CN109517057A - Novel B DFP fluorescin and its fusion protein through genetic modification - Google Patents
Novel B DFP fluorescin and its fusion protein through genetic modification Download PDFInfo
- Publication number
- CN109517057A CN109517057A CN201811009726.6A CN201811009726A CN109517057A CN 109517057 A CN109517057 A CN 109517057A CN 201811009726 A CN201811009726 A CN 201811009726A CN 109517057 A CN109517057 A CN 109517057A
- Authority
- CN
- China
- Prior art keywords
- ala
- val
- leu
- gly
- fluorescin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 title claims abstract description 265
- 102000037865 fusion proteins Human genes 0.000 title description 20
- 108020001507 fusion proteins Proteins 0.000 title description 20
- 238000012239 gene modification Methods 0.000 title description 4
- 230000005017 genetic modification Effects 0.000 title description 4
- 235000013617 genetically modified food Nutrition 0.000 title description 4
- 230000035772 mutation Effects 0.000 claims abstract description 31
- 150000001413 amino acids Chemical class 0.000 claims abstract description 27
- 230000004927 fusion Effects 0.000 claims abstract description 21
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 10
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 10
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 10
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 37
- 235000001014 amino acid Nutrition 0.000 claims description 18
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 8
- 235000004279 alanine Nutrition 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 7
- 239000004473 Threonine Substances 0.000 claims description 7
- 235000018102 proteins Nutrition 0.000 claims description 7
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 5
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 4
- 108091006047 fluorescent proteins Proteins 0.000 claims description 4
- 102000034287 fluorescent proteins Human genes 0.000 claims description 4
- 235000013922 glutamic acid Nutrition 0.000 claims description 4
- 239000004220 glutamic acid Substances 0.000 claims description 4
- 239000005090 green fluorescent protein Substances 0.000 claims description 4
- 239000004474 valine Substances 0.000 claims description 4
- 102000002322 Egg Proteins Human genes 0.000 claims description 3
- 108010000912 Egg Proteins Proteins 0.000 claims description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 235000014103 egg white Nutrition 0.000 claims description 3
- 210000000969 egg white Anatomy 0.000 claims description 3
- 235000013601 eggs Nutrition 0.000 claims description 3
- 229930182817 methionine Natural products 0.000 claims description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 2
- 108010082025 cyan fluorescent protein Proteins 0.000 claims description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 239000004744 fabric Substances 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 2
- 108010073969 valyllysine Proteins 0.000 description 53
- YSUVMPICYVWRBX-VEVYYDQMSA-N Arg-Asp-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YSUVMPICYVWRBX-VEVYYDQMSA-N 0.000 description 29
- CDNPIRSCAFMMBE-SRVKXCTJSA-N Phe-Asn-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CDNPIRSCAFMMBE-SRVKXCTJSA-N 0.000 description 29
- PSOPJDUQUVFSLS-GUBZILKMSA-N Arg-Met-Cys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N PSOPJDUQUVFSLS-GUBZILKMSA-N 0.000 description 28
- VMVUDJUXJKDGNR-FXQIFTODSA-N Asp-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N VMVUDJUXJKDGNR-FXQIFTODSA-N 0.000 description 28
- MHYHLWUGWUBUHF-GUBZILKMSA-N Cys-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N MHYHLWUGWUBUHF-GUBZILKMSA-N 0.000 description 28
- IBSGMIPRBMPMHE-IHRRRGAJSA-N Leu-Met-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(O)=O IBSGMIPRBMPMHE-IHRRRGAJSA-N 0.000 description 28
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 28
- WAIHHELKYSFIQN-XUXIUFHCSA-N Lys-Ile-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O WAIHHELKYSFIQN-XUXIUFHCSA-N 0.000 description 28
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 description 28
- SCZJKZLFSSPJDP-ACRUOGEOSA-N Tyr-Phe-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O SCZJKZLFSSPJDP-ACRUOGEOSA-N 0.000 description 28
- 108010054155 lysyllysine Proteins 0.000 description 28
- OTKUAVXGMREHRX-CFMVVWHZSA-N Asp-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 OTKUAVXGMREHRX-CFMVVWHZSA-N 0.000 description 27
- LPHGXOWFAXFCPX-KKUMJFAQSA-N Glu-Pro-Phe Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O LPHGXOWFAXFCPX-KKUMJFAQSA-N 0.000 description 27
- UWQDKRIZSROAKS-FJXKBIBVSA-N Gly-Met-Thr Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWQDKRIZSROAKS-FJXKBIBVSA-N 0.000 description 27
- CCQLQKZTXZBXTN-NHCYSSNCSA-N Leu-Gly-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CCQLQKZTXZBXTN-NHCYSSNCSA-N 0.000 description 27
- JSGWNFKWZNPDAV-YDHLFZDLSA-N Phe-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JSGWNFKWZNPDAV-YDHLFZDLSA-N 0.000 description 27
- 108010038633 aspartylglutamate Proteins 0.000 description 27
- KXRORHJIRAOQPG-SOUVJXGZSA-N Glu-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O KXRORHJIRAOQPG-SOUVJXGZSA-N 0.000 description 26
- KQFZKDITNUEVFJ-JYJNAYRXSA-N Leu-Phe-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CC=CC=C1 KQFZKDITNUEVFJ-JYJNAYRXSA-N 0.000 description 26
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 25
- PLOUVAYOMTYJRG-JXUBOQSCSA-N Lys-Thr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PLOUVAYOMTYJRG-JXUBOQSCSA-N 0.000 description 25
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 25
- 108010060199 cysteinylproline Proteins 0.000 description 25
- BWMMKQPATDUYKB-IHRRRGAJSA-N Arg-Tyr-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 BWMMKQPATDUYKB-IHRRRGAJSA-N 0.000 description 24
- KKZHXOOZHFABQQ-UWJYBYFXSA-N Cys-Ala-Tyr Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KKZHXOOZHFABQQ-UWJYBYFXSA-N 0.000 description 24
- VKVDRTGWLVZJOM-DCAQKATOSA-N Leu-Val-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O VKVDRTGWLVZJOM-DCAQKATOSA-N 0.000 description 24
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 24
- NFLNBHLMLYALOO-DCAQKATOSA-N Pro-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@@H]1CCCN1 NFLNBHLMLYALOO-DCAQKATOSA-N 0.000 description 24
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 24
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 24
- KIDXAAQVMNLJFQ-KZVJFYERSA-N Pro-Thr-Ala Chemical compound C[C@@H](O)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](C)C(O)=O KIDXAAQVMNLJFQ-KZVJFYERSA-N 0.000 description 21
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 20
- OISWSORSLQOGFV-AVGNSLFASA-N Arg-Met-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCCN=C(N)N OISWSORSLQOGFV-AVGNSLFASA-N 0.000 description 20
- NKLRWRRVYGQNIH-GHCJXIJMSA-N Asn-Ile-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O NKLRWRRVYGQNIH-GHCJXIJMSA-N 0.000 description 20
- QCMVGXDELYMZET-GLLZPBPUSA-N Glu-Thr-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QCMVGXDELYMZET-GLLZPBPUSA-N 0.000 description 20
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 20
- VIIBEIQMLJEUJG-LAEOZQHASA-N Gly-Ile-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O VIIBEIQMLJEUJG-LAEOZQHASA-N 0.000 description 20
- 241000880493 Leptailurus serval Species 0.000 description 20
- QYDKSNXSBXZPFK-ZJDVBMNYSA-N Thr-Thr-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYDKSNXSBXZPFK-ZJDVBMNYSA-N 0.000 description 20
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 20
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 20
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 20
- 108010011559 alanylphenylalanine Proteins 0.000 description 20
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 18
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 238000001514 detection method Methods 0.000 description 12
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 11
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 11
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 11
- 238000003384 imaging method Methods 0.000 description 11
- 230000003834 intracellular effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 230000003595 spectral effect Effects 0.000 description 10
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 9
- OHYQKYUTLIPFOX-ZPFDUUQYSA-N Arg-Glu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OHYQKYUTLIPFOX-ZPFDUUQYSA-N 0.000 description 8
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 8
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 8
- GZBKRJVCRMZAST-XKBZYTNZSA-N Ser-Glu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZBKRJVCRMZAST-XKBZYTNZSA-N 0.000 description 8
