CN115011583A - High-expression high-specific-activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid, and strain screening and purifying method - Google Patents
High-expression high-specific-activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid, and strain screening and purifying method Download PDFInfo
- Publication number
- CN115011583A CN115011583A CN202111419891.0A CN202111419891A CN115011583A CN 115011583 A CN115011583 A CN 115011583A CN 202111419891 A CN202111419891 A CN 202111419891A CN 115011583 A CN115011583 A CN 115011583A
- Authority
- CN
- China
- Prior art keywords
- proteinase
- mutant
- protease
- expression
- pichia pastoris
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010067770 Endopeptidase K Proteins 0.000 title claims abstract description 73
- 238000010276 construction Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims abstract description 14
- 241000235058 Komagataella pastoris Species 0.000 title claims description 19
- 239000013613 expression plasmid Substances 0.000 title claims description 6
- 230000000694 effects Effects 0.000 title abstract description 28
- 238000012216 screening Methods 0.000 title description 7
- 108091005804 Peptidases Proteins 0.000 claims abstract description 23
- 102000035195 Peptidases Human genes 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 235000019833 protease Nutrition 0.000 claims abstract description 7
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 239000013604 expression vector Substances 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 102000008300 Mutant Proteins Human genes 0.000 claims description 5
- 108010021466 Mutant Proteins Proteins 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 230000028327 secretion Effects 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical group C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 claims description 4
- 230000003248 secreting effect Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000005571 anion exchange chromatography Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 239000002808 molecular sieve Substances 0.000 claims description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000012141 concentrate Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 7
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 2
- 239000004365 Protease Substances 0.000 description 14
- 235000019419 proteases Nutrition 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 8
- 229960004441 tyrosine Drugs 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 241000222122 Candida albicans Species 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241001523956 Parengyodontium album Species 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 108010084455 Zeocin Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 229940095731 candida albicans Drugs 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- SBKVPJHMSUXZTA-MEJXFZFPSA-N (2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-5-amino-2-[[2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-indol-3-yl)propanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]-4-methylsulfanylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 SBKVPJHMSUXZTA-MEJXFZFPSA-N 0.000 description 2
- NRTLIYOWLVMQBO-UHFFFAOYSA-N 5-chloro-1,3-dimethyl-N-(1,1,3-trimethyl-1,3-dihydro-2-benzofuran-4-yl)pyrazole-4-carboxamide Chemical compound C=12C(C)OC(C)(C)C2=CC=CC=1NC(=O)C=1C(C)=NN(C)C=1Cl NRTLIYOWLVMQBO-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 108010038049 Mating Factor Proteins 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108010038555 Phosphoglycerate dehydrogenase Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- -1 aromatic amino acids Chemical class 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000010985 leather Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 101150042441 K gene Proteins 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 101710118918 Transcription elongation factor 1 Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
- C12N9/6408—Serine endopeptidases (3.4.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a sequence of a Proteinase K (Proteinase K) mutant, a key technology for constructing a yeast strain Proteinase K-m/pGAPZ alpha-A/GS 115 secreted and expressed by the sequence, a construction result and a purification method for producing high-activity Proteinase K by using the construction result. The invention adopts a gene engineering expression method to produce the high-activity proteinase K mutant, the specific activity reaches 26.8 international units/mg, the expression quantity reaches 366.7 mg/L protein, the target protein proteinase K with the purity of more than 95 percent can be obtained by purification, and a good foundation is laid for the future popularization and use of the proteinase K.
Description
Technical Field
The invention relates to the technical field of enzyme gene function modification, in particular to confirmation of a high-expression high-specific-activity Proteinase K (protease K) sequence, construction of a pichia pastoris expression vector of the sequence, construction of pichia pastoris strain protease K-m/pGAPZ alpha-A/GS 115 for secreting and expressing the protease K, a construction result and a purification method for producing the high-expression high-activity Proteinase K by using the construction result.
Background
Proteinase K (protease K) is a potent proteolytic enzyme isolated from Candida albicans (tritirachium album limber), a serine protease with broad cleavage activity. It cleaves the carboxy-terminal peptide bond of aliphatic and aromatic amino acids. Since proteinase K is stable in urea and SDS and has the ability to degrade native proteins, it has been used in a wide range of applications, including preparation of chromosomal DNA by pulsed electrophoresis, Western blotting and removal of nucleases in DNA and RNA preparations. Proteinase K has been widely used in leather, fur, silk, medicine, food, brewing, etc. The dehairing and softening of the leather industry has made use of a large amount of proteases, both to save time and to improve the labour hygiene conditions. The protease can also be used for degumming silk, tenderizing meat, and clarifying wine. The composition can be used for clinical medicine, such as treating dyspepsia with pepsin, treating bronchitis with acidic protease, treating angiitis with elastase, and cleaning surgical suppurative wound and treating pleural effusion adhesion with trypsin and chymotrypsin. The enzymatic washing powder is a new product in detergent, contains alkaline protease and can remove bloodstain and protein dirt on clothes. And the proteinase K produced by microorganisms from the nature has low content in the thallus, low activity, complex extraction process, high production cost and high market price, thus hindering the popularization and the application of the proteinase K.
Disclosure of Invention
In order to solve the technical problems, the invention provides a high-expression high-specific-activity proteinase K mutant sequence, a construction method of a pichia pastoris expression plasmid, and a strain screening and purifying method.
The invention provides the following technical scheme:
the proteinase K selected for recombinant expression is a strong proteolytic enzyme separated from Candida albicans (tritirachium album limber) and is a serine proteinase with wide cleavage activity, and the mature proteinase K contains 384 amino acids, has the molecular weight of 40299 daltons and has the pH value of 6.68. According to the analysis of the molecular structure and the sequence of the proteinase K, valine (Val) at the 66 th site is mutated into serine (Ser) so as to improve the specific activity of the proteinase K and increase the expression quantity of the proteinase K in a pichia pastoris expression system.
A protease K mutant has an amino acid sequence shown in SEQ ID NO. 1.
The sequence is characterized in that valine (Val) at position 66 is mutated into serine (Ser) so as to improve the specific activity of proteinase K. Meanwhile, the pichia pastoris secretion expression system is adopted, the system has the advantages of high yield, low production cost, eukaryotic expression system posttranslational modification function and the like, and can be used for producing proteinase K in large quantities and reducing the production cost.
The construction method of the proteinase K pichia pastoris recombinant strain comprises the following steps: inserting the modified and mutated protease KProteinase K gene into the downstream site of GAP promoter/alpha-factor signal peptide of pGAPZ alpha-A empty vector, and deleting the downstream site on the vectorKex2After position Lys-ArgSte13The site Glu-Ala-Glu-Ala is constructed to be a protease K gene secretion type expression vector Proteinase K-m/pGAPZ alpha-A; the protease K-m/pGAPZ alpha-A vector is transformed into pichia pastoris (a) by an electrotransformation methodPichia pastoris) Constructing a pichia pastoris engineering strain Proteinase K-m/pGAPZ alpha-A/GS 115 of a secretory expression Proteinase K mutant; obtaining the protease K mutant pichia pastoris recombinant strain with high expression and high activity by screening antibiotics, analyzing SDS-PAGE electrophoresis and determining biological specific activity.
The target protein of the pichia pastoris recombinant strain is as follows: the expression level of Proteinase K mutant (Proteinase K-m) reaches 366.7 mg/L, and the specific activity is 26.8 international units/mg; the proteinase K mutant of the target protein has 7 to 8 times higher specific activity than the natural proteinase K and has high stability.
The purification method of the proteinase K mutant comprises the following steps: fermenting by using the obtained high-activity proteinase K mutant pichia pastoris recombinant strain, centrifuging to collect supernatant, adjusting the pH value by using sodium hydroxide, passing through an anion exchange chromatography column, collecting a target elution peak to obtain a proteinase K mutant crude product with the purity of about 70%, dialyzing to remove salt, performing ultrafiltration concentration, passing through a molecular sieve chromatography column, collecting target protein, and obtaining the proteinase K mutant protein with the purity of more than 95%.
Compared with the prior art, the invention has the beneficial effects that: the invention adopts a gene engineering expression method to produce the high-activity proteinase K mutant, the specific activity reaches 26.8 international units/mg, the expression quantity reaches 366.7 mg/L protein, the target protein proteinase K with the purity of more than 95 percent can be obtained through purification, and a good foundation is laid for the future popularization and use of the proteinase K.
Drawings
FIG. 1 is a diagram showing the construction of an expression vector for Proteinase K mutant (protease K-m).
The molecular size of the protease K-m/pGAPZ alpha-A expression vector is 4.299 kb. Wherein pGAPZ alpha-A is 3.147kb, and the protease K-m is 1152bp (containing a terminator code); proteinase K-m is inserted between 747 and 824bp (XbaI site) of the vector pGAPZ alpha-A.
The pGAPZ alpha-A vector has the structure:
phosphoglycerate dehydrogenase Gene (GAP) promoter region: the length of the gene is 1-483bp,
phosphoglycerate dehydrogenase gene promoter primer sites: the 455-th and 476 bp-th sub-bands,
α -mating factor secretion signal peptide sequence: the 493-th and 759bp,
alpha-mating factor secretion signal peptide primer site: the 696 th channel and the 716bp channel,
vector multiple cloning site: 760 nd and 828bp are added to the reaction solution,
Mycepitope package: the 827-856bp fragment,
3' -ethanol oxidase 1 gene primer site: the 974-994bp of the molecular marker,
ethanol oxidase 1 transcription termination region: the 593-1233bp of the molecular marker,
transcription elongation factor 1 promoter region: the 1234 th and 1644bp of the sequence,
synthesizing a prokaryotic promoter: the length of the 1645-1712bp is less than that of the target,
reading frame of streptomycete zeocin resistance gene: the nucleotide sequence 1713-2087bp is shown in the specification,
cytochrome synthase 1 transcription termination region: 2088, the sub-band with the length of 2405bp,
coli replicon 1 (derived from pUC vector): 2416-3089 bp.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Construction of protease K mutant (protease K-m) pichia pastoris engineering bacteria
1. According to the amino acid sequence of the proteinase K mutant and the preference of the yeast to codons, the whole nucleotide sequence of the proteinase K mutant is designed, corresponding enzyme cutting sites XhoI and XbaI are introduced into two ends of the sequence, and the sequence is synthesized by Dalibao biology company.
2. The protease K mutant gene is subjected to XhoI/XbaI double enzyme digestion by adopting a gene cloning technology and then is inserted into an XhoI/XbaI site at the downstream of a GAP promoter/alpha-factor signal peptide of pGAPZ alpha-A to construct a secretory pichia pastoris expression vector containing the alpha-factor signal peptide; the molecular size of the protease K-m/pGAPZ alpha-A expression vector is 3.876 kb. Wherein pGAPZ alpha-A is 3.147kb, the protease K-m is 732bp (containing terminator code), the Proteinase K mutant is inserted between 747bp and 824bp (XbaI site) of the vector pGAPZ alpha-A, and the specific situation is shown in the attached drawing.
3. Transferring the linearized protease K-m/pGAPZ alpha-A plasmid into Pichia pastoris GS115 competence (purchased from Invitrogen company) by an electrotransformation method, screening by zeocin antibiotics to obtain a positive strain, further identifying the screened positive strain by PCR and Southern blotting technologies, and screening the high-expression and high-activity protease K-m/pGAPZ alpha-A/GS 115 engineering strain by an SDS-PAGE electrophoresis method and an SDS-PAGE activity determination method.
Example 2
Purification method of proteinase K mutant
1. Taking out the engineering bacteria of Proteinase K-m/pGAPZ alpha-A/GS 115 from a seed bank at the temperature of minus 80 ℃, inoculating a flat plate, and activating the bacterial strain;
2. selecting a single colony from the plate, culturing the colony in100 mg/L YPD + zeocin culture medium of 200 ml at 30 ℃ and 250 rpm for 48 hours, wherein the single colony is a first-level seed solution;
3. absorbing 100 ml of the first-stage seed liquid, inoculating the first-stage seed liquid into 2 liters of YPD culture medium, and culturing at 30 ℃ at 250 rpm for 24 hours to obtain a second-stage seed liquid;
4. inoculating 2L of the second-stage seed liquid into a fermentation tank containing 40L of YPD culture medium, fermenting for 72 hours, centrifugally collecting fermentation supernatant, adjusting the pH to 7.5-8.0 by using 1 mol of sodium hydroxide solution, passing through a DEAE Sepharose FF chromatographic column, collecting 0.5 mol/L of sodium chloride solution elution peak, dialyzing overnight to remove salt, obtaining a crude proteinase K mutant protein product with the purity of about 70%, performing ultrafiltration concentration, passing through a G75 chromatographic column, collecting a target protein peak, obtaining a proteinase K mutant protein stock solution with the purity of more than 95%, and freeze-drying and storing the stock solution.
Example 3
Proteinase K mutant activity assay
1. Preparation of a standard curve 0.1000 g of L-tyrosine standard substance which is dried to constant weight at 105 ℃ is accurately weighed, dissolved by 60 mL of 1 mol/L hydrochloric acid solution and then fixed to 100 mL, namely the 1 mg/mL tyrosine solution. Sucking 10.00 mL of 1 mg/mL tyrosine solution, and fixing the volume to 100 mL by using 0.1 mol/L hydrochloric acid solution to obtain 100 mu g/mL L-tyrosine standard stock solution. Standard solutions were prepared at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90. mu.g/mL. And respectively taking 3 mL of tyrosine standard solution into a cuvette, and placing the cuvette in an ultraviolet-visible spectrophotometer to measure the absorbance value of the tyrosine standard solution with each concentration at 275 nm.
2. 2 mL of the protease K mutant protein stock solution obtained in example 2 was pipetted into a 10 mL centrifuge tube and incubated in a 55 ℃ water bath for 2 min. Adding 2 mL of 10 g/L casein solution, mixing, and placing in a water bath kettle at 55 deg.C for warm bath for 5 min. Adding 4 mL of 0.4 mol/L trichloroacetic acid solution, mixing uniformly, stopping the reaction, and standing for 5 min. The mixture was filtered through a quick quantitative filter paper, and 3 mL of the filtrate was taken up to a cuvette at 275 nm to measure the absorbance. After the reaction was carried out by replacing the enzyme solution with 2 mL of the corresponding buffer solution before the measurement, the filtrate was zeroed at 275 nm. The measured specific activity was 26.8 International units/mg.
The invention adopts a gene engineering expression method to produce the high-activity proteinase K mutant, the specific activity reaches 26.8 international units/mg, the expression quantity reaches 366.7 mg/L protein, and the target protein proteinase K mutant has the specific activity 7-8 times higher than that of natural proteinase K and has high stability. The purity of the target protein proteinase K obtained by purification is up to more than 95%, and a good foundation is laid for the future popularization and use of proteinase K.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Henan Hezhi medicine science and technology Co., Ltd
<120> high-expression high-specific activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid and strain screening and purifying method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 296
<212> DNA
<213> Candida albicans (Tritirachium album)
<400> 1
mrsvsagaav rsaaargmva nkyvkkgsas adaamksgkd hsyknvsgaa tdnmvrvrah 60
dvydavvtna atnawgarss tsgtstyyyd saggscvyvd tgashgramv ktyyyssrdg 120
nghgthcagt vgsrtygvak ktgvkvddng sgystagmdv asdknnrnck gvvassgggy 180
sssvnsaaar ssgvmvavaa gnnnadarny sassvctvga sdrydrrsss nygsvdggts 240
stwggstrss gtsmathvag aaymtgktta asacryadta nkgdsngtvn aynnya 296
Claims (8)
1. A protease K mutant sequence characterized by: the amino acid sequence of the proteinase K mutant is shown in SEQ ID NO. 1.
2. A protease K mutant expression vector is characterized in that: the proteinase K mutant gene of claim 1 is transferred into the expression plasmid.
3. The protease K mutant expression vector of claim 2, wherein: expression plasmid pGAPZ alpha-A.
4. The construction of the protease K mutant expression vector is characterized in that: inserting the modified and mutated Proteinase K protease K gene into the downstream site of GAP promoter/alpha-factor signal peptide of pGAPZ alpha-A empty vector, and deleting the downstream site on the vectorKex2After position Lys-ArgSte13The site Glu-Ala-Glu-Ala is used for constructing a protease K gene secretion type expression vector Proteinase K-m/pGAPZ alpha-A.
5. A transformant characterized in that: the host bacterium is transformed with the proteinase K mutant expression vector of claim 2 or 3.
6. A transformant according to claim 5, characterized in that: the host bacterium is Pichia pastoris strain GS 115.
7. A method for producing a proteinase K mutant, which comprises culturing the transformant according to claim 5 or 6 and collecting the secreted product.
8. A method for purifying a protease K mutant pichia pastoris expression product is characterized by comprising the following steps: directly separating and purifying the Proteinase K mutant from the fermentation liquor of the secretory Proteinase K mutant Pichia pastoris strain K-m/pGAPZ alpha-A/GS 115: centrifuging to collect supernatant, adjusting pH value with sodium hydroxide, passing through anion exchange chromatography column, collecting target elution peak to obtain crude proteinase K mutant product with purity of about 70%, dialyzing to remove salt, ultrafiltering to concentrate, passing through molecular sieve chromatography column, collecting target protein to obtain proteinase K mutant protein with purity of more than 95%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111419891.0A CN115011583A (en) | 2021-11-26 | 2021-11-26 | High-expression high-specific-activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid, and strain screening and purifying method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111419891.0A CN115011583A (en) | 2021-11-26 | 2021-11-26 | High-expression high-specific-activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid, and strain screening and purifying method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115011583A true CN115011583A (en) | 2022-09-06 |
Family
ID=83064445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111419891.0A Pending CN115011583A (en) | 2021-11-26 | 2021-11-26 | High-expression high-specific-activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid, and strain screening and purifying method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115011583A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040166560A1 (en) * | 2001-02-09 | 2004-08-26 | Rainer Mueller | Expression of recombinant proteinase k <I> from tritirachium album </I>in yeast |
| CN102660524A (en) * | 2012-05-09 | 2012-09-12 | 海南华研生物科技有限公司 | Sequence of high-expression high-specific-activity H1 Collagenase mutant, construction of Pichia pastoris expression plasmid, and methods for screening and purifying strain |
| CN113215138A (en) * | 2021-06-02 | 2021-08-06 | 武汉瀚海新酶生物科技有限公司 | Proteinase K mutant with improved thermal stability |
| CN113234707A (en) * | 2021-05-31 | 2021-08-10 | 武汉瀚海新酶生物科技有限公司 | Protease K mutant and preparation method thereof |
| CN113481225A (en) * | 2021-07-23 | 2021-10-08 | 武汉瀚海新酶生物科技有限公司 | Construction and application of protease K high-expression engineering strain |
-
2021
- 2021-11-26 CN CN202111419891.0A patent/CN115011583A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040166560A1 (en) * | 2001-02-09 | 2004-08-26 | Rainer Mueller | Expression of recombinant proteinase k <I> from tritirachium album </I>in yeast |
| CN102660524A (en) * | 2012-05-09 | 2012-09-12 | 海南华研生物科技有限公司 | Sequence of high-expression high-specific-activity H1 Collagenase mutant, construction of Pichia pastoris expression plasmid, and methods for screening and purifying strain |
| CN113234707A (en) * | 2021-05-31 | 2021-08-10 | 武汉瀚海新酶生物科技有限公司 | Protease K mutant and preparation method thereof |
| CN113215138A (en) * | 2021-06-02 | 2021-08-06 | 武汉瀚海新酶生物科技有限公司 | Proteinase K mutant with improved thermal stability |
| CN113481225A (en) * | 2021-07-23 | 2021-10-08 | 武汉瀚海新酶生物科技有限公司 | Construction and application of protease K high-expression engineering strain |
Non-Patent Citations (1)
| Title |
|---|
| NCBI: "蛋白酶K氨基酸序列、基因序列以及序列比对结果", GENBANK * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102839165B (en) | Gene mutation type recombined protease K and industrialized production method thereof | |
| JPS61282097A (en) | Separation of fermented product | |
| CN104232611B (en) | A kind of recombination muscardine Proteinase K and its industrialized production and purification process | |
| CN104278017A (en) | Recombinant expression method of human lysozyme | |
| US20230227804A1 (en) | Nucleic Acids, Vectors, Host Cells and Methods for Production of Beta-Fructofuranosidase from Aspergillus Niger | |
| CN103275942B (en) | Glucose oxidase GODJ4A, and gene and application thereof | |
| CN115011583A (en) | High-expression high-specific-activity proteinase K mutant sequence, construction of pichia pastoris expression plasmid, and strain screening and purifying method | |
| JP6991423B2 (en) | Glucose oxidase CnGODA and its genes and their use | |
| CN110894219A (en) | Pichia pastoris transcription factor HAC1, protein, pichia pastoris and preparation and application thereof | |
| CN113736766B (en) | Collagen hydrolase and its coding gene, preparation method and use | |
| US20230220358A1 (en) | Nucleic Acids, Vectors, Host Cells and Methods for Production of Fructosyltransferase from Aspergillus Japonicus | |
| AU2019385785B2 (en) | Glucose oxidase M5GOD and coding genes and applications thereof | |
| CN116478969A (en) | Collagenase mutant, method for promoting secretory expression of recombinant collagenase and application thereof | |
| CN112391367A (en) | Preparation method of Cas9 protein for gene editing of human primary cells | |
| CN117247911B (en) | Coli DNA ligase mutant and expression purification method thereof in pichia pastoris | |
| CN116286755B (en) | Expression and purification method and application of batroxobin | |
| CN110616209A (en) | Mutant RNAseA and expression and purification method thereof in yeast cells | |
| CN113913413B (en) | Salt-tolerant RPK mutant and application thereof | |
| CN117903295B (en) | Kluyveromyces marxianus for secretory expression of lactoferrin and construction method and application thereof | |
| JPH06503718A (en) | Production of human lysothyme and its efficient secretion in methylotrophic yeast cells | |
| CN116656516A (en) | Method for screening recombinant strain of high-expression cobra combined peptide DAMP4-OH30 based on two-copy genes | |
| CA2341760A1 (en) | 17 kda foam protein | |
| CN115369103A (en) | Construction of high specific activity keratinase mutant and pichia pastoris expression plasmid, and strain screening and purifying method | |
| CN118165966A (en) | Preparation method and medical application of human neutrophil elastase | |
| CN116426657A (en) | Method for identifying encoding gene of microbial extracellular antibacterial active protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20220906 |