CN111072783B - Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence - Google Patents
Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence Download PDFInfo
- Publication number
- CN111072783B CN111072783B CN201911373533.3A CN201911373533A CN111072783B CN 111072783 B CN111072783 B CN 111072783B CN 201911373533 A CN201911373533 A CN 201911373533A CN 111072783 B CN111072783 B CN 111072783B
- Authority
- CN
- China
- Prior art keywords
- arg
- gly
- glu
- ser
- ala
- 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
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 title claims abstract description 73
- 230000014509 gene expression Effects 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 34
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 21
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 title claims abstract 14
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 title abstract description 56
- 108090000765 processed proteins & peptides Proteins 0.000 title abstract description 22
- 229920001184 polypeptide Polymers 0.000 title abstract description 10
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 81
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 71
- 102000004190 Enzymes Human genes 0.000 claims abstract description 43
- 108090000790 Enzymes Proteins 0.000 claims abstract description 43
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 24
- 108090000631 Trypsin Proteins 0.000 claims abstract description 9
- 102000004142 Trypsin Human genes 0.000 claims abstract description 9
- 239000012588 trypsin Substances 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 27
- 238000003776 cleavage reaction Methods 0.000 claims description 22
- 230000007017 scission Effects 0.000 claims description 22
- 238000005520 cutting process Methods 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 12
- 150000001413 amino acids Chemical group 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 8
- 230000002255 enzymatic effect Effects 0.000 claims description 7
- 239000007983 Tris buffer Substances 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 102100025101 GATA-type zinc finger protein 1 Human genes 0.000 claims 6
- 239000002028 Biomass Substances 0.000 claims 1
- 230000002934 lysing effect Effects 0.000 claims 1
- 238000001976 enzyme digestion Methods 0.000 abstract description 20
- 230000009465 prokaryotic expression Effects 0.000 abstract description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 abstract description 6
- 239000003398 denaturant Substances 0.000 abstract description 3
- 230000001939 inductive effect Effects 0.000 abstract description 3
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 276
- 108010089804 glycyl-threonine Proteins 0.000 description 133
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 131
- PBSOQGZLPFVXPU-YUMQZZPRSA-N Arg-Glu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PBSOQGZLPFVXPU-YUMQZZPRSA-N 0.000 description 127
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 113
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 112
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 103
- LLWQVJNHMYBLLK-CDMKHQONSA-N Gly-Thr-Phe Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LLWQVJNHMYBLLK-CDMKHQONSA-N 0.000 description 99
- IVDFVBVIVLJJHR-LKXGYXEUSA-N Thr-Ser-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IVDFVBVIVLJJHR-LKXGYXEUSA-N 0.000 description 99
- FBNPMTNBFFAMMH-AVGNSLFASA-N Leu-Val-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-AVGNSLFASA-N 0.000 description 98
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 95
- OLKICIBQRVSQMA-SRVKXCTJSA-N Ser-Ser-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OLKICIBQRVSQMA-SRVKXCTJSA-N 0.000 description 94
- COYSIHFOCOMGCF-WPRPVWTQSA-N Val-Arg-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-WPRPVWTQSA-N 0.000 description 94
- WMBFONUKQXGLMU-WDSOQIARSA-N Trp-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N WMBFONUKQXGLMU-WDSOQIARSA-N 0.000 description 92
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 87
- FJVAQLJNTSUQPY-CIUDSAMLSA-N Ala-Ala-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN FJVAQLJNTSUQPY-CIUDSAMLSA-N 0.000 description 86
- YXXPVUOMPSZURS-ZLIFDBKOSA-N Ala-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 YXXPVUOMPSZURS-ZLIFDBKOSA-N 0.000 description 86
- JZJGEKDPWVJOLD-QEWYBTABSA-N Glu-Phe-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JZJGEKDPWVJOLD-QEWYBTABSA-N 0.000 description 86
- SWSRFJZZMNLMLY-ZKWXMUAHSA-N Ser-Asp-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O SWSRFJZZMNLMLY-ZKWXMUAHSA-N 0.000 description 86
- 108010009298 lysylglutamic acid Proteins 0.000 description 86
- WZPIKDWQVRTATP-SYWGBEHUSA-N Ile-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)=CNC2=C1 WZPIKDWQVRTATP-SYWGBEHUSA-N 0.000 description 82
- DUTMKEAPLLUGNO-JYJNAYRXSA-N Lys-Glu-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DUTMKEAPLLUGNO-JYJNAYRXSA-N 0.000 description 82
- 108010080629 tryptophan-leucine Proteins 0.000 description 82
- UXJCMQFPDWCHKX-DCAQKATOSA-N Arg-Arg-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O UXJCMQFPDWCHKX-DCAQKATOSA-N 0.000 description 79
- XTMZYFMTYJNABC-ZLUOBGJFSA-N Asn-Ser-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N XTMZYFMTYJNABC-ZLUOBGJFSA-N 0.000 description 79
- WDTLNWHPIPCMMP-AVGNSLFASA-N Met-Arg-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O WDTLNWHPIPCMMP-AVGNSLFASA-N 0.000 description 79
- KSCVLGXNQXKUAR-JYJNAYRXSA-N Tyr-Leu-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KSCVLGXNQXKUAR-JYJNAYRXSA-N 0.000 description 79
- 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 76
- NEEOBPIXKWSBRF-IUCAKERBSA-N Leu-Glu-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O NEEOBPIXKWSBRF-IUCAKERBSA-N 0.000 description 74
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 74
- VZFPYFRVHMSSNA-JURCDPSOSA-N Phe-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=CC=C1 VZFPYFRVHMSSNA-JURCDPSOSA-N 0.000 description 74
- 108010010147 glycylglutamine Proteins 0.000 description 74
- 108010068380 arginylarginine Proteins 0.000 description 73
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 72
- WRNAXCVRSBBKGS-BQBZGAKWSA-N Glu-Gly-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O WRNAXCVRSBBKGS-BQBZGAKWSA-N 0.000 description 69
- MFMDKJIPHSWSBM-GUBZILKMSA-N Ala-Lys-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFMDKJIPHSWSBM-GUBZILKMSA-N 0.000 description 64
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 64
- 108010013835 arginine glutamate Proteins 0.000 description 63
- QOJJMJKTMKNFEF-ZKWXMUAHSA-N Asp-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O QOJJMJKTMKNFEF-ZKWXMUAHSA-N 0.000 description 57
- JGAMUXDWYSXYLM-SRVKXCTJSA-N Lys-Arg-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O JGAMUXDWYSXYLM-SRVKXCTJSA-N 0.000 description 54
- 235000018102 proteins Nutrition 0.000 description 54
- UPOJUWHGMDJUQZ-IUCAKERBSA-N Gly-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UPOJUWHGMDJUQZ-IUCAKERBSA-N 0.000 description 53
- CLNSYANKYVMZNM-UWVGGRQHSA-N Gly-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N CLNSYANKYVMZNM-UWVGGRQHSA-N 0.000 description 53
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 52
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 48
- YJIUYQKQBBQYHZ-ACZMJKKPSA-N Gln-Ala-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YJIUYQKQBBQYHZ-ACZMJKKPSA-N 0.000 description 46
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 45
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 45
- PLQWGQUNUPMNOD-KKUMJFAQSA-N Ser-Tyr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O PLQWGQUNUPMNOD-KKUMJFAQSA-N 0.000 description 45
- 108010015792 glycyllysine Proteins 0.000 description 39
- 108010050848 glycylleucine Proteins 0.000 description 38
- 108010005233 alanylglutamic acid Proteins 0.000 description 37
- CGOHAEBMDSEKFB-FXQIFTODSA-N Glu-Glu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O CGOHAEBMDSEKFB-FXQIFTODSA-N 0.000 description 36
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 35
- APFJUBGRZGMQFF-QWRGUYRKSA-N Leu-Gly-Lys Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN APFJUBGRZGMQFF-QWRGUYRKSA-N 0.000 description 34
- 239000002243 precursor Substances 0.000 description 34
- HMRWQTHUDVXMGH-GUBZILKMSA-N Ala-Glu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HMRWQTHUDVXMGH-GUBZILKMSA-N 0.000 description 33
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 33
- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 33
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 32
- LSLIRHLIUDVNBN-CIUDSAMLSA-N Ala-Asp-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LSLIRHLIUDVNBN-CIUDSAMLSA-N 0.000 description 31
- GKWFMNNNYZHJHV-SRVKXCTJSA-N Asp-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC(O)=O GKWFMNNNYZHJHV-SRVKXCTJSA-N 0.000 description 31
- IASNWHAGGYTEKX-IUCAKERBSA-N Arg-Arg-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(O)=O IASNWHAGGYTEKX-IUCAKERBSA-N 0.000 description 29
- GAOJCVKPIGHTGO-UWVGGRQHSA-N Lys-Arg-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O GAOJCVKPIGHTGO-UWVGGRQHSA-N 0.000 description 28
- 108010092854 aspartyllysine Proteins 0.000 description 28
- VMTYLUGCXIEDMV-QWRGUYRKSA-N Lys-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCCCN VMTYLUGCXIEDMV-QWRGUYRKSA-N 0.000 description 27
- UDQBCBUXAQIZAK-GLLZPBPUSA-N Thr-Glu-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UDQBCBUXAQIZAK-GLLZPBPUSA-N 0.000 description 25
- SLNCSSWAIDUUGF-LSJOCFKGSA-N Arg-His-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O SLNCSSWAIDUUGF-LSJOCFKGSA-N 0.000 description 24
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-N 0.000 description 24
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 24
- WSWYMRLTJVKRCE-ZLUOBGJFSA-N Asp-Ala-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O WSWYMRLTJVKRCE-ZLUOBGJFSA-N 0.000 description 23
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 23
- XUUXCWCKKCZEAW-YFKPBYRVSA-N Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N XUUXCWCKKCZEAW-YFKPBYRVSA-N 0.000 description 22
- NAPNAGZWHQHZLG-ZLUOBGJFSA-N Asp-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N NAPNAGZWHQHZLG-ZLUOBGJFSA-N 0.000 description 21
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 20
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 20
- PHHRSPBBQUFULD-UWVGGRQHSA-N Arg-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N PHHRSPBBQUFULD-UWVGGRQHSA-N 0.000 description 19
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 19
- VFUXXFVCYZPOQG-WDSKDSINSA-N Asp-Glu-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VFUXXFVCYZPOQG-WDSKDSINSA-N 0.000 description 18
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 18
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 18
- 108010040030 histidinoalanine Proteins 0.000 description 18
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 description 17
- 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 16
- 108010034529 leucyl-lysine Proteins 0.000 description 16
- 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 15
- 108010038633 aspartylglutamate Proteins 0.000 description 15
- 108010042598 glutamyl-aspartyl-glycine Proteins 0.000 description 15
- VAWNQIGQPUOPQW-ACZMJKKPSA-N Asp-Glu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VAWNQIGQPUOPQW-ACZMJKKPSA-N 0.000 description 13
- RDPOETHPAQEGDP-ACZMJKKPSA-N Glu-Asp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RDPOETHPAQEGDP-ACZMJKKPSA-N 0.000 description 13
- JGDGLDNAQJJGJI-AVGNSLFASA-N Arg-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N JGDGLDNAQJJGJI-AVGNSLFASA-N 0.000 description 12
- JLWZLIQRYCTYBD-IHRRRGAJSA-N Leu-Lys-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JLWZLIQRYCTYBD-IHRRRGAJSA-N 0.000 description 12
- YBXMGKCLOPDEKA-NUMRIWBASA-N Thr-Asp-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YBXMGKCLOPDEKA-NUMRIWBASA-N 0.000 description 11
- LGNBRHZANHMZHK-NUMRIWBASA-N Thr-Glu-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O LGNBRHZANHMZHK-NUMRIWBASA-N 0.000 description 11
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 10
- 238000013461 design Methods 0.000 description 10
- 108020001507 fusion proteins Proteins 0.000 description 9
- 102000037865 fusion proteins Human genes 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- WZZSKAJIHTUUSG-ACZMJKKPSA-N Glu-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O WZZSKAJIHTUUSG-ACZMJKKPSA-N 0.000 description 8
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 8
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 8
- NLQUOHDCLSFABG-GUBZILKMSA-N Ser-Arg-Arg Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NLQUOHDCLSFABG-GUBZILKMSA-N 0.000 description 8
- NNFMANHDYSVNIO-DCAQKATOSA-N Ser-Lys-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NNFMANHDYSVNIO-DCAQKATOSA-N 0.000 description 8
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 7
- FTVRVZNYIYWJGB-ACZMJKKPSA-N Ser-Asp-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O FTVRVZNYIYWJGB-ACZMJKKPSA-N 0.000 description 7
- HJEBZBMOTCQYDN-ACZMJKKPSA-N Ser-Glu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HJEBZBMOTCQYDN-ACZMJKKPSA-N 0.000 description 7
- UOLGINIHBRIECN-FXQIFTODSA-N Ser-Glu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UOLGINIHBRIECN-FXQIFTODSA-N 0.000 description 7
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 6
- NTAZNGWBXRVEDJ-FXQIFTODSA-N Arg-Asp-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NTAZNGWBXRVEDJ-FXQIFTODSA-N 0.000 description 6
- PNQWAUXQDBIJDY-GUBZILKMSA-N Arg-Glu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNQWAUXQDBIJDY-GUBZILKMSA-N 0.000 description 6
- SNDBKTFJWVEVPO-WHFBIAKZSA-N Asp-Gly-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SNDBKTFJWVEVPO-WHFBIAKZSA-N 0.000 description 6
- OHUKZZYSJBKFRR-WHFBIAKZSA-N Gly-Ser-Asp Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O OHUKZZYSJBKFRR-WHFBIAKZSA-N 0.000 description 6
- BRSGXFITDXFMFF-IHRRRGAJSA-N Lys-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N BRSGXFITDXFMFF-IHRRRGAJSA-N 0.000 description 6
- MESDJCNHLZBMEP-ZLUOBGJFSA-N Ser-Asp-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MESDJCNHLZBMEP-ZLUOBGJFSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 108010019598 Liraglutide Proteins 0.000 description 5
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 description 5
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 229960002701 liraglutide Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- BIOCIVSVEDFKDJ-GUBZILKMSA-N Arg-Arg-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O BIOCIVSVEDFKDJ-GUBZILKMSA-N 0.000 description 4
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 4
- DSPQRJXOIXHOHK-WDSKDSINSA-N Glu-Asp-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O DSPQRJXOIXHOHK-WDSKDSINSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- HAPWZEVRQYGLSG-IUCAKERBSA-N His-Gly-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O HAPWZEVRQYGLSG-IUCAKERBSA-N 0.000 description 4
- GQUDMNDPQTXZRV-DCAQKATOSA-N Lys-Arg-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GQUDMNDPQTXZRV-DCAQKATOSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 206010012601 diabetes mellitus Diseases 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 108010056787 lysyl-arginyl-glutamyl-glutamic acid Proteins 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- IIAXFBUTKIDDIP-ULQDDVLXSA-N Arg-Leu-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IIAXFBUTKIDDIP-ULQDDVLXSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- QMVCEWKHIUHTSD-GUBZILKMSA-N Gln-Met-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N QMVCEWKHIUHTSD-GUBZILKMSA-N 0.000 description 3
- NCWOMXABNYEPLY-NRPADANISA-N Glu-Ala-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O NCWOMXABNYEPLY-NRPADANISA-N 0.000 description 3
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- AFPFGFUGETYOSY-HGNGGELXSA-N His-Ala-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AFPFGFUGETYOSY-HGNGGELXSA-N 0.000 description 3
- LEHPJMKVGFPSSP-ZQINRCPSSA-N Ile-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)=CNC2=C1 LEHPJMKVGFPSSP-ZQINRCPSSA-N 0.000 description 3
- WXUOJXIGOPMDJM-SRVKXCTJSA-N Leu-Lys-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O WXUOJXIGOPMDJM-SRVKXCTJSA-N 0.000 description 3
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 3
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 3
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 3
- 238000012772 sequence design Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- WVNFNPGXYADPPO-BQBZGAKWSA-N Arg-Gly-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O WVNFNPGXYADPPO-BQBZGAKWSA-N 0.000 description 2
- UZFHNLYQWMGUHU-DCAQKATOSA-N Asp-Lys-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UZFHNLYQWMGUHU-DCAQKATOSA-N 0.000 description 2
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 description 2
- WOMUDRVDJMHTCV-DCAQKATOSA-N Glu-Arg-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WOMUDRVDJMHTCV-DCAQKATOSA-N 0.000 description 2
- SJJHXJDSNQJMMW-SRVKXCTJSA-N Glu-Lys-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O SJJHXJDSNQJMMW-SRVKXCTJSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- SBVPYBFMIGDIDX-SRVKXCTJSA-N Pro-Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H]2NCCC2)CCC1 SBVPYBFMIGDIDX-SRVKXCTJSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 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 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
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108010031719 prolyl-serine Proteins 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
- 230000007928 solubilization Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- PBAMJJXWDQXOJA-FXQIFTODSA-N Ala-Asp-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PBAMJJXWDQXOJA-FXQIFTODSA-N 0.000 description 1
- NWVVKQZOVSTDBQ-CIUDSAMLSA-N Ala-Glu-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NWVVKQZOVSTDBQ-CIUDSAMLSA-N 0.000 description 1
- KRQSPVKUISQQFS-FJXKBIBVSA-N Arg-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N KRQSPVKUISQQFS-FJXKBIBVSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- 102000016622 Dipeptidyl Peptidase 4 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 description 1
- 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 1
- 239000004471 Glycine Substances 0.000 description 1
- 101001011657 Homo sapiens Myoregulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102100030170 Myoregulin Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- FDMKYQQYJKYCLV-GUBZILKMSA-N Pro-Pro-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 FDMKYQQYJKYCLV-GUBZILKMSA-N 0.000 description 1
- 101710129616 Protein glp-1 Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 229940047296 exenatide injection Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 229940090044 injection Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 108010091617 pentalysine Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Images
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
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Endocrinology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence, and the general formula of recombinant tandem protein of GLP-1 or analog thereof designed by the invention is as follows: x- (Y-Z) n. Wherein: x is leader peptide, Y is trypsin and CPB enzyme double restriction site or Kex2 enzyme and CPB enzyme double restriction site and auxiliary sequence, and Z is GLP-1 or its analogue. n is the number of series repeats. The inclusion body formed by recombinant tandem protein for fermenting, inducing and expressing GLP-1 or the analogue thereof through an escherichia coli prokaryotic expression system can be completely dissolved without a denaturant, and GLP-1 or the analogue thereof is obtained by double enzyme digestion of trypsin and CPB enzyme or double enzyme digestion of Kex2 enzyme and CPB enzyme in a dissolving solution. The GLP-1 or the analog thereof obtained by the method has high expression level, and the GLP-1 target protein after enzyme digestion has high yield and purity after enzyme digestion.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing GLP-1 or analog polypeptide thereof by adopting a series sequence efficiently expressed by escherichia coli.
Background
GLP-1 or an analog thereof usually has a molecular weight of not more than 4000Da and an amino acid number of not more than 40, and can be produced by an Escherichia coli expression system and a yeast expression system. The yeast expression can be secreted outside cells, which is beneficial to the collection and purification of expression products, but the yeast culture cost is relatively high, the fermentation culture period time is long, a powerful promoter which is strictly regulated and controlled is lacked, the secretion efficiency is low, the relative yield is not high, and the comprehensive cost is high. The prokaryotic expression system of the escherichia coli has the characteristics of high yield, low cost, clear genetic background, clear characteristics, simple operation and the like, and can make up for the defects of a yeast expression system. However, the prokaryotic expression system of escherichia coli is not suitable for expressing polypeptide or protein with smaller molecular weight, because the content of small peptide and protein products expressed in escherichia coli is easy to degrade, and in order to enhance the stability of the small protein polypeptide, a section of fusion protein is often required to be connected during design so as to reduce the degradation of the small protein polypeptide. The connected fusion protein needs to be removed by protease enzyme digestion before later purification, so that the target protein accounts for not high amount of total protein after the fusion protein is subjected to enzyme digestion, most of the expression products are removed after enzyme digestion, and as a result, the theoretical yield of the target protein is only 20-30% even if the expression amount of the fusion protein is high. GLP-1 or its analogue has small molecular weight, and in order to increase its expression level in Escherichia coli, a conventional expression strategy is to add a fusion protein to the front end or the rear end of GLP-1 or its analogue to increase its protein size. The fusion protein-GLP-1 or the analog thereof or the GLP-1 or the analog thereof-fusion protein releases GLP-1 or the analog thereof after being enzyme-digested by protease, enterokinase or the like. The yield of GLP-1 or analogues thereof obtained by the expression design is not high, the theoretical yield is only about 25%, the product purity is low and is generally only 40-50%, the subsequent separation and purification are difficult, and the yield is low. Therefore, the exploration of a new GLP-1 or analogue high-expression technology has very significant meaning for developing new drugs and reducing production cost.
At present, no patent or literature about polypeptide or protein tandem expression technology exists, and one reason may be that the enzyme cutting sites among the tandem expression target proteins are difficult to be completely identified, so that the enzyme cutting is incomplete, or residual amino acids exist after the enzyme cutting, so that the yield of the target proteins is low.
Therefore, the need in the art is to provide a design for GLP-1 or its analogue recombinant tandem protein expression and a construction method of prokaryotic expression strain to improve the ratio of GLP-1 or its analogue in the whole expression peptide chain and the yield after enzyme digestion.
Disclosure of Invention
One of the objects of the present invention is to provide recombinant tandem protein sequences of GLP-1 or analogs thereof.
Another object of the present invention is to provide a method for constructing a prokaryotic expression strain of recombinant tandem proteins of GLP-1 or analogues thereof.
The term "GLP-1 or analog thereof" as used herein means all or part of a mutated sequence of the native GLP-1 amino acid sequence, including but not limited to any one of the following (1) to (8):
(1)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG、
(2)HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG、
(3)EGTFTSDVSSYLEGQAAKEFIAWLVRGRG、
(4)HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS、
(5)HAEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS、
(6)HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK、
(7)HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR、
(8)HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG。
in a first aspect of the invention, there is provided a recombinant tandem protein sequence of GLP-1 or an analog thereof, having the general formula: x- (Y-Z) n, wherein:
x is a leader peptide formed from 5-10 amino acids, the amino acid composition including but not limited to: alanine A, leucine L, N asparagine, R arginine, S serine, F phenylalanine, H histidine, M methionine, V valine. The preferred sequence composition is MRLN, MRLNSA or MRLNSAFV, and the sequence numbers are Seq ID No.1-3 respectively;
y is a core sequence of a tandem expression sequence design technology, and the general formula is Y-m-Y; wherein y is an enzyme cleavage site, and the amino acid composition includes but is not limited to: r arginine, K lysine, can be R or K or RR or KK or RK or KR, m is a proprietary linker sequence formed by 4-12 amino acids including but not limited to the combination of S serine, G glycine, E glutamic acid, D aspartic acid, T threonine, R arginine, A alanine; preferred sequence compositions are GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG, with the sequence numbers Seq ID No.4-19, respectively;
z is GLP-1 or an analogue thereof, and the amino acid sequence is selected from any one of Seq ID No.20 to Seq ID No. 27:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(Seq ID No.20)、
HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG(Seq ID No.21)、
EGTFTSDVSSYLEGQAAKEFIAWLVRGRG(Seq ID No.22)、
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(Seq ID No.23)、
HAEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(Seq ID No.24)、
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK(Seq ID No.25)、
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR(Seq ID No.26)、
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG(Seq ID No.27)；
n is a number of repeats, including but not limited to an integer from 2 to 7, preferably 3, 4 or 5.
In some embodiments, the invention provides specific GLP-1 or analog recombinant tandem protein sequences, and the amino acid sequences are shown as SEQ ID NO.28-SEQ ID NO. 111.
The invention also provides a gene for coding the GLP-1 or analogue recombinant tandem protein sequence thereof.
The invention also provides a recombinant plasmid containing a gene encoding the GLP-1 or analogue recombinant tandem protein sequence thereof.
In another preferred embodiment, the recombinant plasmid of the present invention comprises the gene of the present invention encoding the recombinant tandem protein sequence of GLP-1 or its analog, and a vector constituting the recombinant plasmid, preferably a pET series vector.
The invention also provides a host cell containing the gene encoding the GLP-1 or analogue recombinant tandem protein sequence or the recombinant plasmid. In some embodiments, the host cell is e.coli, preferably BL21(DE3), HMS174(DE3), pLysS or pLysE host bacteria.
In the second aspect of the invention, a method for preparing GLP-1 or an analogue thereof by using escherichia coli expression is provided, wherein the escherichia coli expression is adopted to express the recombinant tandem protein sequence of GLP-1 and the analogue thereof.
In a specific embodiment, the invention provides a method for preparing GLP-1 or an analogue thereof by using Escherichia coli expression.
In another preferred embodiment, the method for transforming the expression vector into E.coli is well known in the art, and electroporation, CaCl2 transformation, etc. are preferred; the method for screening positive clones is well known in the art, and a resistance gene method is preferred; the method for inducing the host to express the protein is a technique known to those skilled in the art. Preferably IPTG at a concentration of 0.lmM to 10mM, at a temperature of 25-37 ℃ so that more efficient expression of the protein of interest can be obtained.
In another preferred embodiment, the method for disrupting bacterial cells is preferably high-pressure homogenization, osmotic pressure shock, freeze thawing, or ultrasonic disruption.
In another preferred embodiment, the inclusion body is obtained by centrifugation.
The inclusion body formed by the GLP-1 and the analog recombinant tandem protein sequence thereof has good solubility, can completely dissolve the expressed inclusion body without a denaturant, and is very favorable for subsequent enzyme digestion and separation and purification. The invention provides a preferable method for dissolving inclusion bodies, which is to preferably dissolve 25-250mM Tris solution at the pH of 8.5-11.5 and at the temperature of 12-15 ℃ for 2-6 h.
In another preferred embodiment, the method for obtaining the crude GLP-1 or the analogue recombinant tandem protein lysate is preferably centrifugation or ultrafiltration.
In another preferred embodiment, the method for obtaining crude GLP-1 or its analogue monomer is preferably a trypsin and CPB enzyme double-enzyme cleavage method, and a Kex2 enzyme and CPB enzyme double-enzyme cleavage method.
In another preferred embodiment, the trypsin (or Kex2 enzyme) and CPB enzyme are used in a double enzyme cleavage method under the conditions commonly used for such enzyme cleavage, for example, the buffer may be Tris solution (25-250mM) or phosphate buffer; pH 7.0-10.0, preferably pH 8.0-9.0; the temperature is 15-30 deg.C, preferably 25 deg.C, and both enzymes are added according to the total protein mass in series of 1/400-1/800, preferably 1/500; the enzyme cutting time is 4-12 h. The HPLC purity of the target protein after enzyme digestion is not lower than 80%, which is very beneficial to the subsequent protein purification process. The enzyme cutting yield is not less than 45 percent and is 200 to 300 percent of the enzyme cutting yield of the traditional fusion protein.
An expression technology for designing a novel GLP-1 or a recombinant polypeptide tandem sequence of an analogue thereof by utilizing a genetic engineering means. The recombinant tandem protein can be produced by a prokaryotic expression system, GLP-1 or an analogue thereof with high yield can be obtained by enzyme digestion, and the product can be subjected to side chain modification and is used for treating diabetes and endocrine metabolism related diseases.
The invention designs an effective connecting sequence near the enzyme cutting site between the target genes expressed in series, and the fusion protein is replaced by GLP-1 or analog protein thereof to form series expression, the connecting sequence can fully expose the enzyme cutting site and completely cut off the connecting sequence and the recognition site, thereby obviously improving the enzyme cutting yield and purity of the target product, and leading the main protein after enzyme cutting to be the target protein of GLP-1 or analog thereof, thus greatly reducing the content of useless fusion protein, obviously improving the proportion of GLP-1 or analog protein thereof in the whole expression peptide chain, leading the theoretical yield after enzyme cutting to be close to 70 percent, and being a good method for effectively improving the expression yield of GLP-1 or analog protein thereof. Another advantage of the protein produced by this route is the good solubility of inclusion bodies. The protein product expressed by the traditional escherichia coli is mostly in a compact inclusion body form, the solubility of the inclusion body is poor, and the inclusion body is dissolved by using a denaturant such as high-concentration urea or guanidine hydrochloride.
Drawings
FIG. 1 shows an electrophoresis picture of the recombinant 3 tandem protein 1 engineering bacteria (SEQ ID No.30) induced expression of the somaglutide precursor. Wherein 1 is marker, 2 is post-induction engineering bacteria, and 2 is pre-induction engineering bacteria.
FIG. 2 shows an electrophoresis picture of the recombinant 3 tandem protein 2 engineering bacteria (SEQ ID No.38) induced expression of the somaglutide precursor. Wherein, 1 is engineering bacteria before induction, 2 is marker, and 3 is engineering bacteria after induction.
FIG. 3 shows the electrophoresis picture of the recombinant 4 tandem protein 1 engineering bacterium (SEQ ID No.92) induced expression of the somaglutide precursor. Wherein, 1 is engineering bacteria before induction, 2 is marker, and 3 is engineering bacteria after induction.
FIG. 4 shows an electrophoresis picture of the recombinant 4 tandem protein 2 engineering bacteria (SEQ ID No.109) induced expression of the somaglutide precursor. Wherein 1 is marker, 2 is engineering bacteria before induction, and 3 is engineering bacteria after induction.
FIG. 5 is a HPLC check chart of recombinant 3 tandem protein 1(SEQ ID No.30) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somarglutide precursor peaked at RT6.349min and the purity was 85.06%.
FIG. 6 is a HPLC check chart of recombinant 3 tandem protein 2(SEQ ID No.38) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somalobtide precursor peaked at rt6.323min with a purity of 86.27%.
FIG. 7 is a HPLC check chart of recombinant 4 tandem protein 1(SEQ ID No.92) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somaglutide precursor peaks at RT6.334min and has a purity of 85.33%.
FIG. 8 is a HPLC check chart of recombinant 4 tandem protein 2(SEQ ID No.109) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somalutide precursor peaked at rt6.261min with a purity of 83.89%.
FIG. 9 is a mass spectrum analysis diagram of recombinant 3 tandem protein 1(SEQ ID No.30) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
FIG. 10 is a mass spectrum analysis diagram of recombinant 3 tandem protein 2(SEQ ID No.38) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
FIG. 11 is a mass spectrum analysis diagram of recombinant 4 tandem protein 1(SEQ ID No.92) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
FIG. 12 is a mass spectrum analysis diagram of recombinant 4 tandem protein 2(SEQ ID No.109) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art.
Materials:
strains and plasmids: expression strains BL21(DE3), pLysS and the like were purchased from MERCK, and pET series vectors were purchased from vast Ling Biotech Co., Ltd.
Enzymes and reagents:
the enzymes used in the examples relating to molecular biological manipulations were purchased from Takara and the corresponding procedures were carried out exactly according to the relevant product instructions.
The gene synthesis and sequencing of the nucleotide sequence encoding the recombinant somalutide tandem proteins referred to in the examples was performed by the Nanjing Kinsley Limited.
Other raw materials and auxiliary materials which are not marked are all commercial products.
Example 1:
In order to increase the expression amount of a GLP-1 analogue precursor peptide in prokaryotic expression and improve the proportion of a precursor peptide chain in the whole expression peptide chain, according to the above patent idea, the GLP-1 analogue precursor peptide chain is subjected to triple tandem design (n is 3), and GLP-1 analogue precursors are connected through a trypsin enzyme cutting site or a kex2 enzyme cutting site, so that trypsin or kex2 enzyme can better recognize the trypsin enzyme cutting site or the kex2 enzyme cutting site and the enzyme cutting efficiency is improved, and a key auxiliary sequence (m) is designed at the trypsin enzyme cutting site or the kex2 enzyme cutting site. GLP-1 analogues (somaglutide precursor sequence (9-37) and liraglutide precursor sequence (7-37)) published according to NCBI have the leader peptide MRLNSA at its N-terminus, with kex2 enzymatic cleavage sites and helper sequences incorporated between the GLP-1 native sequence and the leader peptide and between the GLP-1 native sequences: GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG, and n is 3.
Therefore, the sequences of the designed GLP-1 natural analogues (the somaglutide precursor sequence (79-37) and the liraglutide precursor sequence (7-37)) recombinant 3 tandem proteins are shown in Table 1:
[ TABLE 1 ]
According to the above patent design concept, a GLP-1 analog precursor peptide chain is also designed in a quadruple tandem (n ═ 4) using a linker sequence consistent with the triple tandem design. GLP-1 analogues (somaglutide precursor sequence (9-37) and liraglutide precursor sequence (7-37)) published according to NCBI have the leader peptide MRLNSA at its N-terminus, with kex2 enzymatic cleavage sites and helper sequences incorporated between the GLP-1 native sequence and the leader peptide and between the GLP-1 native sequences: GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG, and n is 4.
Therefore, the sequences of the designed GLP-1 natural analogues (the somaglutide precursor sequence (79-37) and the liraglutide precursor sequence (7-37)) recombinant 4 tandem proteins are shown in Table 2:
[ TABLE 2 ]
Example 2: construction of GLP-1 analogue tandem protein recombinant plasmid and construction of engineering bacteria
Nucleotide sequences corresponding to the designed amino acid sequences are optimized according to codons of escherichia coli respectively, the codon-optimized gene synthesis is carried out through a chemical synthesis method, and BamHI and XhoI are respectively connected into pET32a to construct 4 recombinant plasmids. Passing the above recombinant plasmid through CaC12The transformation method is introduced into BL21(DE3), and multiple strains of the somaglutide precursor recombinant 3 and 4 tandem protein engineering bacteria are constructed through resistance screening and monoclone. The sequence in the engineering bacteria is consistent with the design through sequencing.
Example 3: inducible expression of GLP-1 analogue tandem proteins
3.1 LB Medium formulation
TABLE 3 LB medium recipe
Preparing 11L LB medium according to Table 3, dissolving, mixing well, sucking 10ml and 1.7L medium, respectively placing into 50ml and 3L conical flask, preparing 6 bottles, sealing, and sterilizing (121 deg.C, 25min) for use.
3.2 engineering bacteria Resuscitation
One of the somalutide precursor recombinant 3 and 4 tandem protein glycerol strains was taken out, inoculated into 6 sterilized culture media of 10ml in an inoculum size of 1%, and cultured overnight in a shaker at 37 ℃ and 250 rpm.
3.3 Induction culture of engineering bacteria
Taking out the resuscitating bacteria liquid, respectively inoculating to 6 1.7L sterilized culture mediums according to the inoculation amount of 1%, culturing for about 7-8h under the conditions of 37 ℃ of a shaking table and 250rpm, adding 0.lmM IPTG, and carrying out overnight induction expression at 37 ℃.
3.4 centrifugation of the cells
Centrifuging the bacterial liquid at 4000rpm and 8 ℃ for 40min, removing supernatant and collecting thalli. Expression of the target protein in the cells was observed by SDS-PAGE (electrophoresis of the engineered bacteria with partially tandem proteins induced expression is shown in FIGS. 1-4).
Example 4: harvesting and solubilization of GLP-1 analog tandem protein inclusion bodies
4.1 resuspending, crushing and centrifuging the cells
Adding 10L of bacteria breaking buffer (5mmol/L EDTA, 50mmol/L Tris-HCl) into 1kg of bacteria to suspend the bacteria, fully stirring until no blocky bacteria exist, homogenizing the suspension at high pressure to break the bacteria, wherein the operating pressure adopted during the bacteria breaking is 850 +/-50 bar, and continuously breaking twice. The lysate liquid is centrifuged for 40min at 4000rpm and 8 ℃, and the precipitate is collected by centrifugation.
4.2 Inclusion body washing centrifugation
The final volume of the inclusion body wash was calculated according to the weight of inclusion bodies before washing according to the following formula: the final volume (L) of the inclusion body wash was defined as the weight (kg) of the inclusion body multiplied by the dilution factor (10) of the inclusion body, and 0.5L of 1mol/L of the ltis-HCl solution was added to each 10L of the inclusion body wash to prepare a buffer solution. Centrifuging at 6000rpm and 8 deg.C for 30min, and centrifuging to collect precipitate.
4.3 Inclusion body solubilization
Adding 12-15 ml of Tris solution (50mM) into every 1g of the inclusion bodies after washing and centrifugation, dissolving at low temperature (12-15 ℃), adjusting the pH to 11.5 by using NaOH solution after 0.5h, adjusting the pH to 11.5 by using NaOH solution for the second time after 0.5h, and then continuously dissolving for 2-6 h.
Example 5: obtaining of GLP-1 analogue tandem protein precursor
5.1 Ultrafiltration of Inclusion body solutions
After the dissolution of the inclusion bodies is finished, a centrifuge is used for centrifuging for 1h under the condition of 10000g to remove particles. The centrifuged solution was ultrafiltered using a 750K hollow fiber column, and the filtrate was collected by washing with 50mM Tris solution.
5.2 enzyme digestion
And (3) adjusting the pH value of the solution after ultrafiltration to 8.5-9.0 by using 6M HCl, adding 1g of Kex2 enzyme and 1g of CPB enzyme into every 500g of protein after filtration, and adding Kex2 enzyme and CPB enzyme for carrying out enzyme digestion reaction. Slowly stirring the enzyme digestion liquid to control the temperature of the enzyme digestion reaction to be 20-25 ℃, and performing the enzyme digestion reaction for 6-10 h. Samples were taken for HPLC and mass spectrometric detection (results for partial tandem proteins are shown in FIGS. 6-8 and 9-12), and the cleavage yield was calculated.
The results are shown in Table 4, which shows that the purity of the main peak ranges from 80% to 93% and the yield ranges from 45% to 60% by HPLC detection after Kex2 and CPB double enzyme digestion. The graph shows that the HPLC of the somalutide precursor detects that GLP-1 or the analogue thereof generates a peak at RT6.3min and the purity is about 85 percent. Mass spectrometry analysis: the somaglutide precursor has a molecular weight of about 3175, consistent with theory.
[ TABLE 4 ]
And (3) knotting: through the series design, the proportion of GLP-1 or analogues thereof in the whole expression band can be obviously improved, the enzyme digestion efficiency is improved to 40-60 percent, and the enzyme digestion yield is 200-300 percent of that of the fusion protein design scheme. Under the same fermentation conditions, the yield of the target protein of GLP-1 or the analogue thereof can be obviously improved. And the purity of the main peak (GLP-1 or the analogue thereof) after enzyme digestion reaches 80-93%, which is very beneficial to subsequent purification.
Sequence listing
<110> Ten thousand New medicine science and technology (Suzhou) Co., Ltd
Wanbang pharmaceutical science and technology Co Ltd of Jiangsu
<120> method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence
<160> 111
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Arg Leu Asn
1
<210> 2
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Arg Leu Asn Ser Ala
1 5
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Arg Leu Asn Ser Ala Phe Val
1 5
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser
1 5 10
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser
1 5 10
<210> 6
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser
1 5 10
<210> 7
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser
1 5 10
<210> 8
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Glu Ala Glu Lys
1 5
<210> 9
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Asp Asp Ala Asp Lys
1 5
<210> 10
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Thr Glu Glu Ala Glu Lys
1 5
<210> 11
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Thr Asp Asp Ala Asp Lys
1 5
<210> 12
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Thr Glu Asp Ala Glu Lys
1 5
<210> 13
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Thr Asp Glu Ala Asp Lys
1 5
<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly
1 5 10
<210> 15
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly
1 5 10
<210> 16
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly
1 5 10
<210> 17
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly
1 5 10
<210> 18
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly
1 5 10
<210> 19
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly
1 5 10
<210> 20
<211> 31
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 21
<211> 31
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25 30
<210> 22
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
1 5 10 15
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25
<210> 23
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 24
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 25
<211> 44
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys
35 40
<210> 26
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 27
<211> 31
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly
20 25 30
<210> 28
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 29
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 30
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 31
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 32
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 33
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 34
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Met Arg Leu Asn Ser Ala Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Glu Ala
35 40 45
Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210> 35
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Met Arg Leu Asn Ser Ala Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Asp Asp Ala
35 40 45
Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210> 36
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 37
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 38
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 39
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 40
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 41
<211> 130
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly
130
<210> 42
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 43
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 44
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 45
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 46
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 47
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 48
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 49
<211> 134
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
20 25 30
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40 45
Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly
50 55 60
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
65 70 75 80
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly
85 90 95
Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp
100 105 110
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
115 120 125
Leu Val Arg Gly Arg Gly
130
<210> 50
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 51
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 52
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 53
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 54
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Met Arg Leu Asn Ser Ala Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Glu Ala
35 40 45
Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210> 55
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Met Arg Leu Asn Ser Ala Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Asp Asp Ala
35 40 45
Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210> 56
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 57
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 58
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 59
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210> 60
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 61
<211> 130
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly
130
<210> 62
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 63
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 64
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 65
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 66
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 67
<211> 135
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210> 68
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 69
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 70
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 71
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Asp Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 72
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Asp Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 73
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 74
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly
130 135 140
Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210> 75
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly
130 135 140
Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210> 76
<211> 154
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Met Arg Leu Asn Ser Ala Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Glu Ala
35 40 45
Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210> 77
<211> 154
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Met Arg Leu Asn Ser Ala Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Asp Asp Ala
35 40 45
Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210> 78
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Glu Glu Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 79
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Asp Asp Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 80
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Glu Asp Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 81
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Asp Glu Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 82
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 83
<211> 173
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg
130 135 140
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
145 150 155 160
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
165 170
<210> 84
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 84
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Asp Asp Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 85
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 86
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 86
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 87
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Asp Asp Ala Glu
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 88
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 88
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Asp Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 89
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Asp Glu Ala Glu
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 90
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly
130 135 140
Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 91
<211> 177
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
20 25 30
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40 45
Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly
50 55 60
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
65 70 75 80
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly
85 90 95
Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp
100 105 110
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
115 120 125
Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser
130 135 140
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
145 150 155 160
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
165 170 175
Gly
<210> 92
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 92
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Asp Gly
130 135 140
Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 93
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly
130 135 140
Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 94
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 94
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly
130 135 140
Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210> 95
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly
130 135 140
Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210> 96
<211> 154
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 96
Met Arg Leu Asn Ser Ala Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Glu Ala
35 40 45
Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210> 97
<211> 154
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Met Arg Leu Asn Ser Ala Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Asp Asp Ala
35 40 45
Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210> 98
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 98
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Glu Glu Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 99
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Asp Asp Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 100
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 100
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Glu Asp Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 101
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Asp Glu Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210> 102
<211> 177
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 102
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Gly Thr Phe Thr Ser Asp
100 105 110
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
115 120 125
Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys
130 135 140
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
145 150 155 160
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
165 170 175
Gly
<210> 103
<211> 173
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 103
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg
130 135 140
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
145 150 155 160
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
165 170
<210> 104
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 104
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Asp Asp Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 105
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 105
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 106
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 106
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 107
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 107
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Asp Asp Ala Glu
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 108
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 108
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 109
<211> 178
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 109
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210> 110
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 110
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu
130 135 140
Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210> 111
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 111
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu
130 135 140
Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
Claims (11)
1. A recombinant tandem protein of GLP-1 or an analogue thereof is characterized in that the amino acid sequence of the recombinant tandem protein is shown in any one of SEQ ID NO.28-SEQ ID NO. 111.
2. A gene encoding the recombinant tandem GLP-1 protein or an analog thereof according to claim 1.
3. A recombinant plasmid or host cell comprising the gene of claim 2.
4. A method for preparing GLP-1 or analog recombinant tandem protein thereof by using Escherichia coli expression, which is characterized in that the GLP-1 or analog recombinant tandem protein sequence of claim 1 is expressed by using Escherichia coli.
5. The process as claimed in claim 4, wherein the Escherichia coli used is BL21(DE3), HMS174(DE3), pLysS or pLysE host bacteria.
6. The method of claim 4, further comprising disrupting the biomass to obtain inclusion bodies, lysing the inclusion bodies, and performing enzymatic cleavage to obtain GLP-1 or an analog thereof.
7. The method of claim 4, wherein the inclusion body is solubilized using 25-250mM Tris solution at pH 8.5-11.5 at 12-15 ℃ for 2-6 h.
8. The method of claim 4, wherein the recombinant tandem GLP-1 protein or analog thereof is obtained by enzymatic cleavage with trypsin and CPB enzyme or enzymatic cleavage with Kex2 enzyme and CPB enzyme.
9. The method of claim 8, wherein the buffer used for the enzymatic cleavage is a 25-250mM Tris solution or phosphate buffer, pH 7.0-10.0; the temperature is 15-30 ℃, two enzymes in double enzyme cutting are added according to the mass of serial total protein 1/400-1/800, and the enzyme cutting is carried out for 4-12 h.
10. The method of claim 9, wherein the buffer used for the enzymatic cleavage is at a pH of 8.0 to 9.0.
11. The method of claim 9, wherein the temperature of the cleavage is 25 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911373533.3A CN111072783B (en) | 2019-12-27 | 2019-12-27 | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911373533.3A CN111072783B (en) | 2019-12-27 | 2019-12-27 | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111072783A CN111072783A (en) | 2020-04-28 |
| CN111072783B true CN111072783B (en) | 2021-09-28 |
Family
ID=70318296
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911373533.3A Active CN111072783B (en) | 2019-12-27 | 2019-12-27 | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111072783B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113801234B (en) * | 2020-06-11 | 2024-03-01 | 宁波鲲鹏生物科技有限公司 | Sodamide derivative and application thereof |
| WO2021249564A1 (en) * | 2020-06-11 | 2021-12-16 | 宁波鲲鹏生物科技有限公司 | Semaglutide derivative, and preparation method therefor and application thereof |
| CN111718407A (en) * | 2020-08-14 | 2020-09-29 | 北京质肽生物医药科技有限公司 | Purification method of glucagon-like peptide-1 analogue |
| CN113416683B (en) * | 2021-06-01 | 2023-05-02 | 南昌大学 | Escherichia coli Nissle1917 genetically engineered bacterium and preparation method and application thereof |
| JP2024524530A (en) * | 2021-07-06 | 2024-07-05 | スジョウ・アルファマブ・カンパニー・リミテッド | Fusion proteins and uses thereof |
| CN113502310B (en) * | 2021-09-10 | 2022-01-07 | 北京惠之衡生物科技有限公司 | Method for preparing semaglutide precursor through high-density fermentation |
| CN113846124A (en) * | 2021-09-26 | 2021-12-28 | 康霖生物科技（杭州）有限公司 | Nucleic acid construct for gene therapy of diseases related to glycometabolism |
| CN115850385B (en) * | 2022-07-04 | 2023-08-11 | 北京惠之衡生物科技有限公司 | Expression-promoting peptide and application thereof |
| CN116948013B (en) * | 2023-04-28 | 2024-04-09 | 江苏创健医疗科技股份有限公司 | Recombinant micromolecular collagen and expression system and preparation method thereof |
| CN118440968B (en) * | 2024-07-08 | 2024-10-11 | 天津凯莱英生物科技有限公司 | Screening method of multi-copy peptide synthesis system without redundant sequence, multi-copy peptide synthesis system without redundant sequence and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4929553A (en) * | 1987-05-29 | 1990-05-29 | Canadian Patents & Development Ltd. | Protease for specific processing of secreted proteins |
| CN101172996A (en) * | 2006-09-29 | 2008-05-07 | 上海新生源医药研究有限公司 | Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation |
| CN110128552A (en) * | 2019-03-14 | 2019-08-16 | 美药星（南京）制药有限公司 | A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide |
| CN110305223A (en) * | 2019-06-26 | 2019-10-08 | 重庆派金生物科技有限公司 | The method that recombination fused in tandem albumen prepares target polypeptides |
-
2019
- 2019-12-27 CN CN201911373533.3A patent/CN111072783B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4929553A (en) * | 1987-05-29 | 1990-05-29 | Canadian Patents & Development Ltd. | Protease for specific processing of secreted proteins |
| CN101172996A (en) * | 2006-09-29 | 2008-05-07 | 上海新生源医药研究有限公司 | Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation |
| CN110128552A (en) * | 2019-03-14 | 2019-08-16 | 美药星（南京）制药有限公司 | A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide |
| CN110305223A (en) * | 2019-06-26 | 2019-10-08 | 重庆派金生物科技有限公司 | The method that recombination fused in tandem albumen prepares target polypeptides |
Non-Patent Citations (2)
| Title |
|---|
| High-Level Expression of Fusion Protein Containing 10 Tandem Repeated GLP-1 Analogs in Yeast Pichia pastoris and Its Biological Activity in a Diabetic Rat Model;Jianhua HOU,et al.;《Biosci.Biotechnol.Biochem.》;20070630;第71卷(第6期);第1462-1469页 * |
| 重组人胰高血糖素样肽-1类似物的制备及活性分析;李泰明等;《中国药科大学学报》;20110630;第42卷(第6期);第560-564页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111072783A (en) | 2020-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111072783B (en) | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence | |
| CN115975047B (en) | Method for producing polypeptide by recombinant fusion protein and application thereof | |
| AU607973B2 (en) | Process for production of physiologically active peptide containing cysteine residue | |
| CN110724187B (en) | Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof | |
| US10000544B2 (en) | Process for production of insulin and insulin analogues | |
| CN111378027B (en) | Preparation method of somaglutide precursor | |
| JP2019195327A (en) | Method and means for expressing basic fibroblast growth factor that is authentic and biologically active in bacillus subtilis | |
| CN114381471B (en) | Application of auxiliary protein in recombinant protein production and fusion expression system | |
| WO2010062279A1 (en) | Method for producing human recombinant insulin | |
| JP7266325B2 (en) | Fusion proteins containing fluorescent protein fragments and uses thereof | |
| CN114292338B (en) | Fusion protein and method for preparing semaglutide intermediate polypeptide by using same | |
| CN114790474A (en) | Preparation method of Somalutide | |
| AU2019218315A1 (en) | Codon optimized precursor gene and signal peptide gene of human insulin analogue | |
| CN114933658B (en) | Short peptide element and application method thereof | |
| CN101172996A (en) | Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation | |
| JPH09296000A (en) | Cleavage of chimera protein with processing enzyme | |
| CN114380903B (en) | Insulin or analogue precursor thereof | |
| CN113249288B9 (en) | Recombinant bacterium for expressing GLP-1 analogue and application thereof | |
| CN114686504B (en) | Application of Lpp or mutant thereof as molecular chaperone in secretory expression of recombinant protein in escherichia coli | |
| EP0264074A2 (en) | Expression vector for insulin-like growth factor I | |
| EP0076037A2 (en) | Amplified expression of DNA sequences | |
| CN105255932B (en) | A kind of conotoxin variant GMVIIA and its preparation method and application | |
| CN114805610B (en) | Recombinant genetic engineering bacterium for highly expressing insulin glargine precursor and construction method thereof | |
| CN114807205B (en) | Recombinant engineering bacterium for expressing liraglutide precursor and construction method and application thereof | |
| CN114805544B (en) | Insulin lispro precursor, recombinant genetic engineering bacterium thereof and construction method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |