L、K252F+P255D+K438L+I531C、K252F+P255D+K438L+M546L、K252F+P255D+I531C+M546L、K252F+M285L+Q286E+K438L、K252F+M285L+Q286E+I531C、K252F+M285L+Q286E+M546L、K252F+M285L+K438L+I531C、K252F+M285L+K438L+M546L、K252F+M285L+I531C+M546L、K252F+Q286E+K438L+I531C、K252F+Q286E+K438L+M546L、K252F+Q286E+I531C+M546L、K252F+K438L+I531C+M546L、K252F+P255D+M285L+Q286E+K438L、K252F+P255D+M285L+Q286E+I531C、K252F+P255D+M285L+Q286E+M546L、K252F+P255D+M285L+K438L+I531C、K252F+P255D+M285L+K438L+M546L、K252F+P255D+M285L+I531C+M546L, K252F+P255D+Q286E+K438L+I531C, K252F+P255 D+Q286E+K438L+M546L, K252F+P255D+Q286E+I531C+M546L, K252F+P255D+K438L+I5 31C+M546L, K252F+M285L+Q286E+K438L+I531C, K252F+M285L+Q286E+K438L+M546L, K252F+M285L+Q286E+I531C+M546L, K252F+M285L+K438L+I531C+M546L, K252F+Q286 E+K438L+I531C+M546L, K252F+P255D+M285L+Q286E+K438L+I531C, K252F+P255D+M2 85L+Q286E+K438L+M546L, K252F+P255D+M285L+Q286E+I531C+M546L, K252F+P255D+ M285L+K438L+I531C+M546L, K252F+P255D+Q286E+K438L+I531C+M546L, K252F+M285L+Q286E+K438L+I531C+M546L, or K252F+P255D+M285L+Q286E+K438L+I531C+M546L; The positions of the substituted amino acid sites are defined by sequentially arranging the first amino acid of SEQ ID NO: 1 as the first position.

2. A gene characterized in that, The gene encodes the dual-base protease mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the gene as described in claim 2.

4. A host cell, characterized in that, The host cell includes the gene of claim 2 or the expression vector of claim 3.

5. A method for preparing a polypeptide drug, characterized in that, The method includes: using the dual-basic protease mutant of claim 1 to catalyze a polypeptide drug precursor protein to obtain the polypeptide drug; wherein the polypeptide drug precursor protein contains paired basic amino acid recognition sites.

6. The method according to claim 5, characterized in that, The polypeptide drug precursor protein includes any one or more of the following: GLP-1 precursor, GLP-1 derivative precursor, amylin precursor, or proinsulin.

7. The method according to claim 6, characterized in that, The GLP-1 derivative includes any one of the following: smegglutinin or liraglutinin; the amylin is pramlinin.

8. The method according to claim 7, characterized in that, The dual-base protease mutant catalyzes the polypeptide drug precursor protein in the catalytic system. The catalytic system includes the dual-base protease mutant, the polypeptide drug precursor protein, an optional surfactant, and an optional denaturant. The surfactant includes any one or more of the following: SDS, Triton X-100, sodium fatty alcohol polyoxyethylene ether sulfate, or lauroyl sarcosine; The denaturing agent includes any one or more of the following: urea or guanidine hydrochloride.

9. The method according to claim 8, characterized in that, The final concentration of the SDS is 0.05-2%; the final concentration of the Triton X-100 is 0.1-2%; the final concentration of the sodium lauryl polyoxyethylene ether sulfate is 0.5-2%; the final concentration of the lauroyl sarcosine is 0.5-2%; the final concentration of the urea is 0.1-8.0 M; and the final concentration of the guanidine hydrochloride is 0.1-6.0 M.

10. The use of the dual-base protease mutant of claim 1, the gene of claim 2, the expression vector of claim 3, the host cell of claim 4, or the method for preparing a polypeptide drug according to any one of claims 5-9 in the preparation of a polypeptide drug.

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