- CJINPXGSKSZQNE-KBIXCLLPSA-N Ser-Ile-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O CJINPXGSKSZQNE-KBIXCLLPSA-N 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- GOWZVQXTHUCNSQ-NHCYSSNCSA-N Arg-Glu-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GOWZVQXTHUCNSQ-NHCYSSNCSA-N 0.000 description 7
- IBLAOXSULLECQZ-IUKAMOBKSA-N Asn-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC(N)=O IBLAOXSULLECQZ-IUKAMOBKSA-N 0.000 description 7
- HVHRPWQEQHIQJF-AVGNSLFASA-N Leu-Lys-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HVHRPWQEQHIQJF-AVGNSLFASA-N 0.000 description 7
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 7
- LMKSBGIUPVRHEH-FXQIFTODSA-N Met-Ala-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(N)=O LMKSBGIUPVRHEH-FXQIFTODSA-N 0.000 description 7
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 102000011782 Keratins Human genes 0.000 description 6
- 108010076876 Keratins Proteins 0.000 description 6
- 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 6
- KXUZHWXENMYOHC-QEJZJMRPSA-N Phe-Leu-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O KXUZHWXENMYOHC-QEJZJMRPSA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 210000003470 mitochondria Anatomy 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 108010033040 Histones Proteins 0.000 description 5
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 5
- FXGIMYRVJJEIIM-UWVGGRQHSA-N Pro-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FXGIMYRVJJEIIM-UWVGGRQHSA-N 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 108700042769 prolyl-leucyl-glycine Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 108091005962 small ultra red fluorescent proteins Proteins 0.000 description 5
- 210000001082 somatic cell Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- YXTFLTJYLIAZQG-FJXKBIBVSA-N Gly-Thr-Arg Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YXTFLTJYLIAZQG-FJXKBIBVSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 229960000789 guanidine hydrochloride Drugs 0.000 description 4
- 230000008676 import Effects 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- 240000002900 Arthrospira platensis Species 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 102000034342 Calnexin Human genes 0.000 description 3
- 108010056891 Calnexin Proteins 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000003809 bile pigment Substances 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 238000002742 combinatorial mutagenesis Methods 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000007654 immersion Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 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 3
- 238000002372 labelling Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- NNMALANKTSRILL-ZUTFDUMMSA-N 3-[(2z,5z)-2-[[3-(2-carboxyethyl)-5-[(z)-[(3z,4r)-3-ethylidene-4-methyl-5-oxopyrrolidin-2-ylidene]methyl]-4-methyl-1h-pyrrol-2-yl]methylidene]-5-[(4-ethyl-3-methyl-5-oxopyrrol-2-yl)methylidene]-4-methylpyrrol-3-yl]propanoic acid Chemical compound O=C1C(CC)=C(C)C(\C=C/2C(=C(CCC(O)=O)C(=C/C3=C(C(C)=C(\C=C/4\C(\[C@@H](C)C(=O)N\4)=C/C)N3)CCC(O)=O)/N\2)C)=N1 NNMALANKTSRILL-ZUTFDUMMSA-N 0.000 description 2
- 235000016425 Arthrospira platensis Nutrition 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000006830 Luminescent Proteins Human genes 0.000 description 2
- 108010047357 Luminescent Proteins Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- INPDFIMLLXXDOQ-UHFFFAOYSA-N Phycocyanobilin Natural products CCC1=C(C)C(=CC2=NC(=C/c3[nH]c(C=C/4C(C(C(N4)=O)C)=CC)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O INPDFIMLLXXDOQ-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000005056 cell body Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000556 factor analysis Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 108060006184 phycobiliprotein Proteins 0.000 description 2
- 108010072011 phycocyanobilin Proteins 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000002708 random mutagenesis Methods 0.000 description 2
- 102200081529 rs1553259707 Human genes 0.000 description 2
- 102220123567 rs886043442 Human genes 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 1
- CWEAKSWWKHGTRJ-BQBZGAKWSA-N Ala-Gly-Met Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O CWEAKSWWKHGTRJ-BQBZGAKWSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- URAUIUGLHBRPMF-NAKRPEOUSA-N Arg-Ser-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O URAUIUGLHBRPMF-NAKRPEOUSA-N 0.000 description 1
- AOJYORNRFWWEIV-IHRRRGAJSA-N Arg-Tyr-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 AOJYORNRFWWEIV-IHRRRGAJSA-N 0.000 description 1
- BDMIFVIWCNLDCT-CIUDSAMLSA-N Asn-Arg-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O BDMIFVIWCNLDCT-CIUDSAMLSA-N 0.000 description 1
- ZEDBMCPXPIYJLW-XHNCKOQMSA-N Asp-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)N)C(=O)O ZEDBMCPXPIYJLW-XHNCKOQMSA-N 0.000 description 1
- VIRHEUMYXXLCBF-WDSKDSINSA-N Asp-Gly-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O VIRHEUMYXXLCBF-WDSKDSINSA-N 0.000 description 1
- ORRJQLIATJDMQM-HJGDQZAQSA-N Asp-Leu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O ORRJQLIATJDMQM-HJGDQZAQSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000531074 Chroococcidiopsis Species 0.000 description 1
- 241000531075 Chroococcidiopsis thermalis Species 0.000 description 1
- 241001338073 Chroococcidiopsis thermalis PCC 7203 Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- UPURLDIGQGTUPJ-ZKWXMUAHSA-N Cys-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CS)N UPURLDIGQGTUPJ-ZKWXMUAHSA-N 0.000 description 1
- 108091005941 EBFP Proteins 0.000 description 1
- 108091005942 ECFP Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- KHNJVFYHIKLUPD-SRVKXCTJSA-N Gln-Leu-Met Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCC(=O)N)N KHNJVFYHIKLUPD-SRVKXCTJSA-N 0.000 description 1
- ZZLDMBMFKZFQMU-NRPADANISA-N Gln-Val-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O ZZLDMBMFKZFQMU-NRPADANISA-N 0.000 description 1
- GJBUAAAIZSRCDC-GVXVVHGQSA-N Glu-Leu-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O GJBUAAAIZSRCDC-GVXVVHGQSA-N 0.000 description 1
- QNJNPKSWAHPYGI-JYJNAYRXSA-N Glu-Phe-Leu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=CC=C1 QNJNPKSWAHPYGI-JYJNAYRXSA-N 0.000 description 1
- UXJHNZODTMHWRD-WHFBIAKZSA-N Gly-Asn-Ala Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O UXJHNZODTMHWRD-WHFBIAKZSA-N 0.000 description 1
- IALQAMYQJBZNSK-WHFBIAKZSA-N Gly-Ser-Asn Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O IALQAMYQJBZNSK-WHFBIAKZSA-N 0.000 description 1
- ZVXMEWXHFBYJPI-LSJOCFKGSA-N Gly-Val-Ile Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZVXMEWXHFBYJPI-LSJOCFKGSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- ZNOBVZFCHNHKHA-KBIXCLLPSA-N Ile-Ser-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZNOBVZFCHNHKHA-KBIXCLLPSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- QJUWBDPGGYVRHY-YUMQZZPRSA-N Leu-Gly-Cys Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N QJUWBDPGGYVRHY-YUMQZZPRSA-N 0.000 description 1
- NTSPQIONFJUMJV-AVGNSLFASA-N Lys-Arg-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O NTSPQIONFJUMJV-AVGNSLFASA-N 0.000 description 1
- WLCYCADOWRMSAJ-CIUDSAMLSA-N Lys-Asn-Cys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(O)=O WLCYCADOWRMSAJ-CIUDSAMLSA-N 0.000 description 1
- DEFGUIIUYAUEDU-ZPFDUUQYSA-N Lys-Asn-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DEFGUIIUYAUEDU-ZPFDUUQYSA-N 0.000 description 1
- OIQSIMFSVLLWBX-VOAKCMCISA-N Lys-Leu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OIQSIMFSVLLWBX-VOAKCMCISA-N 0.000 description 1
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- HLYIDXAXQIJYIG-CIUDSAMLSA-N Met-Gln-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HLYIDXAXQIJYIG-CIUDSAMLSA-N 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- AQGDXJQRVOCUQX-UHFFFAOYSA-N N.[S] Chemical compound N.[S] AQGDXJQRVOCUQX-UHFFFAOYSA-N 0.000 description 1
- CUMXHKAOHNWRFQ-BZSNNMDCSA-N Phe-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 CUMXHKAOHNWRFQ-BZSNNMDCSA-N 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 108010010522 Phycobilisomes Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- FUMGHWDRRFCKEP-CIUDSAMLSA-N Ser-Leu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O FUMGHWDRRFCKEP-CIUDSAMLSA-N 0.000 description 1
- RHAPJNVNWDBFQI-BQBZGAKWSA-N Ser-Pro-Gly Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O RHAPJNVNWDBFQI-BQBZGAKWSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- CTONFVDJYCAMQM-IUKAMOBKSA-N Thr-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H]([C@@H](C)O)N CTONFVDJYCAMQM-IUKAMOBKSA-N 0.000 description 1
- JMGJDTNUMAZNLX-RWRJDSDZSA-N Thr-Glu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JMGJDTNUMAZNLX-RWRJDSDZSA-N 0.000 description 1
- MECLEFZMPPOEAC-VOAKCMCISA-N Thr-Leu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MECLEFZMPPOEAC-VOAKCMCISA-N 0.000 description 1
- NWECYMJLJGCBOD-UNQGMJICSA-N Thr-Phe-Val 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(C)C)C(O)=O NWECYMJLJGCBOD-UNQGMJICSA-N 0.000 description 1
- KLQPIEVIKOQRAW-IZPVPAKOSA-N Tyr-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O KLQPIEVIKOQRAW-IZPVPAKOSA-N 0.000 description 1
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 1
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 1
- IJGPOONOTBNTFS-GVXVVHGQSA-N Val-Lys-Glu Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O IJGPOONOTBNTFS-GVXVVHGQSA-N 0.000 description 1
- XPKCFQZDQGVJCX-RHYQMDGZSA-N Val-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N)O XPKCFQZDQGVJCX-RHYQMDGZSA-N 0.000 description 1
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 108091005948 blue fluorescent proteins Proteins 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000008932 jinhong Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 210000002306 phycobilisome Anatomy 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of far-red light fluorescins, the far-red light fluorescin includes the amino acid sequence of BDFP near infrared light fluorescin, and including the mutation at the 30th, 101,107,109,113,130,143,151 and 163 amino acid, the far-red light fluorescin does not include the mutation at the amino acids of the 35th, 120 and 122, wherein the amino acid sequence of the BDFP near infrared light fluorescin is as shown in SED ID NO:2.The invention also discloses a kind of fusion fluorescins including far-red light fluorescin of the invention.Far-red light fluorescin or its nucleic acid for merging fluorescin of the invention, and the carrier including the nucleic acid are encoded the present invention further discloses a kind of.
Description
Technical field
The invention belongs to fluorescent marker fields.In particular it relates to a kind of far-red light fluorescin and its fusion
Albumen encodes the nucleic acid and carrier of the far-red light fluorescin.
Background technique
Far-red light (FR) or near infrared light (NIR) light absorption and light scattering in animal tissue are lower, there is higher penetrate
Property, it is the maximum SPECTRAL REGION of ability for penetrating the most tissues such as skin.Fluorescin with this kind of luminescent pigment group
It is more suitable for the imaging deep of animal live soma, is living imaging more preferably fluorescent marker.
At present there are mainly two types of such fluorescent markers, molecular size range is in 35kD or so.One kind being originated from green fluorescence
Albumen (GFP), energy self-catalysis forms chromophore, but spectral region has certain limitation, and maximum emission wavelength generally exists
670nm or so, such as marker TagRFP675.Another kind is originated from the receptor protein being present in bacterium, bacterial typing techniques
Albumen (BphP).BphP is mainly using the biliverdin (BV) of linear tetrapyrrol(e) structure as chromophore；Biliverdin BV is extensive simultaneously
It is present in eucaryote body, it means that the fluorescence marked object of BphP can be applied to zooblast and tissue living, and be not necessarily to
Any enzyme or external source confactor.The representative of BphP class marker has iFP series and iRFP series, fluorescence emission wavelengths range
For 670nm~720nm, such as IFP2.0 maximum emission wavelength 714nm.
But the molecular weight of the existing fluorescin for inspiring far-red light or near infrared light is larger, In vivo detection Shi Yiju
Collection precipitating, and extreme environment is not tolerated, applicable situation is limited.
There are the fluorescent emission of far-red light range, mechanism and bacterial typing techniques for phycobniliprotein (phycobiliprotein)
Albumen (BphP) is similar, is derived mainly from the phycocyanobilin (PCB) in Non-covalent binding.Typical phycobniliprotein is fluorescence marked
Object such as ApcA, smURFP, ApcF2, their maximum emission wavelength are 698nm.
The sequence of core subunit ApcF2 of the DingWL et al. based on phycobilisome has obtained several after progress genetic modification
Kind of new fluorescence phycobniliprotein is simultaneously named as BDFP, these BDFP albumen can covalent bond biliverdin BV, performance ratio
ApcF2 stablizes (Ding W L, Miao D, Hou YN, et al.Small monomeric and highly stable
near-infrared fluorescent markers derived from the thermophilic
Phycobiliprotein, ApcF2 [J] .Biochimica et BiophysicaActa (BBA)-
MolecularCellResearch, 2017,1864 (10): 1877-1886).In addition, the molecular weight of these BDFP albumen compared with
Small, about 15kD, maximum emission wavelength is 710nm or so.
Although the BDFP albumen that Ding W L et al. is obtained compensates for the glimmering of existing far-red light or near infrared light well
The disadvantages mentioned above of photoprotein, but these protein fluorescence launch wavelengths are relatively simple (in 710nm or so), therefore cannot have
Effect it is applied in combination.Therefore it is transformed by genetic engineering, obtains the fluorescence that brightness is higher, spectral property is more various and excellent
Albumen has very important significance.
Summary of the invention
It is an object of the present invention to comparing by homologous sequence, conservative functional site is selected, and determine targeted mutagenesis
Site.
Another object of the present invention is to provide a kind of modified phycobniliprotein for launching far-red light and its fusion eggs
It is white.
Another object of the present invention is to provide a kind of to encode far-red light fluorescin or its fusion protein of the invention
Nucleic acid, or the carrier including the nucleic acid.
In one aspect of the invention, a kind of far-red light fluorescin is provided, the far-red light fluorescin includes
The amino acid sequence of BDFP near infrared light fluorescin, and it is included in the 30th, 101,107,109,113,130,143,151
With the mutation at 163 amino acid.The far-red light fluorescin does not include prominent at the amino acids of the 35th, 120 and 122
Become.The amino acid sequence of the BDFP near infrared light fluorescin is as shown in SED ID NO:2.
Above-mentioned BDFP near infrared light fluorescin derives from truncated phycobniliprotein ApcF2, and the including ApcF2 the 20th~
169 amino acids are made of the 20th~169 amino acids of ApcF2, and including mutation S46T, I51V, N72C, Y82C,
Y92M, N136K, V160I and V161A.
Unless otherwise specified, the amino acid position in the present invention is carried out based on the protein sequence of phycobniliprotein ApcF2
Coding.
The phycobniliprotein ApcF2 of this paper refers to from Chroococcidiopsis thermalisPCC's 7203
ApcF2.Preferably, the amino acid sequence of phycobniliprotein ApcF2 of the invention is as shown in SEQ ID NO.1.
Further, far-red light fluorescin of the invention may include in the 24th, 29,54,57,75,76,127,144
And/or 148 mutation at amino acids.
Specifically, in far-red light fluorescin of the invention, the 24th valine mutation is phenylalanine；29th paddy
Histidine mutations are glycine；30th phenylalanine sports leucine；54th threonine sports alanine；57th
Mutant serine is glycine；75th threonine sports the 76th threonine of glycine and sports alanine；101st
Aspartic acid mutations are glycine；107th glutamic acid mutation is glycine；109th leucine sports first sulphur ammonia
Acid；113rd leucine sports methionine or phenylalanine；127th threonine sports alanine；130th
Mutant serine is glycine；143rd valine mutation is alanine；144th threonine sports alanine；151st
Position threonine sports alanine；Alternatively, the 163rd glutamic acid mutation is valine.
Further, the amino acid sequence of far-red light fluorescin of the invention is as shown in SEQ ID NO:10.Alternatively, this
The far-red light fluorescin of invention is as shown in SEQ ID NO:12.Alternatively, far-red light fluorescin of the invention such as SEQ ID
Shown in NO:13.Alternatively, far-red light fluorescin of the invention is as shown in SEQ ID NO:15.Alternatively, far-red light of the invention
Fluorescin is as shown in SEQ ID NO:16.Alternatively, far-red light fluorescin of the invention is as shown in SEQ ID NO:17