CN117327673A - High-activity mammal urate oxidase mutant - Google Patents
High-activity mammal urate oxidase mutant Download PDFInfo
- Publication number
- CN117327673A CN117327673A CN202311336226.4A CN202311336226A CN117327673A CN 117327673 A CN117327673 A CN 117327673A CN 202311336226 A CN202311336226 A CN 202311336226A CN 117327673 A CN117327673 A CN 117327673A
- Authority
- CN
- China
- Prior art keywords
- mutant
- urate oxidase
- seq
- activity
- 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.)
- Pending
Links
- 108010092464 Urate Oxidase Proteins 0.000 title claims abstract description 125
- 229940005267 urate oxidase Drugs 0.000 title claims abstract description 90
- 230000000694 effects Effects 0.000 title claims abstract description 62
- 241000124008 Mammalia Species 0.000 title claims abstract description 38
- 230000035772 mutation Effects 0.000 claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- 229940088598 enzyme Drugs 0.000 claims abstract description 18
- 241000282465 Canis Species 0.000 claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 210000004027 cell Anatomy 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 22
- 230000014509 gene expression Effects 0.000 claims description 14
- 108091026890 Coding region Proteins 0.000 claims description 13
- 239000003814 drug Substances 0.000 claims description 12
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 229940079593 drug Drugs 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 241000238631 Hexapoda Species 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N lactose group Chemical group OC1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- 210000004962 mammalian cell Anatomy 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 108090000204 Dipeptidase 1 Proteins 0.000 claims description 3
- 241000588914 Enterobacter Species 0.000 claims description 3
- 241000701867 Enterobacteria phage T7 Species 0.000 claims description 3
- 241000701959 Escherichia virus Lambda Species 0.000 claims description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 3
- 102000006635 beta-lactamase Human genes 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims description 3
- 238000002741 site-directed mutagenesis Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 3
- 108091036078 conserved sequence Proteins 0.000 abstract description 2
- 150000001413 amino acids Chemical group 0.000 description 28
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 20
- 229940116269 uric acid Drugs 0.000 description 20
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 17
- 239000002244 precipitate Substances 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 10
- 238000005406 washing Methods 0.000 description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000004475 Arginine Substances 0.000 description 6
- 102000008300 Mutant Proteins Human genes 0.000 description 6
- 108010021466 Mutant Proteins Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- 201000005569 Gout Diseases 0.000 description 4
- 201000001431 Hyperuricemia Diseases 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- POJWUDADGALRAB-UHFFFAOYSA-N allantoin Chemical compound NC(=O)NC1NC(=O)NC1=O POJWUDADGALRAB-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000282708 Aotus <primate> Species 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 241000282898 Sus scrofa Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 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 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 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 2
- POJWUDADGALRAB-PVQJCKRUSA-N Allantoin Natural products NC(=O)N[C@@H]1NC(=O)NC1=O POJWUDADGALRAB-PVQJCKRUSA-N 0.000 description 2
- 241000282709 Aotus trivirgatus Species 0.000 description 2
- 241000228197 Aspergillus flavus Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000235646 Cyberlindnera jadinii Species 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 229960000458 allantoin Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 229960000424 rasburicase Drugs 0.000 description 2
- 108010084837 rasburicase Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- WCTAGTRAWPDFQO-UHFFFAOYSA-K trisodium;hydrogen carbonate;carbonate Chemical compound [Na+].[Na+].[Na+].OC([O-])=O.[O-]C([O-])=O WCTAGTRAWPDFQO-UHFFFAOYSA-K 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000282451 Ailuropoda Species 0.000 description 1
- 241000186074 Arthrobacter globiformis Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000283323 Delphinapterus leucas Species 0.000 description 1
- 241001482566 Enhydra Species 0.000 description 1
- 241000289667 Erinaceus Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241000230491 Gulo Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 241000721701 Lynx Species 0.000 description 1
- 241000283923 Marmota monax Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000282341 Mustela putorius furo Species 0.000 description 1
- 241000288894 Myotis Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 241000283220 Odobenus rosmarus Species 0.000 description 1
- 241000282921 Odocoileus virginianus Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000282515 Papio hamadryas Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000288669 Tupaia Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000002898 library design Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 150000007971 urates Chemical class 0.000 description 1
- 238000012795 verification 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
- C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12N9/0048—Uricase (1.7.3.3)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
According to the evolution trend of urate oxidase, a mutant library is constructed by analyzing the conserved sequences of various mammal urate oxidases, high-activity mammal urate oxidase mutant MU is obtained by screening, site-directed mutagenesis is carried out on the mutant MU, and mutations E208D and R249Q with higher activity are further screened. The invention provides a high-activity mammal urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1.5 or 7. The mutant has significantly improved enzyme specific activity compared to wild-type urate oxidase, e.g., at least 25% improved enzyme specific activity compared to wild-type canine urate oxidase, and has higher thermostability.
Description
Technical Field
The invention relates to the fields of genetic engineering and enzyme engineering, in particular to a high-activity mammal urate oxidase mutant.
Background
Hyperuricemia and gout are serious diseases that seriously jeopardize human health. The existing therapeutic drugs can only control the onset and the continued development of the diseases in the aspects of diminishing inflammation, relieving pain, inhibiting uric acid generation and the like, and the purpose of radical cure cannot be achieved. The urate oxidase medicine can hydrolyze slightly soluble uric acid into soluble allantoin, and has been used in treating tumor radiotherapy and chemotherapy and hyperuricemia and gout caused by metabolic disturbance. At present, no uricase medicines are marketed in China, and the international marketed uricase medicines include Saccharomyces cerevisiae recombinant Aspergillus flavus-derived uricase (Rasburicase) approved by the American FDA in 2002 and polyethylene glycol (PEG) -modified Escherichia coli recombinant swine-derived uricase (Peglotica) approved by the FDA in 2010. The former is low in homology with the estimated human urate oxidase (< 40%), and antibodies are easily produced in a subject after multiple administration, so that the kit can be only used for short-term treatment of acute hyperuricemia; the latter is a PEG modified long-acting biological protein drug, is injected once in two weeks, has the unique effect of rapidly ablating tophus, is honored as a breakthrough therapeutic drug (Pelgotidase is sold in the 2019 year for 3.4 hundred million dollars) for potential refractory gout, but has the defects of high immunogenicity, low long-term injection effective rate and the like.
Mammal-derived urate oxidase is an important source for developing long-acting urate oxidase drugs due to high homology (homology with human-derived urate oxidase > 90%). However, the mammal urate oxidase activity is low, the low activity means high dosage, and the excessive injection dosage (8.0 mg/dose) is one of important factors for causing the super immunogenicity of the Pelgotidase (PEG modified swine-derived recombinant urate oxidase). The construction of the mammal-derived urate oxidase with high specific activity and the reduction of injection dosage are important means for developing the low-immunogenicity urate oxidase. Meanwhile, the injection period of the PEG modified urate oxidase drugs is about 2 weeks, so that the stability of urate oxidase proteins is improved, the in-vivo metabolic rate is prolonged, the initial injection dosage can be reduced, and the potential immunogenicity of mammal urate oxidase drugs is further reduced. Development of highly active, highly stable mammal-derived urate oxidase is an important means for developing highly safe, low-immunogenicity, long-acting urate oxidase drugs.
Patent (WO 2019/010369) discloses recombinant mutant Candida utilis (Candida utilis) urate oxidase with improved pancreatin stability and/or activity; patent (WO 2021/068925) discloses an improved Micrococcus (Arthrobacter globiformis) urate oxidase with improved enzymatic activity and thermostability over the original structure. The urate oxidase proteins of the above patents are all of microbial origin, non-mammalian origin, and may present a risk of too high immunogenicity similar to Aspergillus flavus-derived urate oxidase (Rasburicase) when applied to humans. The patent (WO 2011/050599) discloses humanized recombinant urate oxidase and mutants thereof, the patent (CN 104630168A) discloses a chimeric urate oxidase structure of a human pig, and the humanized degree is improved mainly through chimeric, so that high-activity and high-stability urate oxidase screening is not involved. The methods disclosed in the prior art are not directed to further enhancing enzyme activity and stability based on mammalian urate oxidase structure.
Disclosure of Invention
In order to solve the technical problems, the invention provides a high-activity mammal uricase (also simply called uricase) mutant, wherein the enzyme specific activity of the mutant is obviously improved compared with that of wild type uricase (for example, the enzyme specific activity is improved by about 28.9 percent compared with that of wild type canine uricase).
The invention provides a high-activity mammal urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO:1 or with SEQ ID NO:1 comprises the mutations E208D and/or R249Q.
In some embodiments, the mutant may also comprise other suitable mutations, provided that the mutation does not substantially reduce the specific activity of the enzyme. In some embodiments, the mutant hybridizes to a polypeptide as set forth in SEQ ID NO:3, the specific enzyme activity is improved by at least 25% compared with the wild-type canine urate oxidase.
In some embodiments, the amino acid sequence of the mutant is as set forth in SEQ ID NO: 1.5 or 7.
The invention also provides a nucleic acid molecule comprising the coding sequence of a mutant highly active mammalian urate oxidase according to any of the embodiments of the invention; preferably, the nucleic acid molecule is DNA or RNA.
The invention also provides a gene expression frame, which comprises a promoter and a coding sequence operably connected behind the promoter, wherein the coding sequence is the coding sequence of the high-activity mammal urate oxidase mutant according to any scheme of the invention.
In some embodiments, the promoter is selected from the group consisting of a lactose promoter system, a tryptophan promoter system, a beta lactamase promoter system, or a promoter system derived from phage lambda or T7.
In some embodiments, the nucleotide sequence of the coding sequence is set forth in SEQ ID NO: 2. 4 or 6.
The invention also provides an expression vector comprising the gene expression cassette according to any one of the schemes of the invention.
The present invention provides a host cell comprising a nucleic acid molecule, gene expression cassette or expression vector according to any one of the embodiments of the present invention.
In some embodiments, the host cell is selected from a mammalian cell, an insect, a yeast, or a bacterial cell; preferably an enterobacter cell or a yeast cell.
The invention also provides a method for producing a high activity mammalian urate oxidase mutant according to any of the embodiments of the invention, comprising: expressing the mutant in the host cell, and separating and purifying.
The invention also provides application of the high-activity mammal urate oxidase mutant in preparing urate oxidase medicaments.
Compared with the prior art, the invention has the beneficial effects that at least:
(1) According to the evolution trend of the urate oxidase, the invention constructs a mammal urate oxidase mutant library by analyzing the conserved sequences of various mammal urate oxidases, and screens to obtain the high-activity mammal urate oxidase mutant MU.
(2) The mutant MU is subjected to site-directed mutagenesis, and mutation E208D and R249Q with higher activity are further screened, so that compared with the wild-type canine uric acid oxidase with the highest activity, the specific activity of the mutant MU is improved by at least 25%, and the mutant MU has higher thermal stability.
Drawings
FIG. 1 is an alignment of urate oxidase sequences of different mammals A0A5E4C3I6 Woodchuck; p11645 Rabbit; A0A2Y9P065, beluga whale; p16164, pic; q3MHG7, bovine; A0A6J XAT6: odocoileus virginianus; A0A452G3H7: goat; w5PWL1, sheep; p25688 Mouse; p09118 Rat; l9KW26 Tupaia; A0A1S3WDJ8, erinaceus; p25689 Hamadryas baboon; q8MKJ Aotus; A0A7J7ZXM 4.4 Myotis; G1M4M8: ailuropoda; A0A2U3WVK7:Walrus; A0A3P4NI51:Gulo; M3Y300 Mustela furo; A0A2Y9IZI3:Enhydra patris; A0A673TVJ5: surica; A0A485N2I1 is Lynx; q5FZI9: canine.
FIG. 2 is an identification of recombinant expression of mammalian urate oxidase. M: a high molecular weight pre-dye protein marker;1: before induction; 2: after induction.
FIG. 3 is a graph showing purity identification of mammalian urate oxidase mutants. A: SDS-PAGE identification. M: mid-molecular weight pre-dye protein markers; 1: R291K;2: E208D;3: R249Q. And B, HPLC identification.
FIG. 4 is a comparison of the enzymatic activity of highly active mammalian urate oxidase mutants compared to wild-type canine urate oxidase.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. Examples of which are illustrated in the accompanying drawings. It should be understood that the specific examples described in the following embodiments of the present invention are intended to be illustrative of the specific embodiments of the present invention and are not to be construed as limiting the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass the range or value as being close to the range.
The invention provides a high-activity mammal urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1.
Through systematic studies, the inventor discovers that as evolution progresses, mammal urate oxidase of various species has a trend of lower and lower enzyme activity. By comparing with multiple sequences of mammal urate oxidase with high homology of human urate oxidase, amino acid sequences with high conservation or commonality are selected, missense mutation accumulated in the evolution process of single species mammal urate oxidase can be eliminated, and mammal urate oxidase mutant protein with higher activity can be obtained.
Based on the protein sequences of mammal urate oxidase of dogs, pigs, cattle, rabbits, rats, night monkeys and the like, the wild mammal urate oxidase has been proved to have the activity of degrading uric acid by the inventor, and the total length of the urate oxidase protein sequence is 300-304 amino acids; the sequence Identity (Identity) between the wild-type mammal urate oxidase and the deduced human urate oxidase should be not less than 85.0% by software analysis such as Blast. Based on 6 or more amino acid sequences of mammal urate oxidase, the highly conserved region is reserved through multi-species sequence Alignment (Alignment), and 1 or more alternative amino acid compositions are designed in a non-conserved variable region through consensus analysis.
More specifically, when the amino acid sequence difference of the variable region among multiple species is large, firstly 1-2 amino acids with the highest conservation or commonality are selected, for example, at 52 th site, canine, porcine, rabbit, night monkey, artificial serine, bovine asparagine, rat arginine, serine commonality is obviously higher than other two amino acids, and serine with the highest commonality is selected at 52 th site; if arginine is taken as canine, bovine and porcine sources at 291, and the shareability of arginine and lysine is not obviously different among rabbits, rats, night monkeys and artificial lysine, arginine and lysine are respectively selected at 291, and two alternative mutant sequences are constructed. By analogy, 1 or more amino acids with highest commonality are selected at amino acid positions with sequence differences, and a mammalian urate oxidase mutant library is constructed therefrom. Obtaining MU protein amino acid sequence with highest conservation or commonality of mammal urate oxidase of multiple species, namely SEQ ID NO:1, and a high activity mammalian urate oxidase mutant shown in the specification.
In order to further increase the activity and thermostability, the invention also provides SEQ ID NO:1, and constructing different mammal urate oxidase mutant DNA sequences by means of site-directed mutagenesis. The site-directed mutagenesis method can be achieved by various techniques known to those skilled in the art, such as DNA recombination, PCR, etc., including but not limited to the two-round staggered-extension PCR method and the site-directed mutagenesis method described in quick change of Strantane.
Further, in the sequence set forth in SEQ ID NO:1, and the mutation E208D, R249Q can improve the specific activity and the thermal stability of the enzyme, so the invention also provides a high-activity mammal urate oxidase mutant, which has the same activity as SEQ ID NO:1 comprises the mutations E208D and/or R249Q. The E208D is SEQ ID NO:1 from glutamic acid at position 208 to aspartic acid; R249Q refers to SEQ ID NO:1 to glutamine.
In some embodiments, the mutant may also comprise other suitable mutations, provided that the mutation does not substantially reduce the specific activity of the enzyme. In some embodiments, the mutant hybridizes to a polypeptide as set forth in SEQ ID NO:3, the specific enzyme activity is improved by at least 25% compared with the wild-type canine urate oxidase.
In some embodiments, the amino acid sequence of the mutant is as set forth in SEQ ID NO: 1.5 or 7, wherein the sequence of the mutant is as shown in SEQ ID NO:1 or comprises the single mutation E208D or R249Q.
Based on the highly active mammalian urate oxidase mutant, the amino acid sequence may synthesize a corresponding DNA sequence or mRNA sequence for use in generating the urate oxidase mutant. The invention also provides a nucleic acid molecule comprising a sequence encoding a high activity mammalian urate oxidase mutant according to any of the aspects of the invention; preferably, the nucleic acid molecule is DNA or RNA. When the nucleic acid molecule is DNA, synthesizing the urate oxidase mutant through translation expression of the DNA, and using the urate oxidase mutant for protein production; the RNA may be mRNA for transient expression in a cell.
The invention also provides a gene expression cassette comprising a promoter and a coding sequence operably linked to the promoter, the coding sequence encoding a high activity mammalian urate oxidase mutant sequence according to any of the aspects of the invention.
In some embodiments, the promoter is selected from the group consisting of a lactose promoter system, a tryptophan promoter system, a beta lactamase promoter system, or a promoter system derived from phage lambda or T7.
By the correspondence of amino acids to nucleotides, and codon optimization, one skilled in the art can obtain different nucleotide sequences. In some embodiments, the nucleotide sequence of the coding sequence is set forth in SEQ ID NO: 2. 4 or 6.
The invention also provides an expression vector comprising the gene expression cassette according to any one of the schemes of the invention. The expression vector is preferably a plasmid vector, which can be expressed in mammalian cells, insects, yeasts, bacteria or other cells by cell transduction.
Thus, the present invention provides a host cell comprising a nucleic acid molecule, gene expression cassette or expression vector according to any one of the embodiments of the present invention.
In some embodiments, the host cell is selected from a mammalian cell, an insect, a yeast, or a bacterial cell; preferably an enterobacter cell or a yeast cell.
The invention also provides a method for producing a high activity mammalian urate oxidase mutant according to any of the embodiments of the invention, comprising: expressing the mutant in the host cell, and separating and purifying.
The mammalian urate oxidase mutant protein can be isolated from the inside or outside of host cell (such as culture medium) and purified to obtain high purity homogeneous protein. The method for separating and purifying the protein is not limited to any particular method, such as column chromatography, filtration, ultrafiltration, salting-out, isoelectric precipitation, dialysis, etc. For chromatography, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel exclusion chromatography, reverse phase chromatography, etc., may be applied. These chromatography may be performed using liquid chromatography such as a rapid protein liquid chromatography system. The protein purity and concentration are detected by a general protein detection method, such as HPLC method, SDS-polyacrylamide electrophoresis method, isoelectric point electrophoresis method, BCA method, lowry method, kjeldahl nitrogen method, etc. The specific activity of urate oxidase is detected by uric acid substrate degradation method, for example, based on the characteristic absorption peak of substrate uric acid at 293nm, the uric acid consumption rate is detected by ultraviolet spectrophotometry or high performance liquid chromatography, and the urate oxidase activity per unit volume is calculated. The specific activity of the urate oxidase protein is calculated by the monomer volume urate oxidase activity and the unit volume urate oxidase protein concentration. The thermal stability of the urate oxidase is detected by a thermal stability detection method at 25-37 ℃. More preferably, the thermal stability of the urate oxidase is detected by a method such as 37 ℃ thermal stability detection.
The invention also provides application of the high-activity mammal urate oxidase mutant in preparing urate oxidase medicaments. The urate oxidase medicament can be used for treating hyperuricemia and gout.
Example 1 multiple species mammalian urate oxidase bioinformatics analysis and initial mutant design
Based on the translation of the sequence of the pseudogene of human urate oxidase (GenBank: AB 074326.2) into an amino acid sequence, the nonsense mutations at positions 33 and 187 (nonsense mutantion) were replaced with arginine and arginine, respectively, of night monkey urate oxidase. And (3) performing Blast analysis on the protein sequence to obtain the amino acid sequence of the mammal urate oxidase with the Identity of not less than 85%. The amino acid sequence of the mammal urate oxidase is subjected to multi-sequence Alignment analysis (shown in figure 1) by using software such as Clustal Omega and the like, and the amino acid sequence of MU protein with the highest conservation or commonality of the mammal urate oxidase of multiple species is obtained as follows:
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO：1)
according to the amino acid sequence, DNA design is carried out according to the preferred codons of escherichia coli and saccharomycetes, and corresponding companies are entrusted to carry out total gene synthesis, wherein the DNA sequence corresponds to:
CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTTAAAGGTATTAAATCTATTGAAACCTTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTATTAAAGATCAGTTTACCACCCTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGAAGCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCGTGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATATTCATTATTTTAATATTGATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC(SEQ ID NO：2)。
after amplifying the recombinant plasmid synthesized by the whole gene, the desired fragment was recovered by double digestion with NdeI and BamHI, ligated with plasmid pET-3C (Invitrogen) also recovered by digestion with NdeI and BamHI using T4 DNA ligase, and the ligation mixture was transferred into E.coli clone host strain TOP10 using the standard procedure as described in Current Protocols in Molecular Biology.
And (3) carrying out transformation reaction on an LB plate containing ampicillin, after growing the transformant overnight, picking a monoclonal colony after transformation to prepare a plasmid, screening recombinant plasmid pET-3C-MU by using a method of enzyme digestion and PCR verification, and determining that the MU sequence in the positive recombinant plasmid is completely consistent with the theoretical sequence after DNA sequencing.
EXAMPLE 2 recombinant expression of uric acid oxidase in multiple species of mammals
And (3) transforming the MU recombinant plasmid with correct sequencing into escherichia coli expression host bacteria and expressing. Coli BL21 (DE 3), BL21 Star (DE 3) or BL21 Star (DE 3) plysS was used to express MU proteins. These strains are only some of the many suitable for expressing chimeric proteins, which are commercially available from Novagen, invitrogen and Stratagen, respectively. Transformants were identified by their ability to grow on LB plates containing ampicillin.
The recombinant strain expressing the recombinant strain of the escherichia coli containing the MU recombinant plasmid was cultured overnight in a liquid LB medium containing 50ug/ml of ampicillin, and the culture broth was inoculated with a large-sized culture at an inoculation ratio of 1:100. after the cells were grown to a certain optical density at 600nm, IPTG was added to a final concentration of 0.5mM to induce expression of the target protein, and the cells were further cultured for 3 hours. Cells were then harvested by centrifugation, the pellet washed with 50mM Tris buffer, stored at-20℃by centrifugation, and SDS-PAGE detected, the expressed protein band around 35kDa (as shown in FIG. 2).
EXAMPLE 3 Multispecies mammalian urate oxidase mutant library design
MU protein DNA sequence is used as an original template, and mutation sites are designed according to the degree of commonality, for example, as follows: Q109H (mutation is represented by triplet: letter-number-letter, wherein the number represents the position of the mutated amino acid, the letter before the number corresponds to the amino acid for which the mutation was designed, the letter after the number represents the amino acid for replacing the amino acid before the number, the numbers being ordered by amino acid at position 304 of human uricase), L146M, S148N, E D, T213A, S246T, R249Q, V L.
Specifically, single point mutation is carried out based on MU, a mutant library is constructed, recombinant expression, preparation and enzyme specific activity detection are carried out according to the methods of examples 3, 4 and 5, beneficial mutation with improved enzyme specific activity is screened out, harmful mutation is eliminated, and mammal uricase mutant with optimal enzyme specific activity is screened out. The prior research of the inventor discovers that the wild type canine uricase protease protein (SEQ ID NO: 3) has higher specific activity, so that the uricase mutant and the recombinant wild type canine uricase protein (named wCU) are compared and researched. The amino acid sequence of wild-type canine uricase is as follows:
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSV
QLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSF
NHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIH
SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVD
FEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEIS
LPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO：3)。
the DNA containing the target mutation was prepared by mutation using the staggered-extension PCR method. The following is an example of the preparation of Q109H:
| primer numbering | DNA sequence |
| Primer 1 (SEQ ID NO: 8) | 5'CACGACATATGGCCCATTATCATA3' |
| Primer 2 (SEQ ID NO: 9) | 5'GGATCCTTATCACAGACGAGAGCT3' |
| Primer 3 (SEQ ID NO: 10) | 5'CGTGCCCACGTTTATGTTGAAGAA3' |
| Primer 4 (SEQ ID NO: 11) | 5'ATAAACGTGGGCACGAATAACATG3' |
Preparation of MU Q109H : the first stage PCR is that the template sequence is the total gene synthesis sequence (SEQ ID NO: 2) in example 1, the primers are primer 1 and primer 3 in the table, and the PCR reaction system and method are all set by adopting a PCR reaction kit according to the instruction of a merchant. The PCR reaction conditions were: 94℃1min,56℃1min,72℃1min, 30 cycles, the first cycle denaturation at 94℃for 10min, the last cycle extension at 72℃for 10min. Amplifying under the PCR conditions to obtain the product MU Q109H -a fragment; second stage PCR: the template sequence is the same with the first stage PCR, the primer is primer 2 and primer 4, and MU is obtained by amplifying according to the PCR conditions Q109H -fragment b; third stage PCR: the template is MU Q109H -a fragment and MU Q109H -1 of fragment b: 1, the primer is primer 1 and primer 2, and the product MU is obtained by amplifying the mixture according to the PCR conditions Q109H . The DNA sequence containing the mutated sequence was digested simultaneously with NdeI and BamHI, ligated, transformed, screened and expressed as in example 2.
EXAMPLE 4 purification of mammalian uricase mutant expression
Adding 50g of thallus precipitate into 500ml of bacteria-destroying solution with pH of 8.3 containing 25mM Tris-HCl,5mM EDTA,0.1mg/ml lysozyme, stirring at 37deg.C for 60-80 min, and adding 1mM MgCl 2 ·6H 2 O、1μg·mL -1 Stirring the nuclease overnight; 8500 r.min at 4 ℃ -1 Centrifuging for 20min, and collecting bacterial precipitate. Taking a bacterial breaking precipitate, and mixing the bacterial breaking precipitate with 100g of wet bacteria: 1L of washing liquid(25 mM Tris-HCl,5mM EDTA, 1.5% TritonX-100, pH 8.3) and stirring at 30deg.C for 2h after homogenization; 8500 r.min -1 Centrifuging at 4deg.C for 15min, collecting precipitate, washing with the same washing solution, and centrifuging to collect precipitate. Taking TritonX-100, washing the precipitate, and preparing 100g of wet bacteria: 1L of the washing solution (PBS, 0.34. Mu.g.multidot.mL) -1 Nuclease) is suspended and precipitated, and after being evenly dispersed, the mixture is stirred for 2 hours at 30 ℃ and 8500 r.min -1 Centrifuging at 4 ℃ for 15min, and centrifuging to collect precipitate. The pellet was washed with PBS and nuclease, still at 100 grams wet: 1 liter of washing solution (PBS) is suspended and precipitated, homogenized by a homogenizer, stirred at 30 ℃ for 2 hours, 8500 r.min -1 Centrifuging at 4deg.C for 15min, collecting precipitate, washing with the same washing solution, and centrifuging to collect precipitate. Taking a precipitate after bacterial breaking washing, and precipitating according to 10 g: 3L lysis buffer (0.1M Na 2 CO 3 -NaHCO 3 pH 10.3), and after homogenization with a homogenizer, stirring overnight at room temperature. 8500 r.min -1 Centrifuging at 4 ℃ for 15min, and collecting supernatant. Adding (NH) with final concentration of 10% saturation into supernatant 4 ) 2 SO 4 Standing overnight at 4 ℃ for 8500 r.min -1 Centrifuging at 4deg.C for 15min, and collecting the centrifugal precipitate; dissolving buffer solution, stirring overnight at room temperature, 8500 r.min -1 Centrifuging at 4 ℃ for 15min, and collecting supernatant. Purifying the supernatant by DEAE agarose anion exchange chromatography (GE), and subjecting the target protein to linear gradient of 0-0.2M NaCl (Na with pH of 10.3,0.1M) 2 CO 3 -NaHCO 3 ) Eluting, wherein the target protein is eluted at 0.1M NaCl; concentrating the eluate by DEAE Sepharose anion exchange chromatography column (GE), concentrating with Na (H is 10.3,0.1M) containing 0.2M NaCl 2 CO 3 -NaHCO 3 ) The eluent elutes the target protein. At this time, purity was detected by SDS-PAGE and HPLC and was found to be 95% or more (as shown in FIG. 3).
EXAMPLE 5 detection of mammalian uricase mutant protein Activity
The amount of enzyme that converts 1. Mu. Mol uric acid to allantoin per minute at 37℃and pH8.6 is defined as one International Unit (IU). Uric acid has a characteristic absorption peak at 293nm, and when it is degraded by uricase, the product has no absorption peak in the wavelength rangeThe decrease in uric acid was determined by detecting the change in absorbance at 293nm at regular time, and then the molar extinction coefficient of uric acid was used (1.23X 104M -1 ·CM -1 ) The uric acid concentration is calculated, and the uricase activity can be calculated from the change in uric acid concentration. After the ultraviolet spectrophotometer is regulated to 293nm and the stand-by device is stabilized, the blank is zeroed by using 0.1M sodium tetraborate solution, 3ml of 0.1mM uric acid solution is taken for reaction and dissolution, and then the solution is added into a quartz cuvette, 10ul uricase mutant protein is supplemented, reading is carried out every 30 seconds, and the OD293 change value within 2min is measured. According to the formula C=A/Kb (C is the uric acid concentration of the solution, A is the light absorption value of 293nm, K is the molar extinction coefficient-1.23×104M) -1 ·CM -1 B is the inner diameter of the cuvette), calculate the OD at different time points 293 Corresponding uric acid concentrations; calculating the reduced uric acid substance amount from Δm= Δcv (Δm is the number of moles of uric acid reduction, Δc is the change in uric acid concentration, C is the reaction liquid volume); uricase activity was calculated from u= Δ M/TV1 (U is the uricase activity unit contained per milliliter of plasma, T is the reaction minutes, V1 is the uricase mutant protein volume added to the reaction system). Wherein, MU E208D (SEQ ID NO：5)、MU R249Q (SEQ ID NO: 7) has about 29.8%, 28.9% and 34.3% higher specific activities than wild-type canine uricase, respectively (as shown in FIG. 4).
EXAMPLE 6 mammalian uricase mutant thermal stability assay
Uricase mutant protein was buffered (0.1M Na 2 CO 3 -NaHCO 3 pH 10.3) was diluted to 1mg/ml, placed in an incubator at 37℃and after 0h, 0.5h, 1h, a part of the sample was taken out, and the uricase activity was measured as in example 5, and the enzyme activity retention was compared.
The results show that the thermal stability of all three uricase mutants is better than that of wild-type canine uricase, and MU E208D The thermal stability is best.
Other sequences to which the examples relate
MU E208D Nucleotide sequence:
CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTTAAAGGTATTAAATCTATTGAAACCTTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTATTAAAGATCAGTTTACCACCCTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGATCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCGTGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATATTCATTATTTTAATATTGATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC(SEQ ID NO：4)
the corresponding amino acid sequence:
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFDATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO：5)
MU R249Q nucleotide sequence:
CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTTAAAGGTATTAAATCTATTGAAACCTTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTATTAAAGATCAGTTTACCACCCTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGAAGCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCAGGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATATTCATTATTTTAATATTGATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC(SEQ ID NO：6)。
the corresponding amino acid sequence:
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSQVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO：7)。
finally, the above embodiments are only for illustrating the technical solution of the present invention, and do not limit the present invention. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. A high activity mammal urate oxidase mutant, which is characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO:1 or with SEQ ID NO:1 comprises the mutations E208D and/or R249Q.
2. The mutant of claim 1, wherein the mutant hybridizes to a sequence set forth in SEQ ID NO:3, the specific enzyme activity is improved by at least 25% compared with the wild-type canine urate oxidase.
3. The mutant of claim 1, wherein the amino acid sequence of the mutant is as set forth in SEQ ID NO: 1.5 or 7.
4. A nucleic acid molecule comprising the coding sequence of the high activity mammalian urate oxidase mutant according to any one of claims 1 to 3; preferably, the nucleic acid molecule is DNA or RNA; more preferably, the nucleotide sequence of the coding sequence is as set forth in SEQ ID NO: 2. 4 or 6.
5. A gene expression cassette comprising a promoter and a coding sequence operably linked to the promoter, wherein the coding sequence is that of a mutant highly active mammalian urate oxidase of any one of claims 1 to 3.
6. The gene expression cassette of claim 5, wherein the promoter is selected from the group consisting of lactose promoter system, tryptophan promoter system, beta lactamase promoter system, and phage lambda or T7 derived promoter system; the nucleotide sequence of the coding sequence is shown as SEQ ID NO: 2. 4 or 6.
7. An expression vector comprising the gene expression cassette of claim 5 or 6.
8. A host cell comprising the nucleic acid molecule of claim 4, the gene expression cassette of claim 5 or 6, or the expression vector of claim 7; preferably, the host cell is selected from mammalian cells, insect, yeast or bacterial cells; more preferably an enterobacter cell or a yeast cell.
9. A method of producing a high activity mammalian urate oxidase mutant according to any one of claims 1 to 3 comprising: expressing the mutant in the host cell, and separating and purifying.
10. Use of a highly active mammalian urate oxidase mutant according to any one of claims 1 to 3 for the preparation of a urate oxidase drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311336226.4A CN117327673A (en) | 2023-10-16 | 2023-10-16 | High-activity mammal urate oxidase mutant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311336226.4A CN117327673A (en) | 2023-10-16 | 2023-10-16 | High-activity mammal urate oxidase mutant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117327673A true CN117327673A (en) | 2024-01-02 |
Family
ID=89275242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311336226.4A Pending CN117327673A (en) | 2023-10-16 | 2023-10-16 | High-activity mammal urate oxidase mutant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117327673A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011050599A1 (en) * | 2009-10-27 | 2011-05-05 | 重庆富进生物医药有限公司 | Humanized recombinant uricase and mutants thereof |
| US20140065123A1 (en) * | 2011-02-14 | 2014-03-06 | Chongqing Fagen Biomedical Inc. No. 80 4th Street Keyuan | Pegylated analogue protein or canine urate oxidase, preparation method and use thereof |
| CN103756983A (en) * | 2014-01-10 | 2014-04-30 | 临沂大学 | Preparation method of uricase of natural mammals |
| WO2014170916A2 (en) * | 2013-04-17 | 2014-10-23 | Council Of Scientific & Industrial Research | Novel uricase mutants |
| CN106554948A (en) * | 2015-09-29 | 2017-04-05 | 上海生物制品研究所有限责任公司 | Saltant type uricase, the saltant type uricase of PEG modifications and its application |
-
2023
- 2023-10-16 CN CN202311336226.4A patent/CN117327673A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011050599A1 (en) * | 2009-10-27 | 2011-05-05 | 重庆富进生物医药有限公司 | Humanized recombinant uricase and mutants thereof |
| CN102051348A (en) * | 2009-10-27 | 2011-05-11 | 重庆富进生物医药有限公司 | Humanized recombinant uricase and mutant thereof |
| US20140065123A1 (en) * | 2011-02-14 | 2014-03-06 | Chongqing Fagen Biomedical Inc. No. 80 4th Street Keyuan | Pegylated analogue protein or canine urate oxidase, preparation method and use thereof |
| WO2014170916A2 (en) * | 2013-04-17 | 2014-10-23 | Council Of Scientific & Industrial Research | Novel uricase mutants |
| CN103756983A (en) * | 2014-01-10 | 2014-04-30 | 临沂大学 | Preparation method of uricase of natural mammals |
| CN106554948A (en) * | 2015-09-29 | 2017-04-05 | 上海生物制品研究所有限责任公司 | Saltant type uricase, the saltant type uricase of PEG modifications and its application |
| US20180282707A1 (en) * | 2015-09-29 | 2018-10-04 | Shanghai Institute Of Biological Products Co., Ltd. | Mutant-type uricase, peg modified mutant-type uricase, and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| FENG T 等: "A Practical System for High-Throughput Screening of Mutants of Bacillus fastidiosus Uricase", 《APPL BIOCHEM BIOTECHNOL》, vol. 181, no. 2, 10 September 2016 (2016-09-10), pages 667 - 681 * |
| GUO Y 等: "The effects of free Cys residues on the structure, activity, and tetrameric stability of mammalian uricase", 《 APPL MICROBIOL BIOTECHNOL》, vol. 107, no. 14, 31 May 2023 (2023-05-31), pages 4533 - 4542 * |
| 郭勇: "高稳定性哺乳动物尿酸酶结构筛选及成药性研究", 《临沂大学》, 23 August 2023 (2023-08-23), pages 1 - 67 * |
| 霍晶晶: "哺乳动物尿酸酶重组构建筛选及Cys残基结构功能研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》, no. 1, 15 January 2023 (2023-01-15), pages 016 - 2491 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101865998B1 (en) | Microorganism having l-threonine-producing capability and method of producing l-threonine using the same | |
| EP4242300A1 (en) | Mutant enzyme, use thereof and process for preparing tripeptide by using enzymatic method | |
| WO2021027390A1 (en) | Heparin lyase mutant and recombinant expression method therefor | |
| NZ225798A (en) | Procaryotic xylose isomerase muteins and methods of increasing their stability | |
| CN111269899B (en) | Human urate oxidase with catalytic activity and application thereof | |
| CN110129305B (en) | Cephalosporin C acylase mutant for preparing 7-ACA | |
| CN109852623B (en) | Preparation method, purification method and application of PEG-modified recombinant humanized urate oxidase | |
| CN117327673A (en) | High-activity mammal urate oxidase mutant | |
| CN114181288B (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
| US5496719A (en) | Polypeptide from Humicola insolens possessing protein disulfide isomerase activity gene encoding the same | |
| CN114807087B (en) | Method for improving thermal stability of phytase, mutant and application | |
| CN114349831B (en) | aspA gene mutant, recombinant bacterium and method for preparing L-valine | |
| CN108034642B (en) | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof | |
| US20130330316A1 (en) | Mutants of l-asparaginase | |
| CN109652400B (en) | Molecular modification method of half-life period of anti-cancer drug arginine deiminase and mutant strain | |
| CN114277022B (en) | Nitrile hydratase mutant with high activity and high thermal stability | |
| CN117230034A (en) | High-stability mammal urate oxidase mutant | |
| CN115029327A (en) | Glucose oxidase mutant GOx-MUT 7-11 and coding gene and application thereof | |
| CN108484749A (en) | A kind of recombinant soluble human source Bone targeting gamma interferon 1-b and preparation method thereof | |
| CN115029328A (en) | Glucose oxidase mutant GOx-MUT 1-6 and coding gene and application thereof | |
| Yang et al. | Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris | |
| KR102232837B1 (en) | Novel polypeptides having glucosylglycerol productivity and a method for producing glucosylglycerol using the same | |
| CN108690837B (en) | Method for improving heat stability of polymer protein and alcohol dehydrogenase with improved heat stability | |
| CN112521480A (en) | Human interferon-kappa mutant and preparation method thereof | |
| US6893844B1 (en) | DNA encoding a new human hepatoma derived growth factor and producing method thereof |
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 |