CN114349836B - 一种粘附力强的类贻贝粘蛋白及其制备方法 - Google Patents
一种粘附力强的类贻贝粘蛋白及其制备方法 Download PDFInfo
- Publication number
- CN114349836B CN114349836B CN202210067273.2A CN202210067273A CN114349836B CN 114349836 B CN114349836 B CN 114349836B CN 202210067273 A CN202210067273 A CN 202210067273A CN 114349836 B CN114349836 B CN 114349836B
- Authority
- CN
- China
- Prior art keywords
- mussel
- mucin
- tyr
- recombinant
- lys
- 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.)
- Active
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 102000003425 Tyrosinase Human genes 0.000 claims abstract description 12
- 108060008724 Tyrosinase Proteins 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 10
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 8
- 235000010323 ascorbic acid Nutrition 0.000 claims description 8
- 229960005070 ascorbic acid Drugs 0.000 claims description 8
- 239000011668 ascorbic acid Substances 0.000 claims description 8
- 239000000853 adhesive Substances 0.000 claims description 7
- 230000001070 adhesive effect Effects 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 238000000108 ultra-filtration Methods 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical group CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 229910021538 borax Inorganic materials 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 239000000411 inducer Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 4
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical group [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims description 2
- 230000004151 fermentation Effects 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 230000001590 oxidative effect Effects 0.000 claims description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 241000237536 Mytilus edulis Species 0.000 abstract description 23
- 235000020638 mussel Nutrition 0.000 abstract description 22
- 239000000543 intermediate Substances 0.000 abstract description 16
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 abstract description 9
- 238000009826 distribution Methods 0.000 abstract description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 abstract description 4
- 102000015728 Mucins Human genes 0.000 description 57
- 108010063954 Mucins Proteins 0.000 description 57
- 239000000243 solution Substances 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 108010045397 lysyl-tyrosyl-lysine Proteins 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- YTJFXEDRUOQGSP-DCAQKATOSA-N Lys-Pro-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YTJFXEDRUOQGSP-DCAQKATOSA-N 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- CJEHCEOXPLASCK-MEYUZBJRSA-N Thr-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@H](O)C)CC1=CC=C(O)C=C1 CJEHCEOXPLASCK-MEYUZBJRSA-N 0.000 description 8
- 108010059898 glycyl-tyrosyl-lysine Proteins 0.000 description 8
- 108010003137 tyrosyltyrosine Proteins 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 7
- RCYUBVHMVUHEBM-RCWTZXSCSA-N Pro-Pro-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RCYUBVHMVUHEBM-RCWTZXSCSA-N 0.000 description 6
- KIEIJCFVGZCUAS-MELADBBJSA-N Ser-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CO)N)C(=O)O KIEIJCFVGZCUAS-MELADBBJSA-N 0.000 description 6
- VXFXIBCCVLJCJT-JYJNAYRXSA-N Tyr-Pro-Pro Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N1CCC[C@H]1C(O)=O VXFXIBCCVLJCJT-JYJNAYRXSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 108010051110 tyrosyl-lysine Proteins 0.000 description 6
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 6
- 108010065920 Insulin Lispro Proteins 0.000 description 5
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 5
- FPQMQEOVSKMVMA-ACRUOGEOSA-N Lys-Tyr-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CCCCN)N)O FPQMQEOVSKMVMA-ACRUOGEOSA-N 0.000 description 5
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 5
- 108010010096 glycyl-glycyl-tyrosine Proteins 0.000 description 5
- 229940051875 mucins Drugs 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- CHFFHQUVXHEGBY-GARJFASQSA-N Ala-Lys-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N CHFFHQUVXHEGBY-GARJFASQSA-N 0.000 description 4
- ZZZWQALDSQQBEW-STQMWFEESA-N Arg-Gly-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZZZWQALDSQQBEW-STQMWFEESA-N 0.000 description 4
- YSYTWUMRHSFODC-QWRGUYRKSA-N Asn-Tyr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O YSYTWUMRHSFODC-QWRGUYRKSA-N 0.000 description 4
- KFMBRBPXHVMDFN-UWVGGRQHSA-N Gly-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCNC(N)=N KFMBRBPXHVMDFN-UWVGGRQHSA-N 0.000 description 4
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 4
- IEIHKHYMBIYQTH-YESZJQIVSA-N Lys-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCCCN)N)C(=O)O IEIHKHYMBIYQTH-YESZJQIVSA-N 0.000 description 4
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 4
- WCNVGGZRTNHOOS-ULQDDVLXSA-N Pro-Lys-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O WCNVGGZRTNHOOS-ULQDDVLXSA-N 0.000 description 4
- VVAWNPIOYXAMAL-KJEVXHAQSA-N Pro-Thr-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VVAWNPIOYXAMAL-KJEVXHAQSA-N 0.000 description 4
- IUFQHOCOKQIOMC-XIRDDKMYSA-N Trp-Asn-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N IUFQHOCOKQIOMC-XIRDDKMYSA-N 0.000 description 4
- JAGGEZACYAAMIL-CQDKDKBSSA-N Tyr-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CC=C(C=C1)O)N JAGGEZACYAAMIL-CQDKDKBSSA-N 0.000 description 4
- VTCKHZJKWQENKX-KBPBESRZSA-N Tyr-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O VTCKHZJKWQENKX-KBPBESRZSA-N 0.000 description 4
- 108010062796 arginyllysine Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 108010084389 glycyltryptophan Proteins 0.000 description 4
- 229960004502 levodopa Drugs 0.000 description 4
- 108010064235 lysylglycine Proteins 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 235000002374 tyrosine Nutrition 0.000 description 4
- 108010020532 tyrosyl-proline Proteins 0.000 description 4
- 238000002604 ultrasonography Methods 0.000 description 4
- UDSVWSUXKYXSTR-QWRGUYRKSA-N Asn-Gly-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O UDSVWSUXKYXSTR-QWRGUYRKSA-N 0.000 description 3
- LRCIOEVFVGXZKB-BZSNNMDCSA-N Asn-Tyr-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LRCIOEVFVGXZKB-BZSNNMDCSA-N 0.000 description 3
- PGUYEUCYVNZGGV-QWRGUYRKSA-N Asp-Gly-Tyr Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PGUYEUCYVNZGGV-QWRGUYRKSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- XZRZILPOZBVTDB-GJZGRUSLSA-N Gly-Arg-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)CN)C(O)=O)=CNC2=C1 XZRZILPOZBVTDB-GJZGRUSLSA-N 0.000 description 3
- LURCIJSJAKFCRO-QWRGUYRKSA-N Gly-Asn-Tyr Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LURCIJSJAKFCRO-QWRGUYRKSA-N 0.000 description 3
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- WINFHLHJTRGLCV-BZSNNMDCSA-N Lys-Tyr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=C(O)C=C1 WINFHLHJTRGLCV-BZSNNMDCSA-N 0.000 description 3
- UZWMJZSOXGOVIN-LURJTMIESA-N Met-Gly-Gly Chemical compound CSCC[C@H](N)C(=O)NCC(=O)NCC(O)=O UZWMJZSOXGOVIN-LURJTMIESA-N 0.000 description 3
- UGDMQJSXSSZUKL-IHRRRGAJSA-N Pro-Ser-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O UGDMQJSXSSZUKL-IHRRRGAJSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- UUIYFDAWNBSWPG-IHPCNDPISA-N Trp-Lys-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N UUIYFDAWNBSWPG-IHPCNDPISA-N 0.000 description 3
- CNNVVEPJTFOGHI-ACRUOGEOSA-N Tyr-Lys-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CNNVVEPJTFOGHI-ACRUOGEOSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 3
- 108010015792 glycyllysine Proteins 0.000 description 3
- 108010087823 glycyltyrosine Proteins 0.000 description 3
- 108010036413 histidylglycine Proteins 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 3
- 108010038745 tryptophylglycine Proteins 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- QBQVKUNBCAFXSV-ULQDDVLXSA-N Arg-Lys-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QBQVKUNBCAFXSV-ULQDDVLXSA-N 0.000 description 2
- CVFOYJJOZYYEPE-KBPBESRZSA-N Gly-Lys-Tyr Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CVFOYJJOZYYEPE-KBPBESRZSA-N 0.000 description 2
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 2
- KBBFOULZCHWGJX-KBPBESRZSA-N Gly-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)CN)O KBBFOULZCHWGJX-KBPBESRZSA-N 0.000 description 2
- PNUFMLXHOLFRLD-KBPBESRZSA-N Gly-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 PNUFMLXHOLFRLD-KBPBESRZSA-N 0.000 description 2
- RVOMPSJXSRPFJT-DCAQKATOSA-N Lys-Ala-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RVOMPSJXSRPFJT-DCAQKATOSA-N 0.000 description 2
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 2
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 2
- ZASPELYMPSACER-HOCLYGCPSA-N Lys-Gly-Trp Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ZASPELYMPSACER-HOCLYGCPSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- GFHYISDTIWZUSU-QWRGUYRKSA-N Tyr-Asn-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GFHYISDTIWZUSU-QWRGUYRKSA-N 0.000 description 2
- AUZADXNWQMBZOO-JYJNAYRXSA-N Tyr-Pro-Arg Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=C(O)C=C1 AUZADXNWQMBZOO-JYJNAYRXSA-N 0.000 description 2
- MWUYSCVVPVITMW-IGNZVWTISA-N Tyr-Tyr-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 MWUYSCVVPVITMW-IGNZVWTISA-N 0.000 description 2
- WYOBRXPIZVKNMF-IRXDYDNUSA-N Tyr-Tyr-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(O)=O)C1=CC=C(O)C=C1 WYOBRXPIZVKNMF-IRXDYDNUSA-N 0.000 description 2
- KHPLUFDSWGDRHD-SLFFLAALSA-N Tyr-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O KHPLUFDSWGDRHD-SLFFLAALSA-N 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VIYKYVYAKVNDPS-HKGPVOKGSA-N (2s)-2-azanyl-3-[3,4-bis(oxidanyl)phenyl]propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1.OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 VIYKYVYAKVNDPS-HKGPVOKGSA-N 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
- AOAKQKVICDWCLB-UWJYBYFXSA-N Ala-Tyr-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N AOAKQKVICDWCLB-UWJYBYFXSA-N 0.000 description 1
- PNIGSVZJNVUVJA-BQBZGAKWSA-N Arg-Gly-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O PNIGSVZJNVUVJA-BQBZGAKWSA-N 0.000 description 1
- NGTYEHIRESTSRX-UWVGGRQHSA-N Arg-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NGTYEHIRESTSRX-UWVGGRQHSA-N 0.000 description 1
- UZSQXCMNUPKLCC-FJXKBIBVSA-N Arg-Thr-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UZSQXCMNUPKLCC-FJXKBIBVSA-N 0.000 description 1
- UGJLILSJKSBVIR-ZFWWWQNUSA-N Arg-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)NCC(O)=O)=CNC2=C1 UGJLILSJKSBVIR-ZFWWWQNUSA-N 0.000 description 1
- DXHINQUXBZNUCF-MELADBBJSA-N Asn-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC(=O)N)N)C(=O)O DXHINQUXBZNUCF-MELADBBJSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 1
- ADZGCWWDPFDHCY-ZETCQYMHSA-N Gly-His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 ADZGCWWDPFDHCY-ZETCQYMHSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 1
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 1
- MKIAPEZXQDILRR-YUMQZZPRSA-N Gly-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN MKIAPEZXQDILRR-YUMQZZPRSA-N 0.000 description 1
- JKSMZVCGQWVTBW-STQMWFEESA-N Gly-Trp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(O)=O JKSMZVCGQWVTBW-STQMWFEESA-N 0.000 description 1
- LKJCZEPXHOIAIW-HOTGVXAUSA-N Gly-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN LKJCZEPXHOIAIW-HOTGVXAUSA-N 0.000 description 1
- YJDALMUYJIENAG-QWRGUYRKSA-N Gly-Tyr-Asn Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)CN)O YJDALMUYJIENAG-QWRGUYRKSA-N 0.000 description 1
- LYZYGGWCBLBDMC-QWHCGFSZSA-N Gly-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)CN)C(=O)O LYZYGGWCBLBDMC-QWHCGFSZSA-N 0.000 description 1
- DNAZKGFYFRGZIH-QWRGUYRKSA-N Gly-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 DNAZKGFYFRGZIH-QWRGUYRKSA-N 0.000 description 1
- MWAJSVTZZOUOBU-IHRRRGAJSA-N His-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC1=CN=CN1 MWAJSVTZZOUOBU-IHRRRGAJSA-N 0.000 description 1
- VTMLJMNQHKBPON-QWRGUYRKSA-N His-Gly-His Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 VTMLJMNQHKBPON-QWRGUYRKSA-N 0.000 description 1
- FZKFYOXDVWDELO-KBPBESRZSA-N His-Gly-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FZKFYOXDVWDELO-KBPBESRZSA-N 0.000 description 1
- KPYAOIVPJKPIOU-KKUMJFAQSA-N Leu-Lys-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O KPYAOIVPJKPIOU-KKUMJFAQSA-N 0.000 description 1
- SWWCDAGDQHTKIE-RHYQMDGZSA-N Lys-Arg-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWWCDAGDQHTKIE-RHYQMDGZSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- NNKLKUUGESXCBS-KBPBESRZSA-N Lys-Gly-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NNKLKUUGESXCBS-KBPBESRZSA-N 0.000 description 1
- YUAXTFMFMOIMAM-QWRGUYRKSA-N Lys-Lys-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O YUAXTFMFMOIMAM-QWRGUYRKSA-N 0.000 description 1
- BXPHMHQHYHILBB-BZSNNMDCSA-N Lys-Lys-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BXPHMHQHYHILBB-BZSNNMDCSA-N 0.000 description 1
- RMKJOQSYLQQRFN-KKUMJFAQSA-N Lys-Tyr-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O RMKJOQSYLQQRFN-KKUMJFAQSA-N 0.000 description 1
- PSVAVKGDUAKZKU-BZSNNMDCSA-N Lys-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCCN)N)O PSVAVKGDUAKZKU-BZSNNMDCSA-N 0.000 description 1
- MIMXMVDLMDMOJD-BZSNNMDCSA-N Lys-Tyr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O MIMXMVDLMDMOJD-BZSNNMDCSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 1
- BGWKULMLUIUPKY-BQBZGAKWSA-N Pro-Ser-Gly Chemical compound OC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 BGWKULMLUIUPKY-BQBZGAKWSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- WTUJZHKANPDPIN-CIUDSAMLSA-N Ser-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N WTUJZHKANPDPIN-CIUDSAMLSA-N 0.000 description 1
- PZZJMBYSYAKYPK-UWJYBYFXSA-N Ser-Ala-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O PZZJMBYSYAKYPK-UWJYBYFXSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- QGAHMVHBORDHDC-YUMQZZPRSA-N Ser-His-Gly Chemical compound OC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CN=CN1 QGAHMVHBORDHDC-YUMQZZPRSA-N 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- HQJOVVWAPQPYDS-ZFWWWQNUSA-N Trp-Gly-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQJOVVWAPQPYDS-ZFWWWQNUSA-N 0.000 description 1
- NSTPFWRAIDTNGH-BZSNNMDCSA-N Tyr-Asn-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NSTPFWRAIDTNGH-BZSNNMDCSA-N 0.000 description 1
- JWHOIHCOHMZSAR-QWRGUYRKSA-N Tyr-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JWHOIHCOHMZSAR-QWRGUYRKSA-N 0.000 description 1
- HIINQLBHPIQYHN-JTQLQIEISA-N Tyr-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HIINQLBHPIQYHN-JTQLQIEISA-N 0.000 description 1
- JKUZFODWJGEQAP-KBPBESRZSA-N Tyr-Gly-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O JKUZFODWJGEQAP-KBPBESRZSA-N 0.000 description 1
- ULHJJQYGMWONTD-HKUYNNGSSA-N Tyr-Gly-Trp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ULHJJQYGMWONTD-HKUYNNGSSA-N 0.000 description 1
- ADECJAKCRKPSOR-ULQDDVLXSA-N Tyr-His-Arg Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O ADECJAKCRKPSOR-ULQDDVLXSA-N 0.000 description 1
- PRONOHBTMLNXCZ-BZSNNMDCSA-N Tyr-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PRONOHBTMLNXCZ-BZSNNMDCSA-N 0.000 description 1
- WOAQYWUEUYMVGK-ULQDDVLXSA-N Tyr-Lys-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WOAQYWUEUYMVGK-ULQDDVLXSA-N 0.000 description 1
- ZOBLBMGJKVJVEV-BZSNNMDCSA-N Tyr-Lys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O ZOBLBMGJKVJVEV-BZSNNMDCSA-N 0.000 description 1
- SZEIFUXUTBBQFQ-STQMWFEESA-N Tyr-Pro-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SZEIFUXUTBBQFQ-STQMWFEESA-N 0.000 description 1
- MNWINJDPGBNOED-ULQDDVLXSA-N Tyr-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=C(O)C=C1 MNWINJDPGBNOED-ULQDDVLXSA-N 0.000 description 1
- YYLHVUCSTXXKBS-IHRRRGAJSA-N Tyr-Pro-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YYLHVUCSTXXKBS-IHRRRGAJSA-N 0.000 description 1
- GQVZBMROTPEPIF-SRVKXCTJSA-N Tyr-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GQVZBMROTPEPIF-SRVKXCTJSA-N 0.000 description 1
- QVYFTFIBKCDHIE-ACRUOGEOSA-N Tyr-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCCN)C(=O)O)N)O QVYFTFIBKCDHIE-ACRUOGEOSA-N 0.000 description 1
- RMRFSFXLFWWAJZ-HJOGWXRNSA-N Tyr-Tyr-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 RMRFSFXLFWWAJZ-HJOGWXRNSA-N 0.000 description 1
- 230000010062 adhesion mechanism Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- TUCIXUDAQRPDCG-UHFFFAOYSA-N benzene-1,2-diol Chemical group OC1=CC=CC=C1O.OC1=CC=CC=C1O TUCIXUDAQRPDCG-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 150000004696 coordination complex Chemical class 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 108010004563 mussel adhesive protein Proteins 0.000 description 1
- 239000003988 mussel adhesive protein Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920006112 polar polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
- C09J189/00—Adhesives based on proteins; Adhesives based on derivatives thereof
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Insects & Arthropods (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种粘附力强的类贻贝粘蛋白及其制备方法，属于粘附蛋白领域。本发明提供的类贻贝粘蛋白中间体mlp353，mlp316及mlp319的pI均在10左右，且含有高达23.1％～25.5％摩尔百分数的酪氨酸(Tyr)。本发明将上述类贻贝粘蛋白中间体经酪氨酸酶氧化后得到了新的类贻贝粘蛋白，与天然贻贝粘蛋白相比，本发明的类贻贝粘蛋白具有高粘附力和均匀的分子量分布，可以用来制备高粘附力的粘附材料，在海洋工程、生物医学及表面化学等领域具有广阔的应用前景。
Description
技术领域
本发明属于粘附蛋白领域，具体涉及一种粘附力强的类贻贝粘蛋白及其制备方法。
背景技术
贻贝是一种广泛分布于沿海和近海的甲壳类海洋生物，其足丝腺能分泌足丝，并通过足丝形成的足丝盘将贻贝固定在各种基材表面，研究表明，这与贻贝分泌的足丝有关。其足丝的主要成分为贻贝足丝蛋白(Mytilus edulis foot protein，Mfp)，也称为贻贝粘蛋白(mussel adhesive protein，MAP)，该蛋白具有高强度、高韧性和防水性。贻贝粘蛋白所形成的涂层具有多功能性，对多级别材料均能实现表面改性。贻贝粘蛋白还具有耐腐蚀性强、生物相容性良好、不会产生免疫反应等特点，在海洋工程、生物医学及表面化学等领域具有广泛的应用前景。
目前虽然还没有完全弄清楚贻贝粘蛋白的粘附机理，但大量的研究表明贻贝粘蛋白能粘附于几乎所有的固体材料表面，关键在于其结构中所富含的3，4-二羟基苯丙氨酸(DOPA，多巴)功能元。DOPA是一种侧基为儿茶酚(邻苯二酚)官能团的氨基酸，DOPA的苯酚基团具有很强的金属螯合能力，能在材料表面形成不可逆的有机金属络合物，还能与蛋白质等极性聚合物形成很强的氢键。因此，DOPA对贻贝粘蛋白的防水性黏合及内聚力起关键作用。
天然贻贝粘蛋白均来自于贻贝足丝分泌的一系列含有多巴基团的粘性蛋白，这些蛋白的DOPA含量从2％到28％不等，分子量的分布也从5-7KDa至108KDa不等，因此天然提取获得的贻贝粘蛋白的粘附性较低，而且具有不均一性，导致天然贻贝粘蛋白在实际应用中受限。为了克服天然贻贝粘蛋白的不足，研究者们开始研究人工合成的类贻贝粘蛋白。D.S.Hwang等(Biomaterials 28(2007)3560-3568)公开了一种类贻贝粘蛋白fp-151(即下文所述的类贻贝粘蛋白MLAP151)，它的生物相容性和溶解性优良，同时具有比天然贻贝粘蛋白更强的粘附能力。但是，该类贻贝粘蛋白fp-151的粘附能力还无法满足需求，有待进一步提高。
开发出具有更高粘附力的类贻贝粘蛋白具有重要意义。
发明内容
本发明的目的在于提供一种粘附力强的类贻贝粘蛋白及其制备方法。
本发明提供了一种基因，其核苷酸序列如SEQ ID NO.4、SEQ ID NO.5或SEQ IDNO.6所示。
本发明还提供了一种重组载体，它包括SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列，优选地，所述重组载体是重组pET28a。
本发明还提供了一种重组菌，它包含上述的重组载体；优选地，所述重组菌为重组E.coli。
本发明还提供了一种重组蛋白，其氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQID NO.3所示。
本发明还提供了一种制备上述重组蛋白的方法，它是采用上述的重组菌发酵制得的。
进一步地，所述方法包括以下步骤：
(1)取重组菌，活化，扩增，得种子液；
(2)将种子液接种到培养基中培养，待培养液OD600为0.6～1.0时加入诱导剂诱导表达，收集菌体；所述诱导剂优选为异丙基-β-D-硫代半乳糖苷；
(3)取菌体，破菌，离心，取沉淀纯化后即得。
本发明还提供了一种类贻贝粘蛋白，它是将上述重组蛋白经酪氨酸酶氧化后得到的。
进一步地，它的制备方法包括以下步骤：在包括上述重组蛋白、pH调节剂和抗坏血酸的混合体系中加入酪氨酸酶，反应，提纯，即得。
进一步地，所述混合体系还包括硼酸盐；混合体系中，硼酸盐的浓度为15～25mM，pH调节剂的浓度为0.05～0.15mM，抗坏血酸的浓度为5～15mM；酪氨酸酶的酶活力为10～30U；反应的时间为4～8小时，温度为20～30℃；提纯的方法为：经超滤膜脱盐浓缩；
优选地，所述硼酸盐为硼酸钠，所述pH调节剂为磷酸盐；混合体系中，硼酸盐的浓度为20mM，pH调节剂的浓度为0.1mM，抗坏血酸的浓度为10mM；混合体系的pH为7.0；酪氨酸酶的酶活力为20U；反应的时间为6小时，温度为25℃；所述超滤膜的截留分子量为1000Da。
本发明还提供了上述类贻贝粘蛋白在制备粘附材料中的用途。
本发明提供的类贻贝粘蛋白中间体mlp353，mlp316及mlp319的pI均在10左右，且含有高达23.1％～25.5％摩尔百分数的酪氨酸(Tyr)。mlp353，mlp316及mlp319中，酪氨酸个数及摩尔百分比分别为mlp353:40个(25.5％)；mlp316:52个(24％)；mlp319:64个(23.1％)。
本发明将上述类贻贝粘蛋白中间体mlp353，mlp316及mlp319经酪氨酸酶氧化后得到了新的类贻贝粘蛋白，与天然贻贝粘蛋白相比，本发明的类贻贝粘蛋白具有更高的粘附力和更均匀的分子量分布，与现有技术公开的类贻贝粘蛋白MLAP151相比，本发明的类贻贝粘蛋白也具有更高的粘附力。本发明的类贻贝粘蛋白可以用来制备高粘附力的粘附材料，在海洋工程、生物医学及表面化学等领域具有广阔的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为E.coli单克隆菌体总蛋白SDS-PAGE结果。(a)图：mlp353；(b)图：mlp316；(c)图：mlp319。
图2为超声破碎提取菌体蛋白SDS-PAGE结果。(a)图：泳道1：mlp353超声前；泳道2：mlp353超声上清；泳道3：mlp353超声沉淀。(b)图：泳道1：对照E.coli Rosseta2(DE3)/pET28a；泳道2：mlp316超声前；泳道3：mlp316超声沉淀；泳道4：mlp316超声上清；泳道5：mlp316超声前；泳道6：mlp319超声沉淀；泳道7：mlp319超声上清。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
实施例1：类贻贝粘蛋白中间体的制备
1、构建表达类贻贝粘蛋白中间体的工程菌：E.coli Rosseta2(DE3)/pET28a-mlp353，E.coli Rosseta2(DE3)/pET28a-mlp316，E.coli Rosseta2(DE3)/pET28a-mlp319
分别根据类贻贝粘蛋白中间体mlp353，mlp316及mlp319的氨基酸序列SEQ IDNo.1，SEQ ID No.2及SEQ ID No.3，通过密码子优化获得其核苷酸序列SEQ ID No.4，SEQID No.5，SEQ ID No.6，合成目的基因，并将目的基因片段分别插入到pET28a的多克隆位点Nco I(5’端)和Hind III(3’端)，得到重组表达载体pET28a-mlp353，pET28a-mlp316，pET28a-mlp319和保存重组表达载体的克隆宿主E.coli Top10/pET28a-mlp353，E.coliTop10/pET28a-mlp316，E.coli Top10/pET28a-mlp319。
将pET28a-mlp353，pET28a-mlp316，pET28a-mlp319通过热激法转化感受态细胞E.coli Rosseta2(DE3)，涂布氨苄抗性平板，37℃倒置培养12-16h获得单克隆菌体。具体感受态细胞制备方法和热激转化方法详见《分子克隆实验指南(第3版)》。随机挑取几个单克隆菌体过夜扩增，提取质粒并用Nco I和Hind III进行双酶切验证。琼脂糖凝胶电泳结果显示含有目的基因片段的质粒送出测序，经比对序列正确的单克隆菌体于-20℃保存。
表1.氨基酸序列表
表2.目的基因核苷酸列表
2、mlp353，mlp316，mlp319的重组表达及筛选
种子培养：挑取若干单克隆菌体，接种于含1mL LB培养基的24孔板，并于37℃，700rpm振荡过夜培养。
诱导表达：取样测定种子培养液的OD600，然后吸取20μL新鲜培养的种子液接种于2mL LB培养基，37℃，700rpm振荡培养。待培养液OD600约为0.6-1.0，加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度0.4mM，继续培养6h。培养结束后取样进行SDS-PAGE分析，筛选得到表达水平较好的单克隆菌体。
经过对SDS-PAGE结果分析表明(图1)，接种的单克隆菌体均表达了类贻贝粘蛋白中间体，其中，mlp353占总蛋白(整个泳道蛋白条带)的30％，mlp316占总蛋白的45％，mlp319占总蛋白的35％。
3、mlp353，mlp316，mlp319的可溶性分析
种子培养：将E.coli甘油菌液接种于含1mL LB培养基的24孔板，并于37℃，700rpm振荡过夜培养。
诱导表达：取样测定种子培养液的OD600，然后吸取200μL新鲜培养的种子液接种于50mL LB培养基，37℃，700rpm振荡培养。待培养液OD600约为0.6-1.0，加入IPTG至终浓度0.4mM，继续培养6h。
破胞提取：培养结束的培养液离心4000rpm，10min，菌体重悬于5mL的提取缓冲液(20mM Tris-HCl，pH 8.5)。用超声破碎仪进行破胞，工作条件为：功率200W，工作时间3s，间隔时间7s，60次。超声完成后将菌悬液离心12000rpm，5min，上清和沉淀分别取样进行SDS-PAGE分析。
结果如图2所示，经过对超声样品的SDS-PAGE分析表明，超声后mlp353，mlp316，mlp319绝大部分均位于沉淀，说明其在此表达条件下，几乎以不可溶形式于细胞内表达。
4、mlp353，mlp316，mlp319的提取纯化
取700mL的发酵液，6000rpm离心10min后，弃去上清，用1000mL去离子水重悬菌体，经超声破碎后，6000rpm离心，弃去上清，沉淀用500mL的去离子水重悬洗涤，再次离心后，弃去上清，得包涵体沉淀25g。用200mL5％乙酸溶液浸泡、搅拌4h。溶解完全后经8000rpm离心15min后，弃去沉淀杂质，得到含类贻贝粘蛋白中间体的溶液。将溶液pH调至4.0，用去离子水稀释5倍体积后，用CM Sepharose Fast Flow柱进行离子交换层析。先用10倍柱体积的pH4.0柠檬酸-柠檬酸钠缓冲体系平衡层析柱，然后将上述溶液上样吸附。吸附完全后，用2倍柱体积的柠檬酸-柠檬酸钠缓冲体系冲洗，然后用pH 4.0的柠檬酸-柠檬酸钠缓冲液添加0.8M氯化钠的溶液洗脱。洗脱完毕用后用8倍柱体积的0.5M氢氧化钠洗脱。将含类贻贝粘蛋白中间体的洗脱液用截留分子量为1000Da的超滤膜浓缩，用0.5％的乙酸溶液顶洗脱盐，得纯化后的类贻贝粘蛋白中间体溶液。
实施例2：酶催化氧化获得类贻贝粘蛋白MLAP353，MLAP316，MLAP319
将实施例1将含类贻贝粘蛋白中间体(mlp353，mlp316或mlp319)的洗脱液用截留分子量1000Da的超滤膜浓缩至50mL。取硼酸钠、磷酸盐、抗坏血酸加入其中，配制成抗坏血酸终浓度为10mM，硼酸钠终浓度为20mM，磷酸盐终浓度为0.1mM的反应体系，调pH至7.0，加入20U的酪氨酸酶，25℃下缓慢搅拌反应6h。反应完成后用0.5％的乙酸溶液在截留分子量为1000Da的超滤膜中顶洗脱盐后浓缩。将浓缩蛋白溶液冻干，得类贻贝粘蛋白冻干粉：MLAP353，MLAP316，MLAP319。
以下通过实验例证明本发明的有益效果。
实验例1：类贻贝粘蛋白中间体mlp353，mlp316及mlp319的氨基酸组成
(1)
SEQ ID No.1：mlp353；
氨基酸个数：157；
理论分子量：18801.06；
理论等电点(pI)：10.06。
表3.mlp353的氨基酸组成
(2)
SEQ ID No.2：mlp316；
氨基酸个数：217；
理论分子量：25600.93；
理论等电点(pI)：10.04。
表4.mlp316氨基酸组成
| 氨基酸 | 个数 | Mol％ |
| Ala(A) | 8 | 3.70％ |
| Arg(R) | 11 | 5.10％ |
| Asn(N) | 9 | 4.10％ |
| Asp(D) | 2 | 0.90％ |
| Cys(C) | 0 | 0.00％ |
| Gln(Q) | 0 | 0.00％ |
| Glu(E) | 0 | 0.00％ |
| Gly(G) | 37 | 17.10％ |
| His(H) | 4 | 1.80％ |
| Ile(I) | 0 | 0.00％ |
| Leu(L) | 1 | 0.50％ |
| Lys(K) | 41 | 18.90％ |
| Met(M) | 1 | 0.50％ |
| Phe(F) | 0 | 0.00％ |
| Pro(P) | 26 | 12.00％ |
| Ser(S) | 12 | 5.50％ |
| Thr(T) | 7 | 3.20％ |
| Trp(W) | 6 | 2.80％ |
| Tyr(Y) | 52 | 24.00％ |
| Val(V) | 0 | 0.00％ |
(3)
SEQ ID No.3：mlp319；
氨基酸个数：277；
理论分子量：32400.81；
理论等电点(pI)：10.04。
表5.mlp319氨基酸组成
可以看出，类贻贝粘蛋白中间体的pI均在10左右。mlp353，mlp316及mlp319中，酪氨酸(Tyr)个数及摩尔百分比分别为mlp353:40个(25.5％)；mlp316:52个(24％)；mlp319:64个(23.1％)。
实验例2：类贻贝粘蛋白的粘附力测试
1、实验方法
称取实施例2制得的30mg类贻贝粘蛋白冻干粉样品(MLAP353，MLAP316或MLAP319)，放入200μL离心管，加入90μL去离子水超声并搅拌溶解至均一，透亮。取两块金属板(2024t3板材，长*宽*厚：100mm*25mm*1.6mm，两侧打孔)，在下层板涂抹12μL(250平方毫米)，缓慢盖上上层板，然后尽快压上压板(重600g)，至45℃烘箱烘干。48h后将样品取出，移除压板，静置0.5h冷却至室温，将样品用万能拉力机夹具夹住，保证样品长度方向与拉力方向平行。以5±1mm/min速度拉伸，使剪切力变化在8.3-9.8MPa之间。样品断裂后，用最大剪切力计算最大拉伸剪切强度。
以已知的类贻贝粘蛋白MLAP151(即文献Biomaterials 28(2007)3560-3568报道的类贻贝粘蛋白fp-151)为对照样品。
2、实验结果
表6.类贻贝粘蛋白的粘附力测试结果
可以看出，与对照样品MLAP151相比，本发明的类贻贝粘蛋白具有更高的粘附力。
综上，本发明公开了一种粘附力强的类贻贝粘蛋白及其制备方法。本发明提供的类贻贝粘蛋白中间体mlp353，mlp316及mlp319的pI均在10左右，且含有高达23.1％～25.5％摩尔百分数的酪氨酸(Tyr)。本发明将上述类贻贝粘蛋白中间体经酪氨酸酶氧化后得到了新的类贻贝粘蛋白，与天然贻贝粘蛋白相比，本发明的类贻贝粘蛋白具有更高的粘附力和更均匀的分子量分布，与现有技术公开的类贻贝粘蛋白MLAP151相比，本发明的类贻贝粘蛋白也具有更高的粘附力。本发明的类贻贝粘蛋白可以用来制备高粘附力的粘附材料，在海洋工程、生物医学及表面化学等领域具有广阔的应用前景。
SEQUENCE LISTING
<110> 成都英普博集生物科技有限公司
<120> 一种粘附力强的类贻贝粘蛋白及其制备方法
<130> GYKH1336-2021P0114513CC
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 157
<212> PRT
<213> 人工序列
<400> 1
Met Gly Gly Asn Tyr Tyr Pro Lys Tyr Lys Tyr Pro Arg Gly Tyr Lys
1 5 10 15
Gly Gly Tyr Asn Gly Tyr Pro Arg Gly Asn Tyr Gly Trp Asn Lys Gly
20 25 30
Trp Lys Lys Gly Arg Trp Gly Arg Lys Tyr Tyr Tyr Asp Gly Tyr Ser
35 40 45
Asp Gly Tyr Tyr Pro Gly Ser Ala Tyr Asn Tyr Pro Ser Gly Ser His
50 55 60
Gly Tyr His Gly His Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Gly Lys
65 70 75 80
Lys Tyr Tyr Tyr Lys Tyr Lys Arg Thr Gly Lys Tyr Lys Tyr Leu Lys
85 90 95
Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly
100 105 110
Gly Ser Ser Gly Gly Asn Tyr Tyr Pro Lys Tyr Lys Tyr Pro Arg Gly
115 120 125
Tyr Lys Gly Gly Tyr Asn Gly Tyr Pro Arg Gly Asn Tyr Gly Trp Asn
130 135 140
Lys Gly Trp Lys Lys Gly Arg Trp Gly Arg Lys Tyr Tyr
145 150 155
<210> 2
<211> 217
<212> PRT
<213> 人工序列
<400> 2
Met Gly Gly Asn Tyr Tyr Pro Lys Tyr Lys Tyr Pro Arg Gly Tyr Lys
1 5 10 15
Gly Gly Tyr Asn Gly Tyr Pro Arg Gly Asn Tyr Gly Trp Asn Lys Gly
20 25 30
Trp Lys Lys Gly Arg Trp Gly Arg Lys Tyr Tyr Ala Lys Pro Ser Tyr
35 40 45
Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala
50 55 60
Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Tyr Asp Gly Tyr Ser Asp Gly
65 70 75 80
Tyr Tyr Pro Gly Ser Ala Tyr Asn Tyr Pro Ser Gly Ser His Gly Tyr
85 90 95
His Gly His Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Gly Lys Lys Tyr
100 105 110
Tyr Tyr Lys Tyr Lys Arg Thr Gly Lys Tyr Lys Tyr Leu Lys Lys Ala
115 120 125
Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser
130 135 140
Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr
145 150 155 160
Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly
165 170 175
Gly Asn Tyr Tyr Pro Lys Tyr Lys Tyr Pro Arg Gly Tyr Lys Gly Gly
180 185 190
Tyr Asn Gly Tyr Pro Arg Gly Asn Tyr Gly Trp Asn Lys Gly Trp Lys
195 200 205
Lys Gly Arg Trp Gly Arg Lys Tyr Tyr
210 215
<210> 3
<211> 277
<212> PRT
<213> 人工序列
<400> 3
Met Gly Gly Asn Tyr Tyr Pro Lys Tyr Lys Tyr Pro Arg Gly Tyr Lys
1 5 10 15
Gly Gly Tyr Asn Gly Tyr Pro Arg Gly Asn Tyr Gly Trp Asn Lys Gly
20 25 30
Trp Lys Lys Gly Arg Trp Gly Arg Lys Tyr Tyr Ala Lys Pro Ser Tyr
35 40 45
Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala
50 55 60
Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro
65 70 75 80
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro
85 90 95
Ser Tyr Pro Pro Thr Tyr Lys Tyr Asp Gly Tyr Ser Asp Gly Tyr Tyr
100 105 110
Pro Gly Ser Ala Tyr Asn Tyr Pro Ser Gly Ser His Gly Tyr His Gly
115 120 125
His Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Gly Lys Lys Tyr Tyr Tyr
130 135 140
Lys Tyr Lys Arg Thr Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys
145 150 155 160
Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Ala
165 170 175
Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro
180 185 190
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro
195 200 205
Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr
210 215 220
Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Gly Asn Tyr Tyr
225 230 235 240
Pro Lys Tyr Lys Tyr Pro Arg Gly Tyr Lys Gly Gly Tyr Asn Gly Tyr
245 250 255
Pro Arg Gly Asn Tyr Gly Trp Asn Lys Gly Trp Lys Lys Gly Arg Trp
260 265 270
Gly Arg Lys Tyr Tyr
275
<210> 4
<211> 471
<212> DNA
<213> 人工序列
<400> 4
atgggtggca attattatcc gaaatataaa tacccgcgtg gctataaagg tggttataat 60
ggctatccgc gtggcaatta tggctggaat aagggttgga aaaaaggtcg ctggggtcgc 120
aaatattatt atgatggtta tagtgacggc tattatccgg gtagcgccta taattatccg 180
agtggtagtc atggctatca tggccacggt tataaaggca aatattatgg caaaggtaaa 240
aagtattact acaagtataa gcgcaccggt aaatataaat atctgaaaaa agcgcgcaag 300
tatcatcgca aaggctataa aaaatactac ggcggcggta gcagtggtgg taattattat 360
cctaaatata agtacccgcg cggctataaa ggcggttata atggttatcc gcgtggtaat 420
tatggttgga ataagggctg gaaaaaaggc cgctggggcc gtaaatatta t 471
<210> 5
<211> 651
<212> DNA
<213> 人工序列
<400> 5
atgggcggta attattatcc gaaatataaa tacccgcgcg gctataaagg cggctataat 60
ggttatccgc gtggtaatta tggttggaat aagggctgga aaaaaggtcg ttggggtcgc 120
aaatattatg caaaaccgag ctatccgccg acctataaag caaaaccgag ttatccgccg 180
acatataaag caaagccgag ttatcctccg acctataagt atgatggtta tagtgatggc 240
tattatccgg gtagtgcata taattatccg agcggtagtc atggttatca tggccacggt 300
tataaaggca aatattatgg caaaggtaaa aagtattact acaagtataa gcgtaccggc 360
aaatataaat atctgaaaaa agcacgtaag taccatcgca aaggctataa aaaatattac 420
ggtggtggca gtagcgcaaa accgtcatat ccgccgacgt ataaagccaa accgagttac 480
ccgccgacct acaaagccaa acctagctat ccgcctacct ataaaggtgg caattattat 540
cctaaataca agtatccgcg tggctataaa ggtggttata atggctatcc gcgtggaaat 600
tatggctgga ataagggttg gaaaaaaggc cgctggggtc gcaagtatta t 651
<210> 6
<211> 834
<212> DNA
<213> 人工序列
<400> 6
atgggtggca attattatcc gaaatataaa tacccgcgcg gctataaagg tggctataat 60
ggttatccgc gtggtaatta tggctggaat aagggctgga aaaaaggccg ttggggtcgc 120
aaatattatg caaaaccgag ctatccgccg acctataaag ccaaaccgag ttatccgccg 180
acatataaag caaaaccgtc atatccgccg acgtataaag ccaagccgag ttatcctccg 240
acctataagg caaaaccgtc ttatccgccg acttataaag ccaaacctag ttatccgcct 300
acctataaat atgatggcta tagcgatggt tattatccgg gtagtgcata taattatccg 360
agtggtagcc acggttatca tggccacggt tataaaggta aatattatgg caaaggtaaa 420
aagtactact acaaatataa gcgcaccggt aaatataaat atctgaaaaa agcccgcaag 480
tatcatcgta aaggttataa aaaatactac ggcggtggca gtagtgccaa accgagctac 540
ccgccgacct acaaagccaa accaagctat ccgcctacat ataaagcgaa accgagttac 600
ccgccgacat acaaagccaa gcctagctat ccgccaacct ataaagcgaa gccgagctat 660
cctccgacat ataaggccaa accgtcttac ccgccgacgt acaaaggcgg taattattat 720
cctaaataca agtatccgcg tggctataaa ggcggttata atggttaccc gcgtggtaac 780
tatggttgga ataagggttg gaaaaaaggt cgctggggtc gcaagtatta ttaa 834
Claims (14)
1.一种基因，其特征在于：其核苷酸序列如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示。
2.一种重组载体，其特征在于：它包括SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列。
3.根据权利要求2所述的重组载体，其特征在于：所述重组载体是重组pET28a。
4.一种重组菌，其特征在于：它包含权利要求2或3所述的重组载体。
5.根据权利要求4所述的重组菌，其特征在于：所述重组菌为重组E.coli。
6.一种重组蛋白，其特征在于：其氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQ IDNO.3所示。
7.一种制备权利要求6所述重组蛋白的方法，其特征在于：它是采用权利要求4或5所述的重组菌发酵制得的。
8.根据权利要求7所述的方法，其特征在于：所述方法包括以下步骤：
(1)取重组菌，活化，扩增，得种子液；
(2)将种子液接种到培养基中培养，待培养液OD600为0.6～1.0时加入诱导剂诱导表达，收集菌体；
(3)取菌体，破菌，离心，取沉淀纯化后即得。
9.根据权利要求8所述的方法，其特征在于：所述诱导剂为异丙基-β-D-硫代半乳糖苷。
10.一种类贻贝粘蛋白，其特征在于：它是将权利要求6所述重组蛋白经酪氨酸酶氧化后得到的。
11.根据权利要求10所述的类贻贝粘蛋白，其特征在于：它的制备方法包括以下步骤：在包括权利要求6所述重组蛋白、pH调节剂和抗坏血酸的混合体系中加入酪氨酸酶，反应，提纯，即得。
12.根据权利要求11所述的类贻贝粘蛋白，其特征在于：所述混合体系还包括硼酸盐；混合体系中，硼酸盐的浓度为15～25mM，pH调节剂的浓度为0.05～0.15mM，抗坏血酸的浓度为5～15mM；酪氨酸酶的酶活力为10～30U；反应的时间为4～8小时，温度为20～30℃；提纯的方法为：经超滤膜脱盐浓缩。
13.根据权利要求12所述的类贻贝粘蛋白，其特征在于：所述硼酸盐为硼酸钠，所述pH调节剂为磷酸盐；混合体系中，硼酸盐的浓度为20mM，pH调节剂的浓度为0.1mM，抗坏血酸的浓度为10mM；混合体系的pH为7.0；酪氨酸酶的酶活力为20U；反应的时间为6小时，温度为25℃；所述超滤膜的截留分子量为1000Da。
14.权利要求10～13任一项所述类贻贝粘蛋白在制备粘附材料中的用途。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210067273.2A CN114349836B (zh) | 2022-01-20 | 2022-01-20 | 一种粘附力强的类贻贝粘蛋白及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210067273.2A CN114349836B (zh) | 2022-01-20 | 2022-01-20 | 一种粘附力强的类贻贝粘蛋白及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114349836A CN114349836A (zh) | 2022-04-15 |
| CN114349836B true CN114349836B (zh) | 2024-02-09 |
Family
ID=81091838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210067273.2A Active CN114349836B (zh) | 2022-01-20 | 2022-01-20 | 一种粘附力强的类贻贝粘蛋白及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114349836B (zh) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114853863B (zh) * | 2022-04-28 | 2023-11-21 | 西安德诺海思医疗科技有限公司 | 一种重组类贻贝粘蛋白纯化方法 |
| CN115181169B (zh) * | 2022-05-13 | 2024-01-30 | 南京工业大学 | 一种提高重组贻贝蛋白黏附力的方法 |
| CN114917403A (zh) * | 2022-06-01 | 2022-08-19 | 湖南科妍创美医疗科技有限公司 | 一种类贻贝粘蛋白凝胶及其制备方法和应用 |
| CN114948788B (zh) * | 2022-06-15 | 2023-11-24 | 绽妍生物科技有限公司 | 一种舒缓修护三重蛋白组合物及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989152A (zh) * | 2004-03-26 | 2007-06-27 | Posco公司 | 贝类生物粘合剂 |
| CN108503712A (zh) * | 2018-03-14 | 2018-09-07 | 天津大学 | 具有粘附-抗污双功能的贻贝粘附蛋白/两性离子多肽融合蛋白及合成方法 |
| CN112946041A (zh) * | 2021-02-06 | 2021-06-11 | 自然资源部第一海洋研究所 | 一种基于融合蛋白传感器的重金属离子检测方法 |
-
2022
- 2022-01-20 CN CN202210067273.2A patent/CN114349836B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989152A (zh) * | 2004-03-26 | 2007-06-27 | Posco公司 | 贝类生物粘合剂 |
| CN108503712A (zh) * | 2018-03-14 | 2018-09-07 | 天津大学 | 具有粘附-抗污双功能的贻贝粘附蛋白/两性离子多肽融合蛋白及合成方法 |
| CN112946041A (zh) * | 2021-02-06 | 2021-06-11 | 自然资源部第一海洋研究所 | 一种基于融合蛋白传感器的重金属离子检测方法 |
Non-Patent Citations (1)
| Title |
|---|
| 厚壳贻贝足丝黏附蛋白mfp-3重组表达及黏附功能分析;李楠楠;谭亮;王智平;王信超;廖智;;中国生物化学与分子生物学报;第27卷(第09期);第851-857页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114349836A (zh) | 2022-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114349836B (zh) | 一种粘附力强的类贻贝粘蛋白及其制备方法 | |
| CN106755209B (zh) | 一种酶法制备β-烟酰胺单核苷酸的方法 | |
| CN112831485B (zh) | 一种低温活性改良的外切菊粉酶突变体MutDR121EH9 | |
| CN112813052B (zh) | 一种低温活性提高的外切菊粉酶突变体MutDP121ET6 | |
| CN108085308B (zh) | 一种能提高耐热脂肪酶产量的重组工程菌及其构建方法和应用 | |
| CN113862241B (zh) | 一种壳聚糖酶Csncv及其突变体CsnB和应用 | |
| CN112725310A (zh) | 一种不耐热的低温外切菊粉酶突变体MutG360Δ9 | |
| CN105985968A (zh) | 改进的广谱核酸内切酶及其工业生产方法 | |
| CN112725319B (zh) | polyG底物特异性的褐藻胶裂解酶FaAly7及其应用 | |
| CN112725309A (zh) | 一种中温下稳定的低温外切菊粉酶突变体MutP126R | |
| CN112980814A (zh) | 低温适应性提高的外切菊粉酶突变体MutV268Δ13 | |
| CN112662644B (zh) | 一种甘油磷酸二酯磷酸二酯酶突变体及其应用 | |
| CN107245470B (zh) | 一种脂肪酶重组大肠杆菌表达菌株、重组脂肪酶和应用 | |
| CN110669114B (zh) | 一种羊毛硫肽前体肽amyA6及其制备方法和应用 | |
| CN116554309A (zh) | 一种重组人源ⅲ型胶原蛋白及其制备方法和应用 | |
| CN110791508A (zh) | 密码子优化的片球菌素基因、表达载体及重组工程菌株 | |
| CN114958893B (zh) | 一种乳猪高温教槽料制备所需的乳糖酶的构建方法 | |
| CN112725315B (zh) | 一种壳聚糖酶及其突变体在制备壳寡糖中的应用 | |
| CN113046340B (zh) | 一种木葡聚糖酶及其应用 | |
| CN114573673B (zh) | 双叉犀金龟表皮蛋白，编码核苷酸序列及其应用 | |
| CN108841808A (zh) | 酸性海藻糖酶TreA及其基因和应用 | |
| CN113481187A (zh) | 一种褐藻胶裂解酶突变体及其应用 | |
| CN114854778B (zh) | 一种岩藻多糖酶基因Fcn1及其应用 | |
| CN111286464A (zh) | 一种高效表达几丁质酶的工程菌及植物促生长应用 | |
| CN114214306B (zh) | 一种反刍动物瘤胃原虫特异纤维素酶OCCel1A及其应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |