CN104232611A - Recombinant beauveria brongniartii proteinase K as well as industrial production and purification method thereof - Google Patents
Recombinant beauveria brongniartii proteinase K as well as industrial production and purification method thereof Download PDFInfo
- Publication number
- CN104232611A CN104232611A CN201410472748.1A CN201410472748A CN104232611A CN 104232611 A CN104232611 A CN 104232611A CN 201410472748 A CN201410472748 A CN 201410472748A CN 104232611 A CN104232611 A CN 104232611A
- Authority
- CN
- China
- Prior art keywords
- proteinase
- muscardine
- restructuring
- gene
- sequence
- 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.)
- Granted
Links
- 108010067770 Endopeptidase K Proteins 0.000 title claims abstract description 123
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000000746 purification Methods 0.000 title claims abstract description 17
- 241000248757 Cordyceps brongniartii Species 0.000 title abstract 5
- 238000009776 industrial production Methods 0.000 title abstract 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 58
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 29
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims abstract description 6
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 3
- 102000004190 Enzymes Human genes 0.000 claims description 30
- 108090000790 Enzymes Proteins 0.000 claims description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 230000014509 gene expression Effects 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 19
- 230000004151 fermentation Effects 0.000 claims description 19
- 239000013604 expression vector Substances 0.000 claims description 18
- 210000005253 yeast cell Anatomy 0.000 claims description 17
- 108020004414 DNA Proteins 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 15
- 210000004027 cell Anatomy 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 241000235648 Pichia Species 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 11
- 108020004705 Codon Proteins 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 230000006698 induction Effects 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 108091008146 restriction endonucleases Proteins 0.000 claims description 7
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 150000001413 amino acids Chemical group 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 6
- 238000011020 pilot scale process Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 230000029087 digestion Effects 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 4
- 108060001084 Luciferase Proteins 0.000 claims description 4
- 239000005089 Luciferase Substances 0.000 claims description 4
- 108700008625 Reporter Genes Proteins 0.000 claims description 4
- 235000001014 amino acid Nutrition 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 239000005090 green fluorescent protein Substances 0.000 claims description 4
- 238000012807 shake-flask culturing Methods 0.000 claims description 4
- 230000004071 biological effect Effects 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 2
- 102000053187 Glucuronidase Human genes 0.000 claims description 2
- 108010060309 Glucuronidase Proteins 0.000 claims description 2
- 241000320412 Ogataea angusta Species 0.000 claims description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 claims description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 2
- 229960000723 ampicillin Drugs 0.000 claims description 2
- 238000005277 cation exchange chromatography Methods 0.000 claims description 2
- 239000013522 chelant Substances 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 239000002532 enzyme inhibitor Substances 0.000 claims description 2
- 230000004927 fusion Effects 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 2
- 239000011535 reaction buffer Substances 0.000 claims description 2
- 108091092562 ribozyme Proteins 0.000 claims description 2
- 230000002103 transcriptional effect Effects 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 102000035195 Peptidases Human genes 0.000 claims 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 235000019833 protease Nutrition 0.000 claims 3
- 230000007547 defect Effects 0.000 abstract description 4
- 238000012239 gene modification Methods 0.000 abstract description 2
- 229930014626 natural product Natural products 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract 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 abstract 1
- 241001523956 Parengyodontium album Species 0.000 abstract 1
- 238000003259 recombinant expression Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 19
- 230000000694 effects Effects 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 15
- 241000222122 Candida albicans Species 0.000 description 13
- 229940095731 candida albicans Drugs 0.000 description 13
- 238000001962 electrophoresis Methods 0.000 description 10
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 239000000499 gel Substances 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 102100036826 Aldehyde oxidase Human genes 0.000 description 7
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 4
- 102000057593 human F8 Human genes 0.000 description 4
- 229940047431 recombinate Drugs 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- 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 3
- 235000021240 caseins Nutrition 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 101150061183 AOX1 gene Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 229940093916 potassium phosphate Drugs 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101150069554 HIS4 gene Proteins 0.000 description 1
- 101001104083 Homo sapiens Rabphilin-3A Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102100040040 Rabphilin-3A Human genes 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- -1 aliphatic amino acid Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21064—Peptidase K (3.4.21.64)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a recombinant beauveria brongniartii proteinase K as well as an industrial production and purification method thereof and relates to the technical field of gene modification and protein engineering. Recombinant beauveria brongniartii proteinase K coding genes suitable for the pichia pastoris recombinant expression are obtained with a biological information means, optimization genes are synthesized and successfully expressed in a eukaryotic system, and the recombinant proteinase K with the enzymatic activity obviously higher than that of a traditional tritirachium album limber proteinase K. According to the recombinant beauveria brongniartii proteinase K as well as the industrial production and purification method thereof, the coding genes are synthesized chemically, and GS115/pPIC9K-ProK high-yield engineering bacteria are established, so that defects of the low yield and the high cost of natural products are overcome, the usage cost of the proteinase K in related industries is significantly reduced, and the recombinant beauveria brongniartii proteinase K as well as the industrial production and purification method thereof have broad application prospects.
Description
Technical field:
The present invention relates to genetic modification and protein engineering field, be specifically related to a kind of restructuring muscardine Proteinase K and suitability for industrialized production thereof and purification process.
Background technology:
Proteinase K (Proteinase K, EC 3.4.21.64 has another name called endopeptidase K, tritirachium alkaline proteinase, Candida albicans serine protease, Candida albicans Proteinase K) is the wider serine protease of a kind of nicking activity.Therefore this albumen by cutting carboxy-terminal peptide bond degraded natural keratin (keratin) of aliphatic amino acid and die aromatischen Aminosaeuren, and gain the name.Because Proteinase K all can stable existence have the ability of degrade proteins, the nuclease being thus widely used in the preparation of pulse electrophoresis chromosomal DNA, western blotting and removing in DNA and RNA goods in Guanidinium hydrochloride (3M), urea (4M) and SDS (0.5-1%).
