Method for producing recombinant collagenase type i and its mutants and use thereof

Abstract

The application discloses a preparation method and application of a recombinant collagenase type I and a mutant thereof. A mutant of the collagenase type I is provided in the application, and the mutant has good alkali resistance and can be applied to enzymatic extraction of fish skin waste. The application further provides an expression method of the recombinant collagenase type I and the mutant thereof based on a prokaryotic expression system. The expression method can improve the expression amount of soluble protein, and the product has high activity and is suitable for industrial amplification production.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the preparation method and application of recombinant type I collagenase and its mutants. Background Technology

[0002] Collagenase is an enzyme that specifically hydrolyzes natural collagen or gelatin under suitable pH and temperature conditions without damaging other proteins and tissues. Its specific collagen-hydrolyzing product, collagen tripeptide, significantly improves digestibility, absorption, nutritional value, and functional properties compared to larger collagen molecules. Collagenases are classified into types I, II, III, IV, and V, as well as hepatocyte-specific collagenases. The type of collagenase should generally be selected based on the type of tissue to be isolated and digested.

[0003] One important application of collagenase is in the extraction of collagen peptides from fish skin waste. Fish skin is rich in collagen. Developing and producing novel, highly active collagen peptides using industrial processing waste such as fish skin as raw materials has gradually become a research hotspot. The collagenase degradation method for preparing collagen peptides not only has high yields but is also relatively environmentally friendly, making it one of the most suitable processes for widespread application. In addition, collagenase also has significant medical applications; it can be used to treat lumbar disc herniation, Dupuytren's disease, keloids, and other abnormal hyperplasia, and it also has high application value in cell research and environmental protection.

[0004] However, current domestic methods for producing type I collagenase mostly rely on microbial extraction, with Clostridium histolyticum being the most commonly used microbial source. However, most of these strains are pathogenic, and during collagenase production, they also produce corresponding toxins. Removing these toxins requires complex purification steps. Although the toxin content in the final product is reduced, the process is too complex and unsuitable for industrial-scale production. Furthermore, the inventors also discovered that some recombinant type I collagenases prepared through conventional genetic engineering methods lack sufficient alkali resistance, making them difficult to apply to the degradation of processing wastes such as fish skin.

[0005] In view of this, there is a need in the field to develop a simple process for preparing a low-toxicity, alkali-resistant type I collagenase that is suitable for large-scale industrial production. Summary of the Invention

[0006] The purpose of this invention is to provide a type I collagenase mutant.

[0007] Another object of the present invention is to provide a method for preparing recombinant type I collagenase.

[0008] Another object of the present invention is to provide a multinucleotide sequence encoding recombinant type I collagenase.

[0009] Another object of the present invention is to provide a vector adapted to a multinucleotide sequence encoding recombinant type I collagenase.

[0010] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding recombinant type I collagenase.

[0011] To address the aforementioned technical problems, a first aspect of the present invention provides a type I collagenase mutant, wherein the type I collagenase mutant is mutated at one or more sites selected from the following:

[0012] R929, F952, V978, D966, V973 and D974.

[0013] In some preferred embodiments, the mutation of the type I collagenase mutant is selected from any one or a combination of the following:

[0014] R929A, F952A, V978A, D966A, V973A, and D974A.

[0015] In some preferred embodiments, the amino acid sequence of the type I collagenase mutant is selected from any of the following:

[0016] (i) The amino acid sequence as shown in SEQ ID NO. 5-6; and

[0017] (ii) An amino acid sequence that is more than 95% homologous to the sequence shown in SEQ ID NO. 5-6.

[0018] A second aspect of the invention provides a polynucleotide encoding a recombinant type I collagenase mutant, said polynucleotide being codon-optimized and selected from any of the following:

[0019] (i) Polynucleotides with sequences as shown in SEQ ID NO.1 or SEQ ID NO.3;

[0020] (ii) Polynucleotides with greater than 95% homology to sequences shown in SEQ ID NO.1 or SEQ ID NO.3; and

[0021] (iii) A polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

[0022] In a third aspect, the present invention provides an expression vector comprising the polynucleotides provided in the second aspect of the present invention.

[0023] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0024] In a fourth aspect, the present invention provides a host cell comprising the expression vector provided in the third aspect of the present invention; or

[0025] The host cell genome contains polynucleotides as provided in the second aspect of the present invention.

[0026] In some preferred embodiments, the host cell is Escherichia coli.

[0027] In some preferred embodiments, the host cell is Escherichia coli BL21(DE3) strain.

[0028] The fifth aspect of this invention provides a method for preparing recombinant type I collagenase, the method comprising the steps of: culturing the host cells described in the fourth aspect of this invention to express the target protein; and

[0029] The target protein is isolated to obtain the recombinant type I collagenase;

[0030] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the second aspect of the invention.

[0031] In some preferred embodiments, the host cells are cultured using SB, TB, LB, or SOC media, and more preferably using TB media.

[0032] In some preferred embodiments, the host cells are cultured in an oscillating environment.

[0033] In some preferred embodiments, the host cells are cultured at a temperature of 16 to 19°C or 35 to 39°C, more preferably at a temperature of 16 to 19°C.

[0034] In some preferred embodiments, the culture medium used to culture the host cells contains a kanamycin resistance gene.

[0035] In some preferred embodiments, IPTG is used to induce the expression of the target protein when culturing the host cells.

[0036] In some preferred embodiments, the host cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0037] In some preferred embodiments, the step of isolating the target protein includes:

[0038] The supernatant of the lysed target protein was eluted by passing it through a chromatography column while the flow was constant, and the eluent was collected.

[0039] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column (Ni-NTA).

[0040] A fifth aspect of the present invention provides a kit comprising: a type I collagenase mutant as provided in the first aspect of the present invention; or

[0041] Such as the polynucleotides provided in the second aspect of the present invention; or

[0042] Such as the expression vector provided in the third aspect of the present invention; or

[0043] The host cell as described in the fourth aspect of the present invention.

[0044] Compared with the prior art, the present invention has at least the following advantages:

[0045] (1) The present invention provides a type I collagenase mutant, which has good alkali resistance and is suitable for enzymatic extraction of fish skin waste;

[0046] (2) This invention provides a method for expressing recombinant type I collagenase and its mutants based on a prokaryotic expression system. This method increases the expression level of soluble protein, has low endotoxin content in the product, and has good product activity, making it suitable for industrial-scale production.

[0047] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0048] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0049] Figure 1 This is an SDS-PAGE identification image of the recombinant type I collagenase expression product according to an embodiment of the present invention;

[0050] Figure 2 This is an electrophoresis diagram of recombinant type I collagenase according to an embodiment of the present invention;

[0051] Figure 3 Standard curve for type I collagenase activity assay according to an embodiment of the present invention;

[0052] Figure 4This is a standard curve diagram for endotoxin determination according to an embodiment of the present invention. Detailed Implementation

[0053] In existing technologies, the produced type I collagenase has a high endomycin content. To reduce toxicity, type I collagenase needs to be purified. The purification process is usually extremely cumbersome, time-consuming, and labor-intensive. However, type I collagenase typically does not require extremely high purity in practical applications. Therefore, developing a simple method for preparing low-toxicity type I collagenase is crucial. One aspect of this invention utilizes recombinant protein technology, through synonymous codon preference optimization, to obtain an optimized codon sequence for collagenase. Suitable culture conditions and purification methods were studied, leading to the development of a high-yield, high-activity, and low-toxicity prokaryotic expression system for type I collagenase, and the acquisition of recombinant type I collagenase.

[0054] Furthermore, since existing type I collagenases are often intolerant to strong alkaline environments, limiting their use, the inventors further replaced the optimized type I collagenase codon sequence with the codon sequence encoding a type I collagenase mutant based on the aforementioned expression system, thereby improving the alkalinity tolerance of the expression product.

[0055] In this invention, the construction of a type I collagenase prokaryotic expression system includes the following steps: 1) obtaining the target gene sequence / polynucleotide sequence encoding the target protein from the amino acid sequence of the target protein (type I collagenase or its mutant); 2) performing synonymous codon preference optimization on the obtained polynucleotide sequence to obtain multiple optimized codons; 3) constructing a vector containing the obtained optimized codons; 4) introducing the vector containing the optimized codons into host cells and inducing culture to express the recombinant target protein. Preferably, step 4) further includes a step of purifying the target protein.

[0056] Obtain the nucleic acid sequence related to the target gene / protein.

[0057] In this invention, the full-length nucleotide sequence or fragments of the target protein or its elements can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0058] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0059] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0060] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0061] In one embodiment of the present invention, the target protein is a mature type I collagenase (the ColG gene of Clostridium histolyticum, UniProtKB database number Q9X721, obtained by truncating amino acids aa111-1118). In another embodiment of the present invention, the target protein is a mutant of the aforementioned type I collagenase, said mutant being mutated at one or more sites selected from the following: R929, F952, V978, D966, V973, and D974. More preferably, the type I collagenase mutant is mutated in a manner selected from any one or a combination of the following: R929A, F952A, V978A, D966A, V973A, and D974A. More preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:5 or SEQ ID NO:6.

[0062] Synonymous codon preference optimization

[0063] To overcome the potential problem of reduced yield when expressing heterologous proteins in *E. coli*, this invention relates to polynucleotide sequences optimized by synonymous codon preference. The obtained target gene sequence is optimized by synonymous codon preference, allowing the optimized target gene sequence to express the same amino acid sequence as the target protein. In some embodiments of this invention, *E. coli* synonymous codon preference optimization is performed on the gene sequence encoding type I collagenase from *Clostridium histolyticum* to obtain optimized codon I as shown in SEQ ID NO:1. In some embodiments of this invention, *E. coli* synonymous codon preference optimization is performed on the gene sequence encoding a mutant of type I collagenase from *Clostridium histolyticum* to obtain optimized codon II as shown in SEQ ID NO:2 and optimized codon III as shown in SEQ ID NO:3.

[0064] The present invention also relates to polynucleotides having greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, and more preferably greater than 95% homology with the sequences shown in SEQ ID NO: 1-3; and polynucleotides complementary to the sequences shown in SEQ ID NO: 1-3.

[0065] Vector of the target gene

[0066] This invention also relates to vectors containing the polynucleotides of the invention. In this invention, "vector" refers to a linear or circular DNA molecule containing a fragment encoding a target protein, said target protein being operatively linked to other fragments that provide for its transcription. Such additional fragments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more optional markers, enhancers, polyadenylation signals, vectors, etc. The vector fragment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, either synthetic or readily manipulated with recombinant DNA, and the choice of vector generally depends on the host cell to which the vector is to be introduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that integrates into the host cell genome upon introduction into a host cell and replicates along with the chromosome into which it is integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the invention, pET-28a(+) is selected as the vector to obtain more efficient expression.

[0067] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the vector DNA molecule is cleaved into linear molecules that can be linked to a foreign gene using a DNA endonuclease, and then a codon-optimized target gene fragment is ligated into the vector. The insertion of the foreign DNA fragment can be achieved by using sticky-end ligation at a single restriction enzyme site, directed cloning of double-restricted fragments, sticky-end ligation at different restriction enzyme sites, blunt-end ligation, artificial adapter ligation, or ligation to oligonucleotide ends.

[0068] Transform host cells with a vector containing the target gene

[0069] This invention also relates to host cells genetically engineered using the vector or fusion protein coding sequence of this invention. A vector containing a codon-optimized target gene can be inserted, transfected, or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized target gene of this invention and capable of expressing the target protein. In this invention, "host cell" refers to a cell incorporating exogenous polynucleotides and / or a vector. The host cell can be a eukaryotic or prokaryotic host cell, preferably a bacterium, and more preferably *Escherichia coli* (DE3) strain.

[0070] Methods for preparing target proteins

[0071] This invention also relates to a method for preparing the target protein, which can be used to express or produce recombinant proteins using the polynucleotide sequence of this invention. Generally, the method includes the following steps:

[0072] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0073] (2) Host cells cultured in a suitable culture medium;

[0074] (3) Isolate and purify proteins from culture media or cells.

[0075] In step (1), the recombinant expression vector containing the polynucleotide is transformed or transduced into a suitable host cell by conventional techniques known to those skilled in the art. When the host is Escherichia coli, heat shock and electroconversion methods can be used.

[0076] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media, preferably SB, TB, LB, or SOC media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. In a preferred embodiment of this invention, to promote the expression of the target protein and increase the expression level of soluble proteins, host cells are cultured in TB or LB media containing a kanamycin resistance gene.

[0077] To further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, host cells are cultured to OD0.05. 600After reaching a concentration between 0.6 and 0.8, IPTG was used for induction, and the cells were cultured for approximately 8 to 12 hours at 17 to 19°C or 35 to 39°C. High soluble expression levels were observed at lower temperatures, such as 17 to 19°C.

[0078] The proteins in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, proteins can be separated and purified by various separation methods using their physical, chemical, and other properties. Therefore, in this invention, after successfully culturing the target protein, the steps of separating and purifying it are also involved, for example, in step (3), separating and purifying the protein from the culture medium to obtain the target protein with high purity. Although the methods for purifying the target protein are conventional means well known to those skilled in the art, they include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out method), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods. In a preferred embodiment of the present invention, a simple method for purifying the target protein is provided, comprising the steps of: 1) purifying the lysed recombinant target protein using a Ni-NTA column; 2) concentrating the purified product by ultrafiltration; and 3) collecting the target protein bands of corresponding molecular weight for dialysis to obtain the purified target protein. Preferably, in step 3), a salt containing calcium and / or zinc ions is added to the dialysate. The purified sample can be concentrated using the BCA method to calculate the yield.

[0079] Since collagenase is often used to separate tissues or extract collagen through enzymatic hydrolysis, it is understandable that in the actual use of collagenase, the removal of endotoxins to reduce the toxicity of the product is sufficient, and there is no excessive requirement for enzyme purity. The method of purifying the target protein provided in the embodiments of the present invention works in conjunction with the target protein expression system developed in the present invention, eliminating the need for time-consuming and expensive purification methods, and achieving a low level of endotoxin content in the expression product (below 0.1 EU / mL).

[0080] In this invention, any exemplary or illustrative terminology (e.g., “”) used with respect to certain embodiments herein is merely for the purpose of better presenting the invention and does not limit the scope of the invention as otherwise claimed. No terminology herein should be construed as indicating an element not described in the claims that is indispensable to the implementation of this invention.

[0081] If the definition or use of a term in a cited reference is inconsistent with or inconsistent with the definition of a term described herein, the definition of the term described herein shall be used instead of the definition of the term in the cited reference.

[0082] The various terms used herein are as follows. If a term used in the claims is not defined below, the broadest definition of that term given by a person skilled in the art should be given, as reflected in the publication printed at the time of application or in the published patent.

[0083] As used herein, the term "isolated" refers to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in a solvent, buffer, ion, or other component normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides present in their natural source.

[0084] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

[0085] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0086] As used in this article, the term "codon optimization" refers to the method of improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon utilization exhibited by the actual organisms performing protein expression or production (including E. coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0087] As used herein, the terms “homology” and “identity” are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residue, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0088] As used herein, the terms “sequence complement” and “reverse sequence complement” are used interchangeably and refer to a sequence that is in the opposite direction to the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAT.

[0089] As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. Post-expression can be harvested, i.e., the host cell or the expressed product can be recovered.

[0090] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0091] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0092] Example 1

[0093] In this embodiment, a plasmid containing an optimized codon encoding Clostridium histolytica recombinant collagenase type I was synthesized and introduced into Escherichia coli culture to obtain a single clone.

[0094] (1) Construction of recombinant collagenase type I plasmid

[0095] The gene sequence of Clostridium histolytica collagenase type I was obtained and optimized for Escherichia coli synonymous codon bias to obtain optimized codon I (SEQ ID NO.1), which was ligated into the pET-28a(+) vector and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0096] (2) Recombinant plasmids were introduced into host Escherichia coli.

[0097] Take 1 μL of the expression plasmid prepared in step (1) above, add it to 30 μL of E. coli competent cells BL21(DE3) under ice bath conditions, incubate on ice for 30 min, incubate in water at 42℃ for 45 s, immediately place on ice for 2 min, add 400 μL of antibiotic-free SOC medium, and culture at 37℃ and 230 rpm for 45 min with shaking. Take 100 μL of bacterial culture and spread it evenly on LB agar plates containing 100 μg / mL kanamycin resistance, and incubate overnight at 37℃.

[0098] SEQ ID NO.1

[0099]

[0100] Example 2

[0101] In this embodiment, Clostridium histolytica type I collagenase was randomly mutated to obtain several groups of Clostridium histolytica type I collagenase mutants, such as type I collagenase mutant A and type I collagenase mutant B. The gene sequences encoding each mutant were obtained by analysis, and the codon preference of E. coli was optimized to obtain several optimized codons, such as optimized codon II (SEQ ID NO. 2) corresponding to type I collagenase mutant A and optimized codon III (SEQ ID NO. 3) corresponding to type I collagenase mutant B. These were then ligated into the pET-28a(+) vector, and recombinant plasmids were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0102] The recombinant plasmid obtained was introduced into E. coli and cultured using the same method as in Example 1 to obtain a single clone.

[0103] SEQ ID NO.2

[0104]

[0105] SEQ ID NO.3

[0106]

[0107] Type I collagenase amino acid sequence SEQ ID NO.4

[0108] IANTNSEKYDFEYLNGLSYTELTNIKNIKWNQINGLFNYSTGSQKFFGDKNRVQAIINALQESGRTYTADMKGIETFTEVLRAGFYLGYYNDGLSYLNDRNFQDKCIPAMIAIQKNPNFKLGTAVQDEVITSLGKLIGNASANAEVVNNCVPVLKQFRENLNQYAPDYVKGTAVNELIKGIEFDFSGAAYEKDVKTMPWYGKIDPFINELKALGLYGNITSATEWASDVGIYYLSKFGLYSTNRNDIVQSLEKAVDMYKYGKIAFVAMERITWDYDGIGSNGKKVDHDKFLDDAEKHYLPKTYTFDNGTFIIRAGDKVSEEKIKRLYWASREVKSQFHRVVGNDKALEVGNADDVLTMKIFNSPEEEYKFNTNINGVSTDNGGLYIEPRGTFYTYERTP QQSIFSLEELFRHEYTHYLQARYLVDGLWGQGPFYEKNRLTWFDEGTAEFFAGSTRTSGVLPRKSILGYLAKDKVDHRYSLKKTLNSYDDSDWMFYNYGFAVAHYLYEKDMPTFIKMNKAILNTDVKSYDEIIKKLSDDANKNTEYQNHIQELADKYQGAGIPLVSDDYLKDHGYKKASEVYSEISKAASLTNTSVTAEKSQYFNTFTLRGTYTGETSKGEFKDWDEMSKKLDGTLESLAKNSWSGYKTLTAYFTNYRVTSDNKVQYDVVFHGVLTDNADISNNKAPIAKVTGPSTGAVGRNIEFSGKDSKDEDGKIVSYDWDFGDGATSRGKNSVHAYKKAGTYNVTLKVTDDKGATATESFTIEIKNEDTTTPITKEMEPNDDIKEANGPIVEGVTVKGDLNGSDDADTFYFDVKEDGDVTIELPYSGSSNFTWLVYKEGDDQNHIASGIDKNNSKVGTFKSTKGRHYVFIYKHDSASNISYSLNIKGLGNEKLKEKENND SSDKATVIPNFNTTMQGSLLGDDSRDYYSFEVKEEGEVNIELDKKDEFGVTWTLHPESNINDRITYGQVDGNKVSNKVKLRPGKYYLLVYKYSGSGNYELRVNK

[0109] The amino acid sequence of type I collagenase mutant A (R929A / F952A / V978A) is SEQ ID NO. 5.

[0110]

[0111] Amino acid sequence of type I collagenase mutant B (D966A / V973A / D974A), SEQ ID NO.6

[0112] IANTNSEKYDFEYLNGLSYTELTNLIKNIKWNQINGLFNYSTGSQKFFGDKNRVQAIINALQESGRTYTANDMKGIETFTEVLRAGFYLGYYNDGLSYLNDRNFQDKCIPAMIAIQKNPNFKLGTAVQDEVITSLGKLIGNASANAEVVNNCVPVLKQFRENLNQYAPDYVKGTAVNELIKGIEFDFSGAAYEKDVKTMPWYGKIDPFINELKALGLYGNITSATEWASDVGIYYLSKFG

[0113] LYSTNRNDIVQSLEKAVDMYKYGKIAFVAMERITWDYDGIGSNGKKVDHDKFLDDAEKHYLPKTYTFDNGTFIIRAGDKVSEEKIKRLYWASREVKSQFHRVVGNDKALEVGNADDVLTMKIFNSPEEYKFNTNINGVSTDNGGLYIEPRGTFYTYERTPQQSIFSLEELFRHEYTHYLQARYLVDGLWGQGPFYEKNRLTWFDEGTAEFFAGSTRTSGVLPRKSILGYLAKDKVDHRYS

[0114] LKKTLNSGYDDSDWMFYNYGFAVAHYLYEKDMPTFIKMNKAILNTDVKSYDEIIKKLSDDANKNTEYQNHIQELADKYQGAGIPLVSDDYLKDHGYKKASEVYSEISKAASLTNTSVTAEKSQYFNTFTLRGTYTGETSKGEFKDWDEMSKKLDGTLESLAKNSWSGYKTLTAYFTNYRVTSDNKVQYDVVFHGVLTDNADISNNKAPIAKVTGPSTGAVGRNIEFSGKDSKDEDGKIVS

[0115] YDWDFGDGATSRGKNSVHAYKKAGTYNVTLKVTDDKGATATESFTIEIKNEDTTTPITKEMEPNDDIKEANGPIVEGVTVKGDLNGSDDADTFYFDVKEDGDVTIELPYSGSSNFTWLVY KEGDDQNHIASGIDKNNSKVGTFKSTKGRHYVFIYKHDSASNISYSLNIKGLGNEKLKEKENNDSSDKATVIPNFNTTMQGSLLGDDSRDYYSFEVKEEGEVNIELDKKDEFGVTWTLHP

[0116] ESNINARITYGQAAGNKVSNKVKLRPGKYYLLVYKYSGSGNYELRVNK

[0117] Example 3

[0118] In this embodiment, the monoclonal antibodies obtained in Examples 1 and 2 were aseptically inoculated into TB medium containing 100 μg / mL kanamycin resistance, and cultured at 37°C with shaking at 220 rpm until the OD600 was between 0.6 and 0.8. IPTG induction was then performed, and the cultures were incubated overnight at 18°C ​​with shaking. Samples were taken, ultrasonically disrupted, and analyzed by SDS-PAGE. The analysis results are shown in the figure below. Figure 1 .

[0119] Figure 1 In the diagram, columns 2-3 show the identification results of unmutated type I collagenase; columns 5-6 show the identification results of mutant A; and columns 8-9 show the identification results of mutant B. Figure 1 It can be seen that, at 18℃, both the unmutated type I collagenase and the mutant type I collagenase obtained from TB medium culture can be expressed in the supernatant (coll-1114kDa).

[0120] Example 4

[0121] Following the same scale-up culture method as in Example 3, mutated and non-mutated type I collagenase recombinant cells were obtained. 4g of the non-mutated recombinant cells were weighed and added to 20ml of Lysis Buffer, then dispersed on ice using a disperser. Cell disruption was performed by sonication: Ф10 probe, 10% power, 5.5s on, 9.9s off, for 30min. The cells were centrifuged at 20000rpm, 4℃ for 30min, and the supernatant was collected and filtered through a 0.22µm membrane. Purification was performed using 1ml of Ni-NTA, with the mobile phase composition as shown in Table 1, at a flow rate of 0.5ml / min. After loading, the UV was washed and conductivity was reduced to baseline using 20ml of Lysis Buffer. The elution program included: Step 1: 20% B, 10CV, 1.5ml / min; Step 2: 20-80% B, 20CV, 1.5ml / min; Step 3: 100% B, 15CV, 1.5ml / min.

[0122] Table 1

[0123]

[0124] The collected samples were subjected to electrophoresis, and the results were as follows: Figure 2 Based on the electrophoresis results, the samples containing the target protein were mixed together, concentrated by ultrafiltration, and dialyzed into a solution of 1×PBS, 5mM CaCl, 2mM ZnCl, pH 7.4. Calcium and zinc ions, as coenzymes for collagenase type I, can be added to the enzyme solution to improve the stability and biological activity of the enzyme.

[0125] The purified and concentrated collagenase type I was analyzed for concentration using BCA assay, and the results were as follows: R 2 =0.996, its concentration is 2.693 mg / ml, the volume is 12 ml, the yield is 32.32 mg, and the yield is 8.079 mg / g bacteria.

[0126] Recombinant bacterial cells of mutants A and B were treated in the same manner, purified and dialyzed, and their concentrations were determined using the BCA method. The result for mutant A was: R 2 =0.994, its concentration is 1.947 mg / ml, the volume is 13 ml, the yield is 25.31 mg, and the yield is 6.33 mg / g bacteria; the result of mutant B is: R 2 =0.996, its concentration is 2.826 mg / ml, the volume is 8 ml, the yield is 22.61 mg, and the yield is 5.65 mg / g bacteria.

[0127] Example 5

[0128] In this embodiment, the mutated and non-mutated recombinant type I collagenase obtained from Example 4 were used for enzyme activity detection experiments. The specific steps are as follows:

[0129] (1) Solution preparation

[0130] Working solution: Prepare the working solution (name: Collagenase Activity Assay kit, catalog number: ab196999) using the abcam collagenase activity assay kit. Mix Collagenase Substrate and Collagenase Assay Buffer from the kit at a ratio of 1:1.5 to obtain the enzyme activity assay working solution.

[0131] Preparation of positive enzyme: Dissolve 12500U of type I positive enzyme in 4mL of diluent to prepare a stock solution with a concentration of 3U / μL. Dilute stepwise according to a concentration gradient using PBS pH 7.4 buffer to obtain positive enzymes at various concentration gradients.

[0132] (2) Instrument testing

[0133] Preheat the microplate reader for 30 minutes, setting the temperature to 37℃. Add 100 μL of working solution and measure the absorbance at 345 nm, recording it as A1. Then, add 1 μL of each concentration of enzyme solution to the blank group, react for 10 minutes, and measure the absorbance at 345 nm as A2.

[0134] To plot the standard curve, see Figure 3 Calculate the OD difference A2-A1 between the sample and the blank, and determine the sample concentration based on the difference. The results are shown in Table 2.

[0135] Table 2

[0136]

[0137] Note: coll-1 is the unmutated recombinant type I collagenase, coll-1-A is the recombinant type I collagenase mutant A, and coll-1-B is the recombinant type I collagenase mutant B.

[0138] The concentration of the unmutated type 1 collagenase stock solution was 2.693 mg / mL, and the average activity was 5.33 U / μL. Therefore, the specific activity was (5.33 U / μL). 1000) / 2.693 = 1979.2 U / mg;

[0139] The stock solution concentration of recombinant type I collagenase mutant A was 1.947 mg / mL, and the average activity was 3.58 U / μL. Therefore, the specific activity was (3.58 U / μL). 1000) / 1.947 = 1838.7 U / mg;

[0140] The stock solution concentration of recombinant type I collagenase mutant B was 2.826 mg / mL, and the average activity was 3.80 U / μL. Therefore, the specific activity was (3.80 U / μL). 1000) / 2.826=1344.7U / mg.

[0141] Example 6

[0142] In this embodiment, purified unmutated and mutated recombinant type I collagenase were used for alkali tolerance testing. The specific method is as follows:

[0143] The pH of the 1XPBS solution was adjusted to 5, 6, 7, 8, 9, and 10 using 1M sodium hydroxide or 10% dilute hydrochloric acid. The enzyme activity of the samples was diluted to 1 U / μL using buffers of different pH values ​​and incubated for 1 h. The enzyme activity was then measured according to the method in Example 5. The samples were divided into three groups based on the acidity, neutrality, and alkalinity of the buffer: pH 5-6, pH 7-8, and pH 9-10. The average enzyme activity of each group was calculated. Since the optimal pH range for enzyme activity was 7-8, the average enzyme activity of the pH 7-8 group was taken as 100% and normalized. The percentage of residual enzyme activity of type I unmutated collagenase, mutant A, and mutant B in different pH ranges is shown in Table 3.

[0144] Table 3

[0145]

[0146] Note: coll-1 is the unmutated recombinant type I collagenase, coll-1-A is the recombinant type I collagenase mutant A, and coll-1-B is the recombinant type I collagenase mutant B.

[0147] Experimental results show that coll-1-A has better alkali tolerance and its enzyme activity remains good in strongly alkaline environments, such as around pH 9-10. Wild-type and mutant B, on the other hand, cannot maintain good enzyme activity in strongly alkaline environments.

[0148] Example 7

[0149] In this embodiment, the endomycin content of the purified recombinant collagenase was determined. The specific experimental steps are as follows:

[0150] (1) Pretreatment of instruments

[0151] First, the glassware used in the experiment was baked at 250℃ for 50 minutes, and the plastic centrifuge tubes were soaked in 0.5M NaOH for 1 hour, and then rinsed with ultrapure water.

[0152] (2) Operating steps

[0153] BCA assay for protein sample concentration: 2-10 mg / ml; add 1% Triton X-114, stir at 4°C for 30 min; incubate in a water bath at 30°C for 30 min; preheat centrifuge to 25°C, centrifuge at 12000 rpm for 20 min; carefully aspirate the upper aqueous phase (note aseptic technique). Repeat the above steps twice.

[0154] (3) Endotoxin Assay Kit (Name: Toxin Sensor Chromogenic LAL Endotoxin Assay Kit, Catalog No.: L00350 (16rnx), Manufacturer: Nanjing Genscript Biotech Co., Ltd.) is used to detect endotoxin content. The first step is reagent preparation: 1) LAL: Resuspend in 1.7 mL of LAL reagent water, mix gently, and store at -20℃ for one week; 2) Staining substrate: Resuspend in 1.7 mL of LAL reagent water, and store at 2-8℃ protected from light; 3) 0.46M hydrochloric acid: Add 2 mL of concentrated hydrochloric acid to 50 mL of LAL reagent water, mix thoroughly, and the final concentration is 0.46M hydrochloric acid; 4) Reaction termination solution: Resuspend color-stabilizer #1 in 10 mL of 0.46M hydrochloric acid. The recombinant termination solution is stable for one week after storage at 2-8℃; 5) Color-stabilizer #2 and #3: Resuspend in 10 mL of LAL reagent water and store at 2-8℃; 6) Endotoxin standard: Resuspend in 2 mL of LAL reagent water to a final concentration of 10 EU / mL and store at 2-8℃.

[0155] (4) Endotoxin detection

[0156] Endotoxin standard curve: First, dilute the endotoxin solution to 1 EU / mL, then dilute it to 0.1, 0.05, 0.025, 0.01 EU / mL or 1, 0.5, 0.25, 0.1 EU / mL, respectively. Add 100 μL of standard, sample, and pyrogen-free water to each endotoxin-free tube. Add 100 μL of LAL solution to each tube. Incubate at 37°C for 10 min, then add 100 μL of substrate and incubate at 37°C for 6 min. Add 500 μL of color-stabilizer #1, then 500 μL of color-stabilizer #2, then 500 μL of color-stabilizer #3. Add 200 μL of the solution to a 96-well plate and read the absorbance at 545 nm. Use water as a control. See the endotoxin standard curve diagram below. Figure 4 .

[0157] Sample pretreatment: Take 200uL of each sample tube, add ETDA to a final concentration of 5mM, boil for 20min to inactivate collagenase, centrifuge and collect the supernatant for endotoxin detection.

[0158] The quantitative detection results are shown in Table 4 below. After removing endotoxin, the endotoxin content of type I collagenase was all below 0.25 EU / mL (greater than or equal to this value is positive, less than is negative). It can be determined that the process for removing endotoxin is simple and feasible, and the samples from different batches after removal are all qualified.

[0159] Table 4

[0160]

[0161] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A type I collagenase mutant, characterized in that, The amino acid sequence of the type I collagenase mutant is as shown in SEQ ID NO.

5.

2. An isolated polynucleotide encoding a type I collagenase mutant, characterized in that, The polynucleotide is codon-optimized and is shown in SEQ ID NO.

2.

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

4. The expression vector according to claim 3, characterized in that, The expression vector is an Escherichia coli expression vector.

5. A host cell, characterized in that, The host cell includes the expression vector as described in claim 3 or 4; or The host cell genome contains the polynucleotides as described in claim 2.

6. A method for preparing a type I collagenase mutant, characterized in that, The method includes the following steps: Transform host cells using an expression vector containing the polynucleotide as described in claim 2; The host cells were cultured to express the type I collagenase mutant.

7. The method according to claim 6, characterized in that, The host cells were cultured using TB medium. And / or, the host cells are cultured at a temperature of 16 to 19°C.

8. A reagent kit, characterized in that, The kit comprises: the type I collagenase mutant as described in claim 1; or The polynucleotide as described in claim 2; or The expression vector as described in claim 3 or 4; or The host cell as described in claim 5.

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