CN107056925A - 人fgf21突变体、其制备方法及用途 - Google Patents
人fgf21突变体、其制备方法及用途 Download PDFInfo
- Publication number
- CN107056925A CN107056925A CN201710302732.XA CN201710302732A CN107056925A CN 107056925 A CN107056925 A CN 107056925A CN 201710302732 A CN201710302732 A CN 201710302732A CN 107056925 A CN107056925 A CN 107056925A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- fgf21
- mutant
- gly
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 title claims 3
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 title claims 3
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 208000008589 Obesity Diseases 0.000 claims abstract description 5
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 5
- 235000020824 obesity Nutrition 0.000 claims abstract description 5
- 208000032928 Dyslipidaemia Diseases 0.000 claims abstract description 4
- 208000017170 Lipid metabolism disease Diseases 0.000 claims abstract description 4
- 150000001413 amino acids Chemical class 0.000 claims description 43
- 101000846529 Homo sapiens Fibroblast growth factor 21 Proteins 0.000 claims description 14
- 102000056713 human FGF21 Human genes 0.000 claims description 14
- 230000035772 mutation Effects 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 4
- 208000001145 Metabolic Syndrome Diseases 0.000 claims description 3
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000009465 prokaryotic expression Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 11
- 239000003937 drug carrier Substances 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 9
- 241000282414 Homo sapiens Species 0.000 abstract description 5
- 230000003328 fibroblastic effect Effects 0.000 abstract description 3
- 239000003102 growth factor Substances 0.000 abstract description 2
- 208000030159 metabolic disease Diseases 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 description 19
- 235000001014 amino acid Nutrition 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 13
- 238000012360 testing method Methods 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 108010057821 leucylproline Proteins 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 101000879203 Caenorhabditis elegans Small ubiquitin-related modifier Proteins 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 102000008300 Mutant Proteins Human genes 0.000 description 6
- 108010021466 Mutant Proteins Proteins 0.000 description 6
- 102000051619 SUMO-1 Human genes 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000000692 anti-sense effect Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 4
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 230000000857 drug effect Effects 0.000 description 4
- 238000002270 exclusion chromatography Methods 0.000 description 4
- 229940126864 fibroblast growth factor Drugs 0.000 description 4
- 108010049041 glutamylalanine Proteins 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 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 3
- MCKSLROAGSDNFC-ACZMJKKPSA-N Ala-Asp-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MCKSLROAGSDNFC-ACZMJKKPSA-N 0.000 description 3
- GIVWETPOBCRTND-DCAQKATOSA-N Arg-Gln-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O GIVWETPOBCRTND-DCAQKATOSA-N 0.000 description 3
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- QPTNELDXWKRIFX-YFKPBYRVSA-N Gly-Gly-Gln Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O QPTNELDXWKRIFX-YFKPBYRVSA-N 0.000 description 3
- WCHONUZTYDQMBY-PYJNHQTQSA-N His-Pro-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WCHONUZTYDQMBY-PYJNHQTQSA-N 0.000 description 3
- GLBNEGIOFRVRHO-JYJNAYRXSA-N Leu-Gln-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GLBNEGIOFRVRHO-JYJNAYRXSA-N 0.000 description 3
- ZCXQTRXYZOSGJR-FXQIFTODSA-N Pro-Asp-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZCXQTRXYZOSGJR-FXQIFTODSA-N 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- FKYWFUYPVKLJLP-DCAQKATOSA-N Ser-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FKYWFUYPVKLJLP-DCAQKATOSA-N 0.000 description 3
- OLYXUGBVBGSZDN-ACRUOGEOSA-N Tyr-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 OLYXUGBVBGSZDN-ACRUOGEOSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000001142 circular dichroism spectrum Methods 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 108010042598 glutamyl-aspartyl-glycine Proteins 0.000 description 3
- 230000002218 hypoglycaemic effect Effects 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 2
- YWWATNIVMOCSAV-UBHSHLNASA-N Ala-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YWWATNIVMOCSAV-UBHSHLNASA-N 0.000 description 2
- OILNWMNBLIHXQK-ZLUOBGJFSA-N Ala-Cys-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O OILNWMNBLIHXQK-ZLUOBGJFSA-N 0.000 description 2
- RXTBLQVXNIECFP-FXQIFTODSA-N Ala-Gln-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O RXTBLQVXNIECFP-FXQIFTODSA-N 0.000 description 2
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 2
- NKBQZKVMKJJDLX-SRVKXCTJSA-N Arg-Glu-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NKBQZKVMKJJDLX-SRVKXCTJSA-N 0.000 description 2
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 2
- COWITDLVHMZSIW-CIUDSAMLSA-N Asn-Lys-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O COWITDLVHMZSIW-CIUDSAMLSA-N 0.000 description 2
- HBUJSDCLZCXXCW-YDHLFZDLSA-N Asn-Val-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HBUJSDCLZCXXCW-YDHLFZDLSA-N 0.000 description 2
- SVABRQFIHCSNCI-FOHZUACHSA-N Asp-Gly-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SVABRQFIHCSNCI-FOHZUACHSA-N 0.000 description 2
- 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 2
- AHWRSSLYSGLBGD-CIUDSAMLSA-N Asp-Pro-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AHWRSSLYSGLBGD-CIUDSAMLSA-N 0.000 description 2
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 2
- FTIJVMLAGRAYMJ-MNXVOIDGSA-N Gln-Ile-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(N)=O FTIJVMLAGRAYMJ-MNXVOIDGSA-N 0.000 description 2
- IULKWYSYZSURJK-AVGNSLFASA-N Gln-Leu-Lys Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O IULKWYSYZSURJK-AVGNSLFASA-N 0.000 description 2
- WLRYGVYQFXRJDA-DCAQKATOSA-N Gln-Pro-Pro Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 WLRYGVYQFXRJDA-DCAQKATOSA-N 0.000 description 2
- RWQCWSGOOOEGPB-FXQIFTODSA-N Gln-Ser-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O RWQCWSGOOOEGPB-FXQIFTODSA-N 0.000 description 2
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 2
- UUYBFNKHOCJCHT-VHSXEESVSA-N Gly-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN UUYBFNKHOCJCHT-VHSXEESVSA-N 0.000 description 2
- FNXSYBOHALPRHV-ONGXEEELSA-N Gly-Val-Lys Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN FNXSYBOHALPRHV-ONGXEEELSA-N 0.000 description 2
- RGPWUJOMKFYFSR-QWRGUYRKSA-N His-Gly-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O RGPWUJOMKFYFSR-QWRGUYRKSA-N 0.000 description 2
- OQDLKDUVMTUPPG-AVGNSLFASA-N His-Leu-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OQDLKDUVMTUPPG-AVGNSLFASA-N 0.000 description 2
- FVEWRQXNISSYFO-ZPFDUUQYSA-N Ile-Arg-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FVEWRQXNISSYFO-ZPFDUUQYSA-N 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 2
- XBCWOTOCBXXJDG-BZSNNMDCSA-N Leu-His-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 XBCWOTOCBXXJDG-BZSNNMDCSA-N 0.000 description 2
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 2
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 2
- KWLWZYMNUZJKMZ-IHRRRGAJSA-N Leu-Pro-Leu Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O KWLWZYMNUZJKMZ-IHRRRGAJSA-N 0.000 description 2
- GOFJOGXGMPHOGL-DCAQKATOSA-N Leu-Ser-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(C)C GOFJOGXGMPHOGL-DCAQKATOSA-N 0.000 description 2
- TXKWKTWYTIAZSV-KKUMJFAQSA-N Phe-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N TXKWKTWYTIAZSV-KKUMJFAQSA-N 0.000 description 2
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 2
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 description 2
- LGSANCBHSMDFDY-GARJFASQSA-N Pro-Glu-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O LGSANCBHSMDFDY-GARJFASQSA-N 0.000 description 2
- FEPSEIDIPBMIOS-QXEWZRGKSA-N Pro-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 FEPSEIDIPBMIOS-QXEWZRGKSA-N 0.000 description 2
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 2
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 2
- LCUOTSLIVGSGAU-AVGNSLFASA-N Pro-His-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LCUOTSLIVGSGAU-AVGNSLFASA-N 0.000 description 2
- FYPGHGXAOZTOBO-IHRRRGAJSA-N Pro-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 FYPGHGXAOZTOBO-IHRRRGAJSA-N 0.000 description 2
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- GHPQVUYZQQGEDA-BIIVOSGPSA-N Ser-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N)C(=O)O GHPQVUYZQQGEDA-BIIVOSGPSA-N 0.000 description 2
- XWCYBVBLJRWOFR-WDSKDSINSA-N Ser-Gln-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O XWCYBVBLJRWOFR-WDSKDSINSA-N 0.000 description 2
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 2
- BSXKBOUZDAZXHE-CIUDSAMLSA-N Ser-Pro-Glu Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O BSXKBOUZDAZXHE-CIUDSAMLSA-N 0.000 description 2
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 2
- YOSLMIPKOUAHKI-OLHMAJIHSA-N Thr-Asp-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O YOSLMIPKOUAHKI-OLHMAJIHSA-N 0.000 description 2
- VGYBYGQXZJDZJU-XQXXSGGOSA-N Thr-Glu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VGYBYGQXZJDZJU-XQXXSGGOSA-N 0.000 description 2
- PRTHQBSMXILLPC-XGEHTFHBSA-N Thr-Ser-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PRTHQBSMXILLPC-XGEHTFHBSA-N 0.000 description 2
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 2
- AZGZDDNKFFUDEH-QWRGUYRKSA-N Tyr-Gly-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AZGZDDNKFFUDEH-QWRGUYRKSA-N 0.000 description 2
- OXGVAUFVTOPFFA-XPUUQOCRSA-N Val-Gly-Cys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N OXGVAUFVTOPFFA-XPUUQOCRSA-N 0.000 description 2
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 2
- LAYSXAOGWHKNED-XPUUQOCRSA-N Val-Gly-Ser Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LAYSXAOGWHKNED-XPUUQOCRSA-N 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 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 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 1
- FEGOCLZUJUFCHP-CIUDSAMLSA-N Ala-Pro-Gln Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O FEGOCLZUJUFCHP-CIUDSAMLSA-N 0.000 description 1
- XLWSGICNBZGYTA-CIUDSAMLSA-N Arg-Glu-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XLWSGICNBZGYTA-CIUDSAMLSA-N 0.000 description 1
- UGZUVYDKAYNCII-ULQDDVLXSA-N Arg-Phe-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UGZUVYDKAYNCII-ULQDDVLXSA-N 0.000 description 1
- OVQJAKFLFTZDNC-GUBZILKMSA-N Arg-Pro-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O OVQJAKFLFTZDNC-GUBZILKMSA-N 0.000 description 1
- YFGUZQQCSDZRBN-DCAQKATOSA-N Asp-Pro-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O YFGUZQQCSDZRBN-DCAQKATOSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101000708016 Caenorhabditis elegans Sentrin-specific protease Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102100031734 Fibroblast growth factor 19 Human genes 0.000 description 1
- 102100024802 Fibroblast growth factor 23 Human genes 0.000 description 1
- IKFZXRLDMYWNBU-YUMQZZPRSA-N Gln-Gly-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N IKFZXRLDMYWNBU-YUMQZZPRSA-N 0.000 description 1
- UTKICHUQEQBDGC-ACZMJKKPSA-N Glu-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N UTKICHUQEQBDGC-ACZMJKKPSA-N 0.000 description 1
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 1
- DCBSZJJHOTXMHY-DCAQKATOSA-N Glu-Pro-Pro Chemical compound OC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DCBSZJJHOTXMHY-DCAQKATOSA-N 0.000 description 1
- 102000058063 Glucose Transporter Type 1 Human genes 0.000 description 1
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 1
- UESJMAMHDLEHGM-NHCYSSNCSA-N Gly-Ile-Leu Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O UESJMAMHDLEHGM-NHCYSSNCSA-N 0.000 description 1
- GGLIDLCEPDHEJO-BQBZGAKWSA-N Gly-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)CN GGLIDLCEPDHEJO-BQBZGAKWSA-N 0.000 description 1
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 1
- 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 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-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
- ZVXMEWXHFBYJPI-LSJOCFKGSA-N Gly-Val-Ile Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZVXMEWXHFBYJPI-LSJOCFKGSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- SVHKVHBPTOMLTO-DCAQKATOSA-N His-Arg-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SVHKVHBPTOMLTO-DCAQKATOSA-N 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101000846394 Homo sapiens Fibroblast growth factor 19 Proteins 0.000 description 1
- 101001051973 Homo sapiens Fibroblast growth factor 23 Proteins 0.000 description 1
- FZWVCYCYWCLQDH-NHCYSSNCSA-N Ile-Leu-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N FZWVCYCYWCLQDH-NHCYSSNCSA-N 0.000 description 1
- QKIBIXAQKAFZGL-GUBZILKMSA-N Leu-Cys-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QKIBIXAQKAFZGL-GUBZILKMSA-N 0.000 description 1
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- CSFVADKICPDRRF-KKUMJFAQSA-N Leu-His-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CN=CN1 CSFVADKICPDRRF-KKUMJFAQSA-N 0.000 description 1
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 1
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 1
- RTIRBWJPYJYTLO-MELADBBJSA-N Leu-Lys-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N RTIRBWJPYJYTLO-MELADBBJSA-N 0.000 description 1
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 1
- WFCKERTZVCQXKH-KBPBESRZSA-N Leu-Tyr-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O WFCKERTZVCQXKH-KBPBESRZSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- MQVFHOPCKNTHGT-MELADBBJSA-N Phe-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O MQVFHOPCKNTHGT-MELADBBJSA-N 0.000 description 1
- LXVLKXPFIDDHJG-CIUDSAMLSA-N Pro-Glu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O LXVLKXPFIDDHJG-CIUDSAMLSA-N 0.000 description 1
- UUHXBJHVTVGSKM-BQBZGAKWSA-N Pro-Gly-Asn Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O UUHXBJHVTVGSKM-BQBZGAKWSA-N 0.000 description 1
- VWXGFAIZUQBBBG-UWVGGRQHSA-N Pro-His-Gly Chemical compound C([C@@H](C(=O)NCC(=O)[O-])NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 VWXGFAIZUQBBBG-UWVGGRQHSA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- FHZJRBVMLGOHBX-GUBZILKMSA-N Pro-Pro-Asp Chemical compound OC(=O)C[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1)C(O)=O FHZJRBVMLGOHBX-GUBZILKMSA-N 0.000 description 1
- 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 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091006296 SLC2A1 Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- QGMLKFGTGXWAHF-IHRRRGAJSA-N Ser-Arg-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QGMLKFGTGXWAHF-IHRRRGAJSA-N 0.000 description 1
- SMIDBHKWSYUBRZ-ACZMJKKPSA-N Ser-Glu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O SMIDBHKWSYUBRZ-ACZMJKKPSA-N 0.000 description 1
- IUXGJEIKJBYKOO-SRVKXCTJSA-N Ser-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N IUXGJEIKJBYKOO-SRVKXCTJSA-N 0.000 description 1
- ASGYVPAVFNDZMA-GUBZILKMSA-N Ser-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CO)N ASGYVPAVFNDZMA-GUBZILKMSA-N 0.000 description 1
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 1
- XPKCFQZDQGVJCX-RHYQMDGZSA-N Val-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N)O XPKCFQZDQGVJCX-RHYQMDGZSA-N 0.000 description 1
- DOBHJKVVACOQTN-DZKIICNBSA-N Val-Tyr-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 DOBHJKVVACOQTN-DZKIICNBSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000011808 rodent model Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000011534 wash buffer Substances 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Toxicology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了人FGF21突变体、其制备方法及用途,具体公开了人成纤维细胞生长因子21(FGF21)突变体,其编码基因,以及制备所述突变体的方法和使用所述突变体治疗2型糖尿病、肥胖、异常血脂症、或代谢紊乱的方法。
Description
技术领域
本发明蛋白质工程领域,具体涉及人成纤维细胞生长因子21(FGF21)突变体,其编码基因,以及所述突变体的制备方法和使用所述突变体用于治疗2型糖尿病、肥胖、异常血脂症、或代谢紊乱的方法。
背景技术
FGF21属于成纤维细胞生长因子(FGF)19亚家族的分泌蛋白,所述成纤维细胞生长因子(FGF)亚家族包括FGF19、FGF21、和FGF23(Itoh等,2004,Trend Genet.20:563-69)。FGF21是一种非典型的FGF,其独立于肝素,并在葡萄糖、脂类和能量代谢中发挥功能。其通过上调GLUT1表达,促进脂肪细胞中的葡萄糖摄取,其机制不同于胰岛素。在患有糖尿病的啮齿动物、猴子和人中,FGF21降低葡萄糖空腹血清浓度,并降低甘油三酯、胰岛素和胰高血糖素的空腹血清浓度。此外,在饮食诱导的肥胖啮齿类动物模型中,施用FGF21导致累计体重呈剂量依赖性丧失。实验研究提供了FGF21具有用于治疗糖尿病、肥胖、异常血脂症、代谢综合症的药物学给药的支持。
人FGF21易降解,用量大,稳定性及药效较差,因此需要对野生型FGF21进行改造,提高人FGF21的有效性和稳定性,以减少对患者给药的剂量。
发明概述
为了解决上述问题,本发明提供了替代性的人FGF21突变体,其编码基因以及所述突变体的制备方法和用途。
具体而言,本发明一方面提供了一种人FGF21突变体,其在野生型人FGF21蛋白氨基酸序列的基础上具有以下氨基酸改变:
1)在野生型人FGF21蛋白氨基酸序列的第31位Ala和第32位His之间增加一个Cys,并且第43位Gly、第44位Ala中的任意1个或2个位置处的氨基酸突变为Cys,
2)野生型人FGF21蛋白氨基酸序列的第171位Pro突变为Gly;和/或
3)野生型人FGF21蛋白氨基酸序列的第24位至31位的氨基酸片段用5-15个氨基酸,优选6-14个氨基酸,更优选7-13个氨基酸,还优选8-10个氨基酸,最优选8个氨基酸的片段替换。
在优选的实施方案中,用于所述替换的氨基酸片段是任意氨基酸的组合。
在优选的实施方案中,用于所述替换的氨基酸片段为8个氨基酸的片段,用式X1X2X3X4X5X6X7X8表示。
在优选的实施方案中,X1-X8各自独立地选自任意氨基酸,优选X1是Ser或Asp,X2是Gly或Asp,X3是Pro或Ala,X4是Ala或Gln,X5是Gly或Gln,X6是Leu或Tyr,X7是Ser或His,X8是Ser或Ala。
本发明的方案中,基于FGF21的空间结构,本发明人发现,FGF21C端的第171位的氨基酸P容易暴露在表面易被酶所降解,本发明将其突变为G,从而增加了FGF21稳定性,但不影响FGF21的活性。进一步地,本发明人基于结构发现,FGF21的N端的第24-31的空间结构为一段loop环,loop环的长度在对FGF21的稳定性有显著影响的情况下仍然保持FGF21的活性。此外,基于FGF21的空间结构,本发明人还发现野生型FGF21的第31位和第32位与第43位和第44位的氨基酸在空间上是靠近的,但是结构不够稳定。经过深入研究,本发明人出人意料地发现:在不影响第23-32位间loop环的长度的情况下,在第31位和第32位之间插入了一个半胱氨酸Cys后,该半胱氨酸通过与第43位或第44位突变的半胱氨酸形成二硫键,从而稳定了其结构;另外,突变loop环上的氨基酸,在插入在第31-32位间的Cys与第43位或与第44位突变的Cys形成二硫键并稳定结构的基础上改变第24-31位Loop环的氨基酸组成,能够充分提高FGF21的活性。
在本发明的方案中,野生型人FGF21蛋白氨基酸序列在31-32位增加的Cys和第43位或44位的Cys之间形成稳定二硫键时,在野生FGF21第23-32位间的氨基酸的长度对于FGF21的稳定性影响效果明显。因此,在本说明书公开内容的基础上,本领域技术人员能够任意选择所述片段的长度和氨基酸种类来对本发明进行效果可以预期的改变或改进。
在优选的实施方案中,用于所述替代的氨基酸片段是DDAQQTEA(其相应的突变体在下文中也称为FGF21-AG),编码该突变体的核酸序列如SEQ ID NO:3所示,氨基酸序列如SEQ ID NO:4所示。
在另一个优选的实施方案中,用于所述替代的氨基酸片段是SGPHGLSS(其相应的突变体在下文中也称为FGF21-LG),编码该突变体的核酸序列如SEQ ID NO:5所示,氨基酸序列如SEQ ID NO:6所示。
本发明中“野生型FGF21”是从http://www.uniprot.org/中搜索FGF21,在得到的结果中选择物种(organism)为人类(Homo sapiens)而得到的。人源FGF21在uniprot的编号为(Q9NSA1),我们得到FGF21的蛋白质序列有209个残基。去除N端28个氨基酸的信号肽,得到的FGF21的序列有181个残基,即为野生型FGF21序列,其核酸序列为SEQ ID NO:2,相应氨基酸序列为SEQ ID NO:1。
因此,本发明中所述的“野生型人FGF21蛋白”是指天然存在于人中的成熟FGF21蛋白(下文中也称为FGF21),在典型的情况下,其氨基酸序列如SEQ ID NO:1所示,或与SEQ IDNO:1具有95%以上,优选98%以上,更优选99%以上的同源性的氨基酸序列。
另一方面,本发明提供一种核酸,其编码以上所述的任一突变体。
本发明还提供一种载体,其包含以上所述的核酸。在优选的实施方案中,所述载体为表达载体,更优选为原核表达载体。
本发明还提供一种宿主细胞,其包含以上所述的载体。
在优选的实施方案中,所述宿主细胞为大肠杆菌(E.coli)细胞。
本发明还提供一种制备人FGF21突变体的方法,所述方法包括将所述人FGF21突变体的编码基因与所述表达载体可操作性的连接,得到重组表达载体;将重组表达载体转化宿主细胞;培养重组宿主细胞,诱导重组蛋白表达,收集并纯化表达的蛋白。
在优选的实施方案中,所述方法包括将SUMO标签基因(SUMO标签基因N端有6个his标签)和编码所述人FGF21突变体的核酸序列依次连接在一起。将该基因与表达载体可操作性的相连接,得到重组表达载体;将重组表达载体转化宿主细胞,培养重组宿主细胞,诱导重组蛋白表达,收集并纯化所表达的蛋白,SUMO酶切割,再纯化。
所述的宿主细胞为大肠杆菌(E.coli)细胞。
本发明人发现,对于本发明所述的突变体,诱导表达条件为:10-37℃培养细胞到OD600达到0.2-1.0时加入终浓度为0.1-1mM的IPTG,4-37℃诱导表达2-20h。在更优选的实施方案中,条件为37℃培养细胞到OD600达到0.8时加入终浓度为1mM的IPTG,25℃诱导表达6h。
本发明的纯化方法是将收集到的菌体用裂解缓冲液(20Mm Tris-HCl 100MmNaCl,pH 8.0)重悬,进行细胞破碎,离心,收集上清液,将上清液与纯化填料混合后孵育0.1-10个小时,将混合物转移到层析柱中,进行蛋白的纯化,将纯化得到的SUMO融合的人FGF21突变体蛋白用SUMO酶酶切,酶切条件包括:温度为0-37℃,缓冲液为20Mm Tris-HCl100mMNaCl pH,8.0PBS,Tris-HCl,pH值为6.8-8.0,切割0-24小时,利用Ni金属螯合亲和层析,得到人FGF21突变体,再利用分子排阻层析对FGF21突变体蛋白进行再纯化。
另一方面,本发明提供所述突变体在制备药物中的用途,所述药物用于治疗2型糖尿病、肥胖、异常血脂症、或代谢综合征。
本发明中所述的“突变”包括氨基酸的置换、缺失、添加。
综上所述,本发明的突变体具有野生型FGF21没有的优点。这种优点包括改善的药理学效力和/或改善的药物稳定性。本发明的FGF21突变体蛋白具有一种或多种有利的生理学特征,包括在体内具有更显著的药效,具有更强热稳定性。另外,本发明的FGF21变体对2型糖尿病、肥胖、异常脂血症、或代谢综合症、或其任意的治疗具有潜在的作用。
附图简述
图1.SUMO-FGF21突变体表达载体示意图
图2.纯化后SUMO-FGF21-LG核酸电泳图,M是DL5000DNA Marker,1是SUMO-FGF21-LG基因的纯化产物
图3.纯化后SUMO-FGF21-AG核酸电泳图,M是DL5000DNAMarker,1是SUMO-FGF21-AG基因的纯化产物
图4.纯化后pET22b(DE3)SUMO-FGF21-LG蛋白的SDS-PAGE图
图5.纯化后FGF21-LG蛋白的SDS-PAGE图,M是蛋白分子量标准,1是FGF21-LG
图6.纯化后FGF21-AG蛋白的SDS-PAGE图,M是蛋白分子量标准,1是FGF21-AG
图7.FGF21-LG蛋白动物药效试验(降低血糖)结果图。
图8.FGF21-AG蛋白动物药效试验(降低血糖)结果图。
图9.FGF21-LG蛋白动物药效试验(对体重的影响)结果图。
图10.FGF21-AG蛋白动物药效试验(对体重的影响)结果图。
图11.FGF21-LG蛋白的热稳定性试验。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施中实验主要试剂耗材及仪器:克隆所用引物均为上海生工合成。聚合酶链式反应(PCR)中DNA聚合酶(primer star)、限制性内切酶、连接酶、DpnI均购于Takara公司;DNA marker购于Thermo公司;普通DNA产物回收试剂盒、琼脂糖凝胶回收试剂盒购于TIANGEN公司。E.coliDH5α菌株,E.coliBL21菌株,pET22b-SUMO-FGF21质粒,SUMO蛋白酶购置北京索莱宝科技有限公司。Tris、咪唑购于上海生工,其他盐类购于国药,浓缩管购于Millipore。FPLC仪器使用的是GE公司的系统,使用的Ni-sepharose层析柱和分子排阻层析柱Hiload 16/60 Superdex75pg均购于GE公司。BCA试剂盒购于Thermo。
表1.FGF21分子克隆所使用的引物
a第1-8位为保护碱基,第9至14位为限制性内切酶位点;
b第1-3位为保护碱基,第4至9位为限制性内切酶位点。
实施例1:重组FGF21突变体表达载体的构建
以pET22b-SUMO-FGF21质粒为模板,第一次突变:利用上游引物171G-F和下游引物171G-R进行PCR定点突变。得到FGF21-P171G突变体。第二次突变利用FGF21-P171G突变体为模板,以43C-F为上游引物和43C-R为下游引物进行PCR定点突变,得到FGF21-G43C-P171G突变体。第三次突变:利用FGF21-G43C-P171G突变体为模板,以31-32C-F为上游引物和31-32C-R为下游引物进行PCR定点突变,得到FGF21-AG突变体。第四次突变:以FGF21-AG为模板,以FGF21-LG-F为上游引物,以FGF21-LG-R为下游引物进行突变。得到FGF21-LG突变体。PCR反应条件如下。以上突变过程的PCR反应体系及反应程序如下:
PCR反应体系:2×Prime STAR HS(Premix)25μl,上游引物(10μM)1μl,下游引物(10μM)1μl,模板μl,ddH2O22μl,总体积50μl。
PCR反应程序:第1步、预变性温度98℃10mins;第2步、变性温度98℃10secs;3、退火温度57℃5secs;第4步、延伸温度72℃,1min。从第2到4步重复30个循环。
每次突变后的产物用Dpn I酶在37℃酶切反应1h。将酶切产物进行琼脂糖凝胶电泳,并利用胶回收试剂盒回收质粒,取1ul质粒放入100ul的感受态细胞中,将细胞放入冰上30分钟,42℃热激90秒,加入400ul LB培养基,37℃振荡培养45分钟后涂平板,37℃倒置过夜培养。挑去单克隆做为模板,利用SUMO-F为上游引物和FGF21-R为下游引物进行PCR鉴定,其中FGF21-LG的鉴定结果如图2所示,FGF21-AG的鉴定结构如图3所示。经初步鉴定成功的FGF21突变体进行基因的测序与结果分析。样品由生工生物工程(上海)有限公司进行序列测定。采用DNAMAN软件进行序列分析。序列测定的结果:突变体FGF21-AG的核酸序列如SEQID NO.3所示,相应氨基酸序列如SEQ ID NO.4所示;突变体FGF21-LG的核酸序列如SEQID NO.5所示,相应氨基酸序列如SEQ ID NO.6所示。其构建载体示意图见图1。
将测序成功的含有编码FGF21突变体的核酸序列的质粒转化到BL21(DE3)菌株中,挑取单克隆菌株进行扩大培养,并将菌株保存在-80℃冰箱中。
实施例2:人重组FGF21突变体的表达
将FGF21突变体菌株按2%的接种量接种于5ml LB培养基(含100μg/ml氨苄青霉素)中,37℃、220rpm振荡培养过夜,取过夜培养菌液按2%接种于每瓶中,每2000ml三角瓶装500mlLB培养基(含100μg/ml氨苄青霉素)。培养到OD600为0.8时加入1.0MIPTG 500ul,在25℃诱导表达4个小时。4℃,8000r/min离心10分钟,弃上清,收集菌体,将菌体放于-80℃冻存。
实施例3:重组人FGF21突变体纯化
(1)重组人FGF21突变体初纯化
将表达得到的菌体用裂解缓冲液(50mM Tris,300mMNaCl,pH8.5)重悬,然后进行超声破碎(30%功率,超2秒,停5秒,总时间45分钟),4℃14000rpm离心40分钟,收集上清液。将上清液加入已经用缓冲液预平衡的Ni-sepharose层析柱,4℃旋转混合30分钟。将溶液穿出后,用5倍柱体积的缓冲液冲洗以去除未结合的蛋白和杂质,再用5倍柱体积的含30mM咪唑的缓冲液(50mM Tris,300mMNaCl,pH8.5)将非特异性的杂蛋白洗脱下来,再用5倍柱体积的含300mM咪唑的缓冲液(50mM Tris,300mMNaCl,pH8.5)将目的蛋白洗脱下来,用SDS-PAGE检测纯化结果(结果见图4)。
(2)重组人FGF21突变体酶切纯化
检测透析后的融合蛋白:以分子摩尔比为0.1%的比例加入SUMO酶,在4℃下酶切2h,向酶切后的蛋白溶液加入终浓度为20mM咪唑,然后加入到已经用缓冲液(50mM Tris,300mMNaCl,pH8.5)预平衡的Ni-sepharose层析柱上,4℃旋转混合30分钟。收集流穿的溶液,用浓缩管将样品浓缩到10mg/ml,再经Superdex 75分子排阻层析柱纯化,分子排阻层析缓冲液(20mMPBS,100mM NaCl,pH7.2),得到的蛋白即为FGF21突变体蛋白,实验中的FGF21及突变体如FGF21-LG和FGF21-AG蛋白都是用上述方法纯化。并经12%SDS-PAGE电泳检测,其中FGF21-LG蛋白的SDS-PAGE凝胶电泳结果如图5,FGF21-AG蛋白的SDS-PAGE凝胶电泳结果见图6。
实施例4:FGF21突变体降血糖实验
使用突变体蛋白进行动物降血糖实验。将ob/ob小鼠模型分成3组,每组8只,分别为载体对照组,即注射生理盐水,FGF21组和FGF21-LG组,实施方式为背部皮下注射,0.1mg/kg/天,连续给药七天,并且记录每天给药前的血糖值,如图7所示,FGF21-LG(如图7)组相对于野生型FGF21蛋白而言表现出更强的药效。同样,以FGF21-AG进行相同的实验,实施方式为0.6mg/kg/天,并记录每天给药前的血糖值。实验说明FGF21-AG同样有很明显的降血糖作用,结果如图8所示。
实施例5:FGF21突变体降体重实验
使用FGF21突变体蛋白进行动物的降体重试验。将ob/ob小鼠分成4组,分别为载体对照组,即注射生理盐水,FGF21组和人FGF21-LG组,FGF21-AG组,每组8只。将纯化后的蛋白按照0.6mg/kg/天给药,连续给药6天,并且记录6天后体重变化,实验证实FGF21-LG(如图9)和FGF21-AG(如图10)组相对于野生型FGF21蛋白具有更明显的减轻体重作用,具有更强的药效。
实施例6:热稳定性试验
使用人FGF21突变蛋白进行实验,利用圆二色谱(CD)对FGF21及FGF21-LG进行变温实验,20-95℃,每5℃作为一个测试点进行测试。测试结果为FGF21的转化温度在69℃,而FGF21-LG的转化温度在98℃,实验证明人FGF21-LG相对于野生型FGF21具有更强的热稳定性(如图11)。同样,利用FGF21-AG,得到类似的CD结果。
可见,根据本发明的突变体与野生型相比具有更强的热稳定性。
SEQUENCE LISTING
<110> 中国科学院合肥物质科学研究院
<120> 人FGF21突变体、其制备方法及用途
<130> IB177786
<160> 18
<170> PatentIn version 3.1
<210> 1
<211> 181
<212> PRT
<213> Homo sapiens
<400> 1
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Ala Ser
180
<210> 2
<211> 543
<212> DNA
<213> Homo sapiens
<400> 2
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120
gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180
ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240
gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300
gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360
aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 420
ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 480
ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 540
tcc 543
<210> 3
<211> 546
<212> DNA
<213> 人工序列
<400> 3
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacag atgatgccca gcagacagaa gcttgccacc tggagatcag ggaggatggg 120
acggtggggt gcgctgctga ccagagcccc gaaagtctcc tgcagctgaa agccttgaag 180
ccgggagtta ttcaaatctt gggagtcaag acatccaggt tcctgtgcca gcggccagat 240
ggggccctgt atggatcgct ccactttgac cctgaggcct gcagcttccg ggagctgctt 300
cttgaggacg gatacaatgt ttaccagtcc gaagcccacg gcctcccgct gcacctgcca 360
gggaacaagt ccccacaccg ggaccctgca ccccgaggac cagctcgctt cctgccacta 420
ccaggcctgc cccccgcact cccggagcca cccggaatcc tggcccccca gccccccgat 480
gtgggctcct cggaccctct gagcatggtg ggaggttccc agggccgaag ccccagctac 540
gcttcc 546
<210> 4
<211> 182
<212> PRT
<213> 人工序列
<400> 4
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala Cys
20 25 30
His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Cys Ala Ala Asp Gln
35 40 45
Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile
50 55 60
Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
65 70 75 80
Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
85 90 95
Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
100 105 110
His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp
115 120 125
Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
130 135 140
Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp
145 150 155 160
Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Gly Ser Gln Gly Arg
165 170 175
Ser Pro Ser Tyr Ala Ser
180
<210> 5
<211> 546
<212> DNA
<213> 人工序列
<400> 5
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacat caggacctca tgggctctca agttgccacc tggagatcag ggaggatggg 120
acggtggggt gcgctgctga ccagagcccc gaaagtctcc tgcagctgaa agccttgaag 180
ccgggagtta ttcaaatctt gggagtcaag acatccaggt tcctgtgcca gcggccagat 240
ggggccctgt atggatcgct ccactttgac cctgaggcct gcagcttccg ggagctgctt 300
cttgaggacg gatacaatgt ttaccagtcc gaagcccacg gcctcccgct gcacctgcca 360
gggaacaagt ccccacaccg ggaccctgca ccccgaggac cagctcgctt cctgccacta 420
ccaggcctgc cccccgcact cccggagcca cccggaatcc tggcccccca gccccccgat 480
gtgggctcct cggaccctct gagcatggtg ggaggttccc agggccgaag ccccagctac 540
gcttcc 546
<210> 6
<211> 182
<212> PRT
<213> 人工序列
<400> 6
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys
20 25 30
His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Cys Ala Ala Asp Gln
35 40 45
Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile
50 55 60
Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
65 70 75 80
Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
85 90 95
Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
100 105 110
His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp
115 120 125
Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
130 135 140
Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp
145 150 155 160
Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Gly Ser Gln Gly Arg
165 170 175
Ser Pro Ser Tyr Ala Ser
180
<210> 7
<211> 32
<212> DNA
<213> 人工序列
<400> 7
gggaattcca tatgcatcat catcatcatc ac 32
<210> 8
<211> 24
<212> DNA
<213> 人工序列
<400> 8
ccgctcgagt caggaagcgt agct 24
<210> 9
<211> 38
<212> DNA
<213> 人工序列
<400> 9
cagagaacag attggtggtc accccatccc tgactcca 38
<210> 10
<211> 38
<212> DNA
<213> 人工序列
<400> 10
tggagtcagg gatggggtga ccaccaatct gttctctg 38
<210> 11
<211> 27
<212> DNA
<213> 人工序列
<400> 11
catggtggga ggttcccagg gccgaag 27
<210> 12
<211> 27
<212> DNA
<213> 人工序列
<400> 12
cttcggccct gggaacctcc caccatg 27
<210> 13
<211> 27
<212> DNA
<213> 人工序列
<400> 13
gggacggtgg ggtgcgctgc tgaccag 27
<210> 14
<211> 27
<212> DNA
<213> 人工序列
<400> 14
ctggtcagca gcgcacccca ccgtccc 27
<210> 15
<211> 28
<212> DNA
<213> 人工序列
<400> 15
cagcagacag aagcttgcca cctggaga 28
<210> 16
<211> 28
<212> DNA
<213> 人工序列
<400> 16
tctccaggtg gcaagcttct gtctgctg 28
<210> 17
<211> 58
<212> DNA
<213> 人工序列
<400> 17
agcggtacct ctacacatca ggacctcatg ggctctcaag ttgccacctg gagatcag 58
<210> 18
<211> 58
<212> DNA
<213> 人工序列
<400> 18
ctgatctcca ggtggcaact tgagagccca tgaggtcctg atgtgtagag gtaccgct 58
Claims (10)
1.一种人FGF21突变体,其在野生型人FGF21蛋白氨基酸序列的基础上具有以下氨基酸改变:
1)在野生型人FGF21蛋白氨基酸序列的第31位Ala和第32位His之间增加一个Cys,并且第43位Gly、第44位Ala中的任意1个或2个位置处的氨基酸突变为Cys,
2)野生型人FGF21蛋白氨基酸序列的第171位Pro突变为Gly;和/或
3)野生型人FGF21蛋白氨基酸序列的第24位至第31位的氨基酸片段用5-15个氨基酸,优选6-14个氨基酸,更优选7-13个氨基酸,还优选8-10个氨基酸,最优选8个氨基酸的片段替换。
2.根据权利要求1所述的突变体,用于所述替换的氨基酸片段是任意氨基酸的组合。
3.根据权利要求1所述的突变体,用于所述替换的氨基酸片段为用式X1X2X3X4X5X6X7X8表示的8个氨基酸的片段,其中X1-X8各自独立地选自任意氨基酸,优选X1是Ser或Asp,X2是Gly或Asp,X3是Pro或Ala,X4是Ala或Gln,X5是Gly或Gln,X6是Leu或Tyr,X7是Ser或His,X8是Ser或Ala。
4.根据权利要求1所述的突变体,用于所述替换的氨基酸片段的氨基酸序列是DDAQQTEA或SGPHGLSS,相应的人FGF21突变体的氨基酸序列如SEQ ID NO:4或SEQ ID NO:6所示。
5.根据权利要求1所述的突变体,所述野生型人FGF21蛋白氨基酸序列如SEQ ID NO:1所示或是与SEQ ID NO:1具有95%以上,优选98%以上,更优选99%以上的同源性的氨基酸序列。
6.核酸,其编码权利要求1-5中任一项所述的突变体。
7.载体,其包含权利要求6所述的核酸,所述载体优选为表达载体,更优选为原核表达载体。
8.宿主细胞,其包含权利要求7所述的载体。
9.药物组合物,其包含权利要求1-5中任一项所述的突变体以及药用载体。
10.权利要求1-5中任一项所述的突变体、权利要求6所述的核酸、权利要求7所述的载体或权利要求8所述的宿主细胞在制备药物中的用途,所述药物用于治疗2型糖尿病、肥胖、异常血脂症、或代谢综合征。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710302732.XA CN107056925B (zh) | 2017-04-28 | 2017-04-28 | 人fgf21突变体、其制备方法及用途 |
US16/608,706 US11746134B2 (en) | 2017-04-28 | 2018-04-11 | Human FGF21 mutant with improved effectiveness and stability and pharmaceutical composition thereof |
PCT/CN2018/082669 WO2018196616A1 (zh) | 2017-04-28 | 2018-04-11 | 人fgf21突变体、其制备方法及用途 |
JP2020509145A JP7097434B2 (ja) | 2017-04-28 | 2018-04-11 | ヒトfgf21変異体、その製造方法、及びその使用 |
EP18791988.1A EP3636663A4 (en) | 2017-04-28 | 2018-04-11 | FGF21 MUTANT HUMAN, ITS PREPARATION PROCESS AND ITS USE |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710302732.XA CN107056925B (zh) | 2017-04-28 | 2017-04-28 | 人fgf21突变体、其制备方法及用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107056925A true CN107056925A (zh) | 2017-08-18 |
CN107056925B CN107056925B (zh) | 2022-01-14 |
Family
ID=59595925
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710302732.XA Active CN107056925B (zh) | 2017-04-28 | 2017-04-28 | 人fgf21突变体、其制备方法及用途 |
Country Status (5)
Country | Link |
---|---|
US (1) | US11746134B2 (zh) |
EP (1) | EP3636663A4 (zh) |
JP (1) | JP7097434B2 (zh) |
CN (1) | CN107056925B (zh) |
WO (1) | WO2018196616A1 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018196616A1 (zh) * | 2017-04-28 | 2018-11-01 | 中国科学院合肥物质科学研究院 | 人fgf21突变体、其制备方法及用途 |
CN110128525A (zh) * | 2018-02-08 | 2019-08-16 | 广东东阳光药业有限公司 | Fgf21变体、融合蛋白及其应用 |
WO2023035817A1 (zh) * | 2021-09-08 | 2023-03-16 | 北京志道生物科技有限公司 | 一种fgf21突变蛋白及其应用 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102495299B1 (ko) * | 2020-11-03 | 2023-02-06 | 토드제약 주식회사 | Fgf21의 열탄력성을 이용한 fgf21 생산 방법 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102655877A (zh) * | 2009-05-05 | 2012-09-05 | 安姆根有限公司 | Fgf21突变体及其用途 |
CN103923207A (zh) * | 2014-04-08 | 2014-07-16 | 东北农业大学 | 一种fgf-21突变体蛋白的制备及其在治疗非酒精性脂肪肝中的应用 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JOP20190083A1 (ar) * | 2008-06-04 | 2017-06-16 | Amgen Inc | بولي ببتيدات اندماجية طافرة لـfgf21 واستخداماتها |
WO2010042747A2 (en) | 2008-10-10 | 2010-04-15 | Amgen Inc. | Fgf21 mutants and uses thereof |
RU2573896C2 (ru) * | 2009-10-15 | 2016-01-27 | Дженентек, Инк. | Химерные факторы роста фибробластов с измененной рецепторной специфичностью |
MX2014002260A (es) | 2011-08-31 | 2014-08-18 | Amgen Inc | Factor de crecimiento de fibroblasto 21 para usar en el tratamiento de diabetes tipo 1. |
EP2859014B1 (en) * | 2012-06-11 | 2017-04-26 | Eli Lilly and Company | Fibroblast growth factor 21 variants |
AU2017382038A1 (en) * | 2016-12-22 | 2019-08-08 | Sanofi | FGF21 compound / GLP-1R agonist combinations with optimized activity ratio |
CN107056925B (zh) * | 2017-04-28 | 2022-01-14 | 中国科学院合肥物质科学研究院 | 人fgf21突变体、其制备方法及用途 |
-
2017
- 2017-04-28 CN CN201710302732.XA patent/CN107056925B/zh active Active
-
2018
- 2018-04-11 WO PCT/CN2018/082669 patent/WO2018196616A1/zh unknown
- 2018-04-11 EP EP18791988.1A patent/EP3636663A4/en active Pending
- 2018-04-11 JP JP2020509145A patent/JP7097434B2/ja active Active
- 2018-04-11 US US16/608,706 patent/US11746134B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102655877A (zh) * | 2009-05-05 | 2012-09-05 | 安姆根有限公司 | Fgf21突变体及其用途 |
CN103923207A (zh) * | 2014-04-08 | 2014-07-16 | 东北农业大学 | 一种fgf-21突变体蛋白的制备及其在治疗非酒精性脂肪肝中的应用 |
Non-Patent Citations (2)
Title |
---|
HUANG Z.F: "A better anti-diabetic recombinant human fibroblast growth factor 21 (rhFGF21) modified by polyethylene glycol", 《PLOS ONE》 * |
陈秋池等: "FGF21 (L59R)突变体基因工程菌发酵表达条件的优化", 《基因组学与应用生物学》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018196616A1 (zh) * | 2017-04-28 | 2018-11-01 | 中国科学院合肥物质科学研究院 | 人fgf21突变体、其制备方法及用途 |
US11746134B2 (en) | 2017-04-28 | 2023-09-05 | Heifei Zhongke Longwood Biotechnology Co., Ltd. | Human FGF21 mutant with improved effectiveness and stability and pharmaceutical composition thereof |
CN110128525A (zh) * | 2018-02-08 | 2019-08-16 | 广东东阳光药业有限公司 | Fgf21变体、融合蛋白及其应用 |
CN110128525B (zh) * | 2018-02-08 | 2022-08-26 | 广东东阳光药业有限公司 | Fgf21变体、融合蛋白及其应用 |
WO2023035817A1 (zh) * | 2021-09-08 | 2023-03-16 | 北京志道生物科技有限公司 | 一种fgf21突变蛋白及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN107056925B (zh) | 2022-01-14 |
EP3636663A1 (en) | 2020-04-15 |
US11746134B2 (en) | 2023-09-05 |
JP2020518281A (ja) | 2020-06-25 |
WO2018196616A1 (zh) | 2018-11-01 |
JP7097434B2 (ja) | 2022-07-07 |
EP3636663A4 (en) | 2021-01-13 |
US20210163560A1 (en) | 2021-06-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107056925A (zh) | 人fgf21突变体、其制备方法及用途 | |
Moody et al. | Structure and function of a bacterial Fasciclin I Domain Protein elucidates function of related cell adhesion proteins such as TGFBIp and periostin | |
CN110546161B (zh) | 对胰岛素受体具有降低的结合力的胰岛素类似物及其用途 | |
CN100579986C (zh) | 一种生产促胰岛素分泌肽glp-1(7-36)及glp-1类似物的方法 | |
CN106459222A (zh) | Mic‑1融合蛋白及其用途 | |
SK171492A3 (en) | Polypeptide, method of its preparation its use and pharmaceutical agent | |
CN1807456B (zh) | 重组人甲状旁腺激素pth1-34的制备方法 | |
TW211585B (zh) | ||
CN111793126A (zh) | Glp-1类似物多肽的制备方法及在ⅱ型糖尿病中应用 | |
CN101875700B (zh) | 一种增加促胰岛素分泌肽融合蛋白生物活性的方法 | |
CN103834623B (zh) | 具有催化活性的人源尿酸氧化酶 | |
US9469674B2 (en) | α-conotoxin peptide, pharmaceutical composition and use thereof | |
CN113735959B (zh) | 一种治疗nash的fgf类似物 | |
CN102584966A (zh) | 一种重组花生过敏原与突变体及其制备方法和应用 | |
CN101897953B (zh) | 无创性高穿透性表皮生长因子及其应用 | |
Kashima et al. | cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland | |
US6893844B1 (en) | DNA encoding a new human hepatoma derived growth factor and producing method thereof | |
CN101168740B (zh) | 三分三托品酮还原酶ⅰ基因及其编码的蛋白质和应用 | |
Nikaidou et al. | Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonasmarginalis N6301 | |
CN109750021A (zh) | 一种扇贝类胡萝卜素氧化裂解酶基因及其应用 | |
CN101481689A (zh) | 新序列人胰岛素样生长因子-ⅰ基因的克隆及其高效表达 | |
JPH0380096A (ja) | モチリン様ポリペプチドの製法並びにそのための組換えdna及び発現用プラスミド | |
CN108117599A (zh) | Ssm6a的重组表达和纯化方法及其使用的融合蛋白 | |
CN107217069B (zh) | 原核表达载体及rbFGF-2表达方法与工程菌和应用 | |
CN108085329A (zh) | 大肠杆菌表达系统表达人淀粉样Aeta蛋白及纯化的方法 |
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 | ||
TR01 | Transfer of patent right |
Effective date of registration: 20220818 Address after: 230088 706-710, building 2, Hefei Innovation Institute, 2666 Xiyou Road, high tech Zone, Hefei City, Anhui Province Patentee after: Hefei Zhongke Changmu Biotechnology Co.,Ltd. Address before: 230031 Shushan Lake Road, Shushan District, Hefei, Anhui 350 Patentee before: HEFEI INSTITUTES OF PHYSICAL SCIENCE, CHINESE ACADEMY OF SCIENCES |
|
TR01 | Transfer of patent right |