Proteinase K early than 1974 from woods Bai Shi Candida albicans purifying obtain.But Natural strains cultivates difficult, that output is too low problem seriously hinders Proteinase K practical application all the time.After within 1985, measuring its natural acid sequence, the investigator of multiple country and associated companies have dropped into very large energy and have attempted realizing the recombinant expressed on a large scale of this albumen, recombinant expressed in E. coli system of this albumen is realized before and after 2000, but this method can only obtain inclusion body protein, refolding strategy process causes production cost to increase, production loss is serious; Until within about 2008, just achieve the Bichi yeast system secreting, expressing of this albumen, so far, the production of woods Bai Shi Candida albicans Proteinase K and application enter fast-developing period.Meanwhile; the expression and purification patent of woods Bai Shi Candida albicans Proteinase K wild-type and various mutant is at very short time Nei Beige great international drugmaker monopolization (Patent No.:WO 02/072634A2; US 7368274B2), as after the Chinese Enterprise of the person of entering be difficult to get around Patents protection and join in the research and development of this enzyme, production and selling process and go.
For solving the problem, the present inventor has carried out deep bioinformatic analysis to the Proteinase K homologous sequence in open gene database, in conjunction with existing Proteinase K crystalline structure and conserved positions comparison, find that its coded product of a kind of proteinase gene in muscardine may have complete Proteinase K biologic activity.Express obtain this recombinant protein enzyme through encoding gene Optimizing Reconstruction, eukaryotic cell-yeast cell external secretion, prove that this albumen has complete Proteinase K through biological chemistry and zymetology measuring active, and its protease activity comparatively wild-type woods Bai Shi Candida albicans Proteinase K exceed 50%.By optimization expression system, the present inventor utilizes this recombinant protein enzyme of yeast cell external secretion expression technology large scale fermentation high expression K, and improves activity and the output of this enzyme further; Adopt efficient rabphilin Rab way of purification, improve the rate of recovery of Proteinase K in purge process, realize mass-producing and the continuous prodution of this enzyme, complete the present invention.Up to now, all do not find in patent document and non-patent document about the industrial report of muscardine recombinant protein enzyme K.
Summary of the invention:
The restructuring muscardine Proteinase K that technical problem to be solved by this invention is to provide a kind of activity good, reasonable in design, simple to operate and suitability for industrialized production thereof and purification process.
Technical problem to be solved by this invention adopts following technical scheme to realize:
A kind of restructuring muscardine Proteinase K, containing full length amino acid sequence, particularly containing the 99 to the 380 amino acids sequence or with this section amino acid sequence homology higher than the aminoacid sequence of 95%, and there is the biological activity of Proteinase K.
The DNA encoding method of described restructuring muscardine Proteinase K is the DNA sequence dna encoding sequence of wild-type muscardine Proteinase K being replaced with host cell preferred codons composition, deduct original signal peptide sequence and after adding histidine mark and restriction enzyme site, then prepared by the mode of chemosynthesis.
Described host cell preference codon is yeast biased codons.
The expression vector of described restructuring muscardine Proteinase K has such structure: the DNA upstream promoter sequence that the DNA of described restructuring muscardine Proteinase K expresses being connected to coding Chong Zushi muscardine Proteinase K, also can be connected to the DNA downstream of Chong Zushi muscardine Proteinase K by terminator.
The expression vector of described restructuring muscardine Proteinase K can select any one in pPICZ, pPICZ Q, pGAPZ, pGAPZ Q, pGAPZ Q A and pPIC9K, preferred pPIC9K.
The selectable marker gene for selecting recombinant chou or the reporter gene for detecting quiding gene expression can be inserted in the expression vector of described restructuring muscardine Proteinase K.
Described selectable marker gene includes but not limited to hygromycin gene, kalamycin resistance gene and ampicillin resistance gene, and reporter gene includes but not limited to beta-Glucuronidase (GUS) gene, E.C. 2.3.1.28 (CAT) gene, luciferase (LUC) gene and green fluorescent protein (GFP) gene.
Can appended sequence be comprised in the expression vector of described restructuring muscardine Proteinase K, restructuring muscardine Proteinase K is expressed with the form of fusion rotein (albumen of encoding with appended sequence or peptide merge).
Described appended sequence includes but not limited to the nucleotide sequence of coded signal peptide or propetide, and the nucleotide sequence of coding His label or GST label.
The transformant of described restructuring muscardine Proteinase K can be prokaryotic cell prokaryocyte or eukaryotic cell, preferred eukaryotic cell.
Described eukaryotic cell includes but not limited to yeast cell, preferred yeast cell.
Described yeast cell is the one in Pichia pastoris, candida cell, Hansenula polymorpha cell, torulopsis cell, fission yeast cell and Crewe dimension ferment loam cell, preferred Pichia pastoris.
Described Pichia pastoris is GS115 yeast cell, and adopt electroporated method to obtain the transformant of pichia spp: first linearizing expression vector is transformed in competent yeast cells, coating on flat board by thalline suspension again, until there is single bacterium colony in culture plate.
The test kit of described restructuring muscardine Proteinase K comprise in Chong Zushi muscardine Proteinase K, the DNA of coding Chong Zushi muscardine Proteinase K, expression vector and transformant one or more.
The test kit of described restructuring muscardine Proteinase K also can comprise one or more components in addition, component is made up of test tube, reaction buffer, PCR primer, dNTP, Taq polysaccharase, reversed transcriptive enzyme, DNA enzymatic, RNA enzyme inhibitors, DEPC water and sterilized water, but is not always limited to this.
To recombinate the industrialized preparing process of muscardine Proteinase K, comprise the steps:
1) from the transformant of picking, obtain 20 bacterial strains that restructuring muscardine Proteinase K expression amount is the highest, shake-flask culture, centrifugally obtain fermented supernatant fluid, then carry out enzyme and live, express the test such as output, digestion BSA, choose the best bacterial strain of a strain further and carry out enlarged experiment test;
2) carry out optimal culture condition exploration at 14L fermentation tank level, comprise the consumption of culture medium prescription, dissolved oxygen amount, induction time and inductor methyl alcohol;
3) fermentation condition that 14L fermentor tank obtains is transplanted to the test of 500L fermentor tank industrial scale, copy pilot scale fermentation condition completely, continuously ferment cultivation 48,72,108h, the every fermentation index of on-line monitoring and cataphoretic determination protein expression level, lower tank centrifugation thalline and fermented liquid.
Described pilot scale fermentation condition adopts YPD substratum and MM substratum, and the methanol induction time is 48,108h, and dissolved oxygen amount is 20%, 30%, 40%, and methanol usage is 0.5%, 3% (v/v).
A kind of purification process of muscardine Proteinase K of recombinating, comprise the steps: to add hexahistine label at the C end of restructuring muscardine Proteinase K, fermented liquid is first through centrifugation, supernatant liquor is again through metal ion-chelant affinitive layer purification, and then through cation exchange chromatography, concentrated, last freeze-drying becomes finished product enzyme.
The invention has the beneficial effects as follows:
1) optimize first and obtain being suitable for the recombinant expressed muscardine Proteinase K coding DNA of pichia spp, build GS115/pPIC9K-ProK high production bacteria by the above-mentioned encoding gene of chemosynthesis, overcome that natural product yield poorly, defect costly;
2) the active restructuring muscardine Proteinase K product higher than existing commercially available wild-type woods Bai Shi Candida albicans Proteinase K is obtained in a large number by shaking flask and fermentor tank;
3) avoiding multi-step in conventional purification methods uses ultrafiltration to cause most of albumen precipitation and the defect that causes yield to reduce, thus makes comprehensive recovery improve 80% compared to existing technology.
4) significantly reduce the Proteinase K use cost of related industries, be with a wide range of applications.
Accompanying drawing illustrates:
Fig. 1 is Yeast expression carrier PIC9K Vector map of the present invention;
Fig. 2 is that the present invention utilizes the expression (laboratory scale of SDS-PAGE electrophoresis detection restructuring muscardine Proteinase K in GS115 bacterial strain; Shake flask fermentation);
Fig. 3 is that muscardine Proteinase K precursor self splicing of recombinating under the present invention utilizes SDS-PAGE electrophoresis detection different condition forms the result of ripe Proteinase K; Fig. 3 is not mentioned in embodiment!
Fig. 4 is the effect of muscardine Proteinase K and woods Bai Shi Candida albicans proteinase K digestion calf serum BSA of recombinating under the present invention utilizes SDS-PAGE electrophoresis detection same protein concentration conditions;
Fig. 5 is F-C analytical method tyrosine typical curve of the present invention;
Fig. 6 is that the present invention utilizes the expression (industrial scale of SDS-PAGE electrophoresis detection restructuring muscardine Proteinase K under pilot scale fermentation condition; Ferment tank).
Embodiment:
The technique means realized to make the present invention, creation characteristic, reaching object and effect is easy to understand, below in conjunction with specific embodiment, setting forth the present invention further.
1) acquisition of the recombinant expressed Optimized Coding Based gene of muscardine Proteinase K pichia spp:
From GeneBank database, inquiry obtains muscardine Proteinase K original cDNA sequence (Gene ID:AY520815, SEQ ID NO.2), according to tRNA abundance data in Pichia pastoris, rare triplet codon in muscardine Proteinase K original coding gene is replaced with the corresponding codon nucleotides that in Pichia pastoris, tRNA abundance is the highest, and final acquisition is suitable for the recombinant expressed muscardine Proteinase K optimized gene encoding sequence of pichia spp.In this optimizing process, in muscardine Proteinase K original cDNA sequence, signal peptide coding region section is removed, and final A+T base number reaches 50% of sequence, meets the recombinant expressed requirement of pichia spp (SEQ ID NO.3).
2) structure of restructuring muscardine Proteinase K Expression vector pPIC9K-ProK:
The encoding sequence (SEQ ID NO.2) of wild-type muscardine Proteinase K is replaced with the DNA sequence dna be made up of pichia spp preferred codons, EcoR I restriction enzyme site GAATTC is added at its 5 ' end, 3 ' end adds hexahistine label C ACCATCACCACCATCAC (SEQ ID NO.4), terminator codon TAA and Not I restriction enzyme site GCGGCCGC in turn, utilizes chemical process to synthesize the sequence (SEQ ID NO.5) (Sangon Biotech (Shanghai) Co., Ltd.) of gained.The sequence of above-mentioned chemosynthesis oneself be cloned into (Sangon Biotech (Shanghai) Co., Ltd.) in pUC57 plasmid.
Amplification pUC57/ProK plasmid, with restriction enzyme EcoR I, Not I, ProK encoding sequence is cut respectively, connect with the pPIC9K carrier (American I nvitrogen company) carrying out same double digestion, product conversion DH5 α competent cell will be connected, picked clones, amplification plasmid, identifies plasmid with restriction enzyme EcoR I, Not I respectively.
By aforesaid method, by the DNA fragmentation subclone of encoding wild type muscardine Proteinase K to (as shown in Figure 1) in Yeast expression carrier pPIC9K, thus construct wild-type protease K Expression vector pPIC9K-ProK.
Sequencing result shows, target fragment is correctly inserted in above-mentioned expression vector.
3) conversion of restructuring muscardine Proteinase K Expression vector pPIC9K-ProK:
Restriction enzyme Sal I enzyme is utilized to cut recombinant plasmid constructed in step 2, make it linearizing, then TE damping fluid (pH 8.0) is used to be dissolved to the concentration of l μ g/ μ L, plasmid and the GS115 competent yeast cells of getting 20 μ L dissolvings mix, be transferred in the electric revolving cup (two clearance between poles 0.1cm) of ice precooling, ice bath 5min.Adopt electroporated method, the pichia spp Transformation Parameters utilizing electric conversion instrument built-in shocks by electricity, and is transformed in GS115 competent yeast cells by above-mentioned expression vector.
After electric shock, be rapidly in electric revolving cup the 1M Sorbitol Solution USP adding the precooling of 1mL ice, mix gently, go in 1.5mL EP pipe.Coated by thalline suspension on MD flat board (13.4g/L yeast basic nitrogen source, 0.4mg/L sit thing element, 20g/L glucose, 1.5% agar), every 500 μ L are coated with one flat plate.Flat board is placed in 30 DEG C of environment to cultivate until there is single bacterium colony, obtains GS115/pPIC9K-ProK.
4) high copy number and Mut
+the screening of the GS115/pPIC9K-ProK of phenotype:
Prepare the YPD screening dull and stereotyped (yeast extract 10g/L, peptone 20g/L, glucose 20g/L, 1.5% agar) of four G418 concentration gradients (0.75mg/mL, 1mg/mL, 1.75mg/mL, 2.0mg/mL) respectively.Dull and stereotyped for the MD carrying out in step 3 cultivating, with each picking of sterile toothpick 50 yeast list bacterium colonies, put and screen in flat board at the YPD of above-mentioned four G418 concentration gradients, and make corresponding numbering.Each clone is all cooked the screening of 4 gradients.
PPIC9K expression vector can give pichia spp geneticin resistant, and its resistance level depends on the copy number of the kan gene of integration.The pPIC9K of single copy is incorporated into pichia spp postgenome, can give the geneticin resistant level that pichia spp is about 0.25mg/mL.Owing to there is genetic linkage between kan gene and expression cassette (pAOX1 and goal gene), thus can roughly infer according to the resistance level of Geneticin the target protein encoding gene copy number that this clone comprises.
Dull and stereotyped by observing above-mentioned screening, whether preliminary judgement GS115/pPIC9K-ProK yeast clone contains the target protein encoding gene of high copy number.In screening flat board, the content of G418 is higher, and yeast colony growth profile is larger, shows the copy containing more target protein gene in this yeast mono-clonal, can tolerate the G418 of higher concentration.
Meanwhile, PCR qualification is carried out to above-mentioned mono-clonal.Primer is 5'AOX1 (SEQ ID NO.6), 3'AOX1 (SEQ ID NO.7).
5'AOX1:GACTGGTTCCAATTGACAAGC
3'AOX1:GCAAATGGCATTCTGACATCC
PCR system (50 μ L):
PCR reaction conditions is: 98 DEG C of denaturation 10min, 98 DEG C of sex change 50s, and 55 DEG C of annealing 50s, 72 DEG C extend 2min, and 30 circulations are carried out in reaction.
Agarose gel electrophoresis is utilized to detect the size of PCR primer.After transforming GS115 with Sal I linearization plasmid, mostly recombinate on HIS4 site, most of transformant is Mut
+phenotype; But contain AOX1 gene order due to plasmid, likely recombinate in AOX1 site, destroy wild-type AOX1 gene, produce Mut
stransformant.Therefore, in theory, if object fragment inserts successfully and phenotype is Mut
+, two band of about 2200bp, about 1632bp should be had; If only have 1632bp mono-band, then phenotype may be Mut
s.
The display of PCR result can amplify the band of about 2200bp, show that object fragment is inserted successfully, and phenotype is Mut
+.
The sequence table of SEQ ID NO.1-NO.7 is as follows:
1. SEQ ID NO.1 muscardine Proteinase K aminoacid sequence
2. SEQ ID NO.2 muscardine Proteinase K DNA sequences encoding (1143bp)
3. SEQ ID NO.3 codon optimized after restructuring muscardine Proteinase K DNA sequences encoding (1098bp)
4. SEQ ID NO.4 hexahistine label dna sequence (18bp)
caccatcacc accatcac 18
5. the DNA sequence dna (1130bp) of restructuring muscardine Proteinase K prepared of SEQ ID NO.5 chemosynthesis mode
6. SEQ ID NO.65'AOX1 primers DNA sequences (21bp)
gactggttcc aattgacaag c 21
7. SEQ ID NO.73'AOX1 primers DNA sequences (21bp)
gcaaatggca ttctgacatc c 21
5) to recombinate the expression in yeast cell of muscardine Proteinase K and detection (laboratory scale):
Step 4 is screened the high copy number and Mut that obtain
+the yeast list colony inoculation of the GS115/pPIC9K-ProK of phenotype is in 20mL YPD substratum (yeast extract 10g/L, peptone 20g/L, glucose 20g/L), and 30 DEG C, 250rpm cultivates 24h.1500g collected by centrifugation thalline.With 10mL MM substratum (13.4g/L YNB, 4 × 10
-4g/L vitamin H, 5mL/L methyl alcohol) resuspended thalline.Subsequently at 30 DEG C, shaking flask (2L) abduction delivering under 250rpm.Every 4-8h samples in order to detecting from fermented liquid, and in the fermented liquid of abduction delivering, add the methyl alcohol that final concentration is 1% (v/v).Continuous 5d, Dual culture 108h.
After cultivation, centrifugal substratum, thus obtain the fermented liquid supernatant containing above-mentioned restructuring muscardine Proteinase K, and be placed in 4 DEG C of preservations.
Utilize SDS-PAGE to detect the expression of restructuring muscardine Proteinase K of the present invention in yeast cell, SDS-PAGE formula is as follows:
In small beaker, add solution composition needed for 12% separation gel successively, pour in the gap of the double glazing unit be pre-assembled, room temperature places more than 20min, until gel polymerisation is complete.In small beaker, add solution composition needed for 5% concentrated glue more successively, pour into the gap above the own separation gel condensed between double glazing unit, room temperature places more than 20min, until gel polymerisation is complete.
2 × SDS albumen loading buffer fills a prescription: 1.5M Tris-HCL pH 6.8 10ml, and 4g SDS, tetrabromophenol sulfonphthalein 0.2g, glycerine 40ml, adds distilled water and be settled to 100ml.
The substratum supernatant of GS115/pPIC9K-ProK is taken out by 4 DEG C of refrigerators, get 20 μ L supernatant liquors mixing isopyknic 2 × SDS albumen loading buffer after with boiling water bath process 10min, the centrifugal 1min of 12000g, drawing supernatant joins in the sample duct of the gel as above prepared, applied sample amount is 10 μ L, add protein molecular weight standard, 200V constant voltage electrophoresis 1.5h, unloads gel simultaneously.Prepare staining fluid and destainer by shown in following formula, carry out coomassie brilliant blue staining, to observe the target protein band in sample.
Prescription of its dyeing liquor: distilled water 500ml; Ethanol 400ml; Acetic acid 100ml; 1g coomassie brilliant blue R_250.
Destainer is filled a prescription: distilled water 500ml; Ethanol 400ml; Acetic acid 100ml.
Result as shown in Figure 2, is cultivated under shaking flask condition and can be observed obvious target protein band in fermented liquid supernatant (shake flask fermentation) to 66h.
Wherein, 5 visible protein bands of protein molecular weight standard (Unstained Protein Molecular Weight Marker, Fermentas) represent the albumen size of 45kD, 35kD, 25kD, 18.4kD and 14.4kD respectively.
From electrophoresis result, restructuring muscardine Proteinase K of the present invention can in yeast cell effective expression.
6) Activity determination of restructuring muscardine Proteinase K:
Utilize above-mentioned obtained shake-flask culture fermented liquid supernatant, measure protein concn by BCA method, with PBS, the protein concentration of fermented liquid supernatant is adjusted to 1mg/mL.With PBS, commercially available woods Bai Shi Candida albicans Proteinase K (precious biotechnology (Dalian) company limited) is mixed with 1mg/mL enzyme solution simultaneously.
Reaction substrate is the BSA of guanidine hydrochloride denaturation: 5mg BSA is dissolved in 6M Guanidinium hydrochloride, 50mM Tris-CI (pH8.0), 5mM DTT, final volume is 1mL.95 DEG C of water-bath 20min; After being down to room temperature, add 50mM Tris-CI (pH 7.5) and 5mM CaCI
2, make the final concentration of Guanidinium hydrochloride be down to below 2M.
Temperature of reaction: 37 DEG C, reaction system (10 μ L) is: BSA solution 4 μ L, enzyme solution 6 μ L.
After mixed reaction solution adds rapidly 2 × SDS albumen loading buffer 10 μ L after 37 DEG C of reactions 30s, 1min, 5min, 10min and 30min, boiling water bath process 10min.The centrifugal 1min of 12000g, draw supernatant and join in the sample duct of the gel as above prepared, applied sample amount is 10 μ L.Add protein molecular weight standard (Unstained Protein Molecular Weight Marker, Fermentas) simultaneously.After 200V constant voltage electrophoresis 1.5h, unload gel.Carry out coomassie brilliant blue staining, decolouring, to observe the target protein band in sample.
As shown in Figure 4, under same protein concentration, same reaction time conditions, restructuring muscardine Proteinase K is significantly better than wild-type woods Bai Shi Candida albicans Proteinase K to the degradation capability of BSA to result.
Result show restructuring muscardine Proteinase K of the present invention there is obvious protease activity and activity higher than wild-type woods Bai Shi Candida albicans Proteinase K.
7) the specific activity detection by quantitative of restructuring muscardine Proteinase K:
Detect the enzymic activity of the restructuring muscardine Proteinase K of the present invention utilizing the experimental program in step 5 to obtain by the following method.
With 50mM potassium phosphate solution preparation 0.65% (w/v) casein food grade solution, slowly heating is until form single uniform dispersion, at 37 DEG C, regulate pH to 7.5 by 1M HCl solution or NaOH solution.Preparation is containing 10mM NaAc and 5mM Ca (Ac)
2solution, at 37 DEG C, regulate pH to 7.5 with 0.1M HCI or NaOH.Be cooled to room temperature containing 10mM NaAc and 5mM Ca (Ac)
2solution preparation concentration be the restructuring muscardine Proteinase K Solution of 0.1 ~ 0.2U/mL, this solution is use front preparation.
Each reagent (mL) is added successively according to following order:
Hatch 30min manually mixing off and on for 37 DEG C, collect clear liquor and carry out next step with No. 50 filter paper filterings and test.
1. typical curve:
10mL forint phenol reagent is diluted with water to 40mL, preparation 0.5N F-C reagent.Slow heating is until tyrosine dissolves and is cooled to room temperature, and preparation 1.1mM TYR standard solution, then adds following reagent (mL) successively.
2. sample determination:
In 4 bottles, add following reagent (mL) respectively successively:
Hatch and manually mix off and on for 37 DEG C, taking out bottle after 30min and be cooled to room temperature.If solution presents muddy shape, measure with being conducive to absorbance after 0.45 μm of membrane filtration.Reaction solution is transferred in Glass Containers, measures the absorbance of Stdl ~ 5, blank, sample and contrast with spectrophotometer at 660nm place.
Result calculates:
Typical curve: △
660nmstd=A
660nm std-A
660 is blank
Use △
660nmamount (μm ol) the drawing standard graphic representation of Std and TYR.
Gained typical curve as shown in Figure 5.
Sample determination: △
660nmsample=A
660nm sample-A
660nm is blank
Typical curve is utilized to calculate the amount (μm ol) of the TYR of release.
Enzymic activity (U/mL)=(amount (μm ol) of the TYR of release) × 11/ (1 × 10 × 2).
Wherein: the cumulative volume (mL) during 11=reaction terminating, 10=reaction times (min), enzyme reagent volume (mL) during 1=reaction, reaction solution volume (mL) used during 2=colorimetric.
U/mg solid=(U/mL enzyme)/(mg solid/mL enzyme)
U/mg albumen=(U/mL enzyme)/(mg albumen/mL enzyme)
Enzymic activity defines: 1 unit of enzyme activity is defined as, and at pH 7.5, under 37 DEG C of conditions, per minute decomposites the TYR of 1.0 μm of ol (181 μ g) from casein food grade.(Forint phenol method carries out colorimetric)
In the reaction system of 6mL, Reagents Final Concentration is: 42mM potassiumphosphate, 0.54% (w/v) casein food grade, 1.7mM NaAc, 0.8mM Ca (Ac)
2with the Proteinase K Solution of 0.1 ~ 0.2U/mL, result is as shown in the table:
Visible, the restructuring muscardine Proteinase K that the present invention prepares can demonstrate the enzymic activity being obviously better than wild-type woods Bai Shi Candida albicans Proteinase K, and two kinds of commercially available woods Bai Shi Candida albicans Proteinase K specific activity that its activity comparatively uses in this experiment all exceed about 50%
8) industrial mass production of muscardine Proteinase K of recombinating and purifying:
20 bacterial strains that genic mutation type recombinant protein enzyme K expression amount is the highest are obtained from the transformant of picking.Utilize step 5) described in experimental program, shake-flask culture 20 strain engineering strain, purifying protein enzyme K from fermented liquid also carries out enzyme to live, expresses the test such as output, digestion BSA, chooses the best bacterial strain of a strain further and carries out enlarged experiment test.
The exploration of engineering strain optimal culture condition is carried out at 14L fermentation tank level, with reference to the pichia spp culture scheme of American I nvitrogen company, adopt the substratum identical with shake flask fermentation, the methanol induction time is 108h, dissolved oxygen amount controls 40%, the consumption of inductor methyl alcohol is 1.5% (v/v), and after induction, interval 8h samples, electrophoretic analysis Proteinase K expression amount.
By the technical scheme that 14L fermentor tank obtains, be transplanted to the test of 500L tank industrial scale, copy pilot scale fermentation condition completely, continuously ferment and cultivate 72h, the every fermentation index of on-line monitoring at any time and cataphoretic determination protein expression level, lower tank centrifugation thalline and fermented liquid.
The restructuring muscardine Proteinase K bacterial strain fermentation liquor supernatant getting the rear different time sections (16h, 24h, 32h, 40h, 48h, 56h, 64h, 72h) of induction, as detected object, carries out SDS-PAGE electrophoresis detection.Result as shown in Figure 6.
Wherein, 7 visible protein bands of protein molecular weight standard (Unstained Protein Molecular Weight Marker, Fermentas) represent the albumen size of 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD and 14.4kD respectively.
As seen from Figure 6, utilize this industrialization mass production method, effectively can improve the productive rate (higher than 200mg/L fermented liquid) of restructuring muscardine Proteinase K of the present invention.This productive rate is better than the restructuring muscardine Proteinase K (20mg/L fermented liquid) under shaking flask condition, thus make later-period purification, the high yield of freeze-drying becomes possibility, produce for large-scale industrial and provide condition.
Utilize the hexahistine label added at the C end of restructuring muscardine Proteinase K, greatly reduce non-specific binding to the irrelevant foreign protein in purification column and nucleic acid and other non-specific binding molecule, fermented liquid is separated through 10000rpm high speed centrifugation, metal-chelating column chromatography is separated with ion-exchange chromatography and becomes finished product enzyme after freeze-drying, avoiding multi-step in conventional purification methods uses ultrafiltration to cause most of albumen precipitation and the defect that causes yield to reduce, thus makes comprehensive recovery improve 80% compared to existing technology.
SDS-PAGE is utilized to detect the purity of the Proteinase K after above-mentioned technique purifying.Result as shown in Figure 3.Can find out, the recombinant protein enzyme K after purifying only has the maturation protein enzyme K band after total length precursor protein enzyme K and self splicing, and both total amounts exceed 99% of total protein concentration.4 DEG C, 25 DEG C and add or do not add CaCl
2after placing 24h under condition, most precursor protein enzyme K can self splicing be all maturation protein enzyme K.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (13)
1. a restructuring muscardine Proteinase K, it is characterized in that: containing full length amino acid sequence, particularly containing the 99 to the 380 amino acids sequence or with this section amino acid sequence homology higher than the aminoacid sequence of 95%, and there is the biological activity of Proteinase K.
2. one restructuring muscardine Proteinase K according to claim 1, it is characterized in that: the DNA encoding method of described restructuring muscardine Proteinase K is the DNA sequence dna encoding sequence of wild-type muscardine Proteinase K being replaced with host cell preferred codons composition, deduct original signal peptide sequence and after adding histidine mark and restriction enzyme site, prepared by the mode of chemosynthesis, described host cell preference codon is yeast biased codons again.
3. one restructuring muscardine Proteinase K according to claim 1, it is characterized in that, the expression vector of described restructuring muscardine Proteinase K has such structure: the DNA upstream promoter sequence that the DNA of described restructuring muscardine Proteinase K expresses being connected to coding Chong Zushi muscardine Proteinase K, also can be connected to the DNA downstream of Chong Zushi muscardine Proteinase K by terminator.
4. one restructuring muscardine Proteinase K according to claim 3, it is characterized in that: the expression vector of described restructuring muscardine Proteinase K can select pPICZ, pPICZ Q, pGAPZ, pGAPZ Q, pGAPZ Q A and pPIC9K, preferred pPIC9K.
5. one restructuring muscardine Proteinase K according to claim 3, it is characterized in that: the selectable marker gene for selecting recombinant chou or the reporter gene for detecting quiding gene expression in the expression vector of described restructuring muscardine Proteinase K, can be inserted, described selectable marker gene comprises hygromycin gene, kalamycin resistance gene and ampicillin resistance gene, reporter gene comprises beta-Glucuronidase (GUS) gene, E.C. 2.3.1.28 (CAT) gene, luciferase (LUC) gene and green fluorescent protein (GFP) gene.
6. one restructuring muscardine Proteinase K according to claim 3, it is characterized in that: in the expression vector of described restructuring muscardine Proteinase K, can appended sequence be comprised, restructuring muscardine Proteinase K is expressed with the form of fusion rotein (albumen of encoding with appended sequence or peptide merge), described appended sequence comprises the nucleotide sequence of coded signal peptide or propetide, and the nucleotide sequence of coding His label or GST label.
7. one restructuring muscardine Proteinase K according to claim 1, is characterized in that: the transformant of described restructuring muscardine Proteinase K can be prokaryotic cell prokaryocyte or eukaryotic cell, preferred eukaryotic cell, described eukaryotic cell preferred yeast cell.
8. one restructuring muscardine Proteinase K according to claim 7, it is characterized in that: described yeast cell can select Pichia pastoris, candida cell, Hansenula polymorpha cell, torulopsis cell, fission yeast cell and Crewe to tie up ferment loam cell, preferred Pichia pastoris.
9. one restructuring muscardine Proteinase K according to claim 8, it is characterized in that: described Pichia pastoris is GS115 yeast cell, and adopt electroporated method to obtain the transformant of pichia spp: first linearizing expression vector is transformed in competent yeast cells, coating on flat board by thalline suspension again, until there is single bacterium colony in culture plate.
10. one restructuring muscardine Proteinase K according to claim 1, it is characterized in that: the test kit of described restructuring muscardine Proteinase K comprise in Chong Zushi muscardine Proteinase K, the DNA of coding Chong Zushi muscardine Proteinase K, expression vector and transformant one or more, also can comprise one or more components in addition, component is made up of test tube, reaction buffer, PCR primer, dNTP, Taq polysaccharase, reversed transcriptive enzyme, DNA enzymatic, RNA enzyme inhibitors, DEPC water and sterilized water.
The industrialized preparing process of 11. 1 kinds of muscardine Proteinase Ks of recombinating, is characterized in that, comprise the steps:
1) from the transformant of picking, obtain 20 bacterial strains that restructuring muscardine Proteinase K expression amount is the highest, shake-flask culture, centrifugally obtain fermented supernatant fluid, then carry out enzyme and live, express the test such as output, digestion BSA, choose the best bacterial strain of a strain further and carry out enlarged experiment test;
2) carry out optimal culture condition exploration at 14L fermentation tank level, comprise the consumption of culture medium prescription, dissolved oxygen amount, induction time and inductor methyl alcohol;
3) fermentation condition that 14L fermentor tank obtains is transplanted to the test of 500L fermentor tank industrial scale, copy pilot scale fermentation condition completely, continuously ferment cultivation 48,72,108h, the every fermentation index of on-line monitoring and cataphoretic determination protein expression level, lower tank centrifugation thalline and fermented liquid.
The industrialized preparing process of 12. a kind of muscardine Proteinase Ks of recombinating according to claim 11, it is characterized in that, described pilot scale fermentation condition adopts YPD substratum and MM substratum, the methanol induction time is 48,108h, dissolved oxygen amount is 20%, 30%, 40%, and methanol usage is 0.5%, 3% (v/v).
The purification process of 13. 1 kinds of muscardine Proteinase Ks of recombinating, it is characterized in that, comprise the steps: to add hexahistine label at the C end of restructuring muscardine Proteinase K, fermented liquid is first through centrifugation, supernatant liquor is again through metal ion-chelant affinitive layer purification, and then through cation exchange chromatography, concentrated, last freeze-drying becomes finished product enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410472748.1A CN104232611B (en) | 2014-09-16 | 2014-09-16 | A kind of recombination muscardine Proteinase K and its industrialized production and purification process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410472748.1A CN104232611B (en) | 2014-09-16 | 2014-09-16 | A kind of recombination muscardine Proteinase K and its industrialized production and purification process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104232611A true CN104232611A (en) | 2014-12-24 |
| CN104232611B CN104232611B (en) | 2018-06-08 |
Family
ID=52221512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410472748.1A Active CN104232611B (en) | 2014-09-16 | 2014-09-16 | A kind of recombination muscardine Proteinase K and its industrialized production and purification process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104232611B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878036A (en) * | 2015-04-29 | 2015-09-02 | 南京肽德生物技术有限公司 | Method for improving protein expression efficiency by employing model fitting and gene modification and application thereof |
| CN108603187A (en) * | 2015-11-24 | 2018-09-28 | 诺维信公司 | Polypeptide with proteinase activity and encode their polynucleotides |
| CN109207460A (en) * | 2018-10-23 | 2019-01-15 | 大连博格林生物科技有限公司 | Recombinate muscardine Proteinase K mutant PK-M2 and preparation method |
| CN109280656A (en) * | 2018-10-23 | 2019-01-29 | 大连博格林生物科技有限公司 | Recombinate muscardine Proteinase K mutant PK-M1 and preparation method |
| CN117247922A (en) * | 2023-11-06 | 2023-12-19 | 仲恺农业工程学院 | Low-temperature neutral protease and preparation method and application thereof |
| CN118165964A (en) * | 2024-04-11 | 2024-06-11 | 铭诚惠众(江苏)药物研究有限公司 | A method for purifying recombinant proteinase K and its application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106160B (en) * | 2019-05-29 | 2021-07-20 | 中国农业科学院北京畜牧兽医研究所 | Alkaline protease mutant PROK-G with improved catalytic activity and its encoding gene and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839165A (en) * | 2012-09-26 | 2012-12-26 | 金普诺安生物科技（苏州）有限公司 | Gene mutation type recombined protease K and industrialized production method thereof |
-
2014
- 2014-09-16 CN CN201410472748.1A patent/CN104232611B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839165A (en) * | 2012-09-26 | 2012-12-26 | 金普诺安生物科技（苏州）有限公司 | Gene mutation type recombined protease K and industrialized production method thereof |
Non-Patent Citations (2)
| Title |
|---|
| JUZHENG SHENG等: "Cloning a Cuticle-Degrading Serine Protease Gene with Biologic Control Function from Beauveria brongniartii and Its Expression in Escherichia coli", 《CURRENT MICROBIOLOGY》 * |
| 生举正: "布氏白僵菌遗传转化体系的建立及稳定高效表达Pr1蛋白酶工程菌株的构建", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878036A (en) * | 2015-04-29 | 2015-09-02 | 南京肽德生物技术有限公司 | Method for improving protein expression efficiency by employing model fitting and gene modification and application thereof |
| CN104878036B (en) * | 2015-04-29 | 2018-06-08 | 南京肽德生物技术有限公司 | A kind of models fitting and genetic modification improve method and the application of protein expression efficiency |
| CN108603187A (en) * | 2015-11-24 | 2018-09-28 | 诺维信公司 | Polypeptide with proteinase activity and encode their polynucleotides |
| US10829753B2 (en) * | 2015-11-24 | 2020-11-10 | Novozymes A/S | Polypeptides having protease activity and polynucleotides encoding same |
| CN109207460A (en) * | 2018-10-23 | 2019-01-15 | 大连博格林生物科技有限公司 | Recombinate muscardine Proteinase K mutant PK-M2 and preparation method |
| CN109280656A (en) * | 2018-10-23 | 2019-01-29 | 大连博格林生物科技有限公司 | Recombinate muscardine Proteinase K mutant PK-M1 and preparation method |
| CN109280656B (en) * | 2018-10-23 | 2021-06-15 | 大连博格林生物科技有限公司 | Recombinant beauveria bassiana proteinase K mutant PK-M1 and preparation method thereof |
| CN109207460B (en) * | 2018-10-23 | 2021-06-15 | 大连博格林生物科技有限公司 | Recombinant beauveria bassiana proteinase K mutant PK-M2 and preparation method thereof |
| CN117247922A (en) * | 2023-11-06 | 2023-12-19 | 仲恺农业工程学院 | Low-temperature neutral protease and preparation method and application thereof |
| CN118165964A (en) * | 2024-04-11 | 2024-06-11 | 铭诚惠众(江苏)药物研究有限公司 | A method for purifying recombinant proteinase K and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104232611B (en) | 2018-06-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104232611A (en) | Recombinant beauveria brongniartii proteinase K as well as industrial production and purification method thereof | |
| CN102839165B (en) | Gene mutation type recombined protease K and industrialized production method thereof | |
| US4914027A (en) | Process for the microbiological preparation of human serum albumin | |
| NO840200L (en) | GLUCOAMYLASE CDNA. | |
| Filho et al. | Stable Yeast Transformants that Secrete Functional α–Amylase Encoded by Cloned Mouse Pancreatic cDNA | |
| CN111118018B (en) | Cat serum albumin recombinant protein and expression method thereof in pichia pastoris | |
| CN110358754B (en) | A method for improving the activity of Pichia pastoris surface-displayed β-glucuronidase | |
| CN109280656B (en) | Recombinant beauveria bassiana proteinase K mutant PK-M1 and preparation method thereof | |
| CN106337042B (en) | Double alkali yl enzyme kex2 variant and its high-efficiency expression method | |
| CN115873836A (en) | A kind of nerolidol synthase and its application | |
| US5037744A (en) | Process for the microbiological preparation of human serum albumin | |
| CN101182475B (en) | Pichia yeast highly-effective expression of elastase as well as construction method and uses thereof | |
| CN104278017A (en) | Recombinant expression method of human lysozyme | |
| CN106701787A (en) | Pichia pastoris for expressing foreign proteins, construction method of pichia pastoris and induced expression method of pichia pastoris | |
| CN107778365A (en) | The recombinant-protein expression that a kind of multiple spot is integrated | |
| CN101748077B (en) | Pichia yeast for expressing chymosin as well as construction method and application thereof | |
| CN111304182A (en) | β -glucosidase and preparation method and application thereof | |
| CN116103267B (en) | Medium-temperature amylase mutant and application thereof | |
| CN106636174B (en) | Induced expression method of Pichia pastoris expressing foreign protein | |
| CN113755474B (en) | Carboxypeptidase, and coding gene and application thereof | |
| CN105802989A (en) | Vector, gene and method for expressing recombinant protein in pichia pastoris and application of recombinant protein | |
| CN110819609B (en) | A kind of mutant lipase with improved thermostability and preparation method and application thereof | |
| CN104232804B (en) | A method for increasing the expression level of hyaluronidase induced by gradient temperature changes | |
| Chen et al. | Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system | |
| CN102827784B (en) | Construction method of recombination crucian modified block sum pyramid (MBSP) expression bacterial strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Ding Haiping Inventor after: Li Kun Inventor after: Liang Liang Inventor before: Li Xu Inventor before: Teng Maikun |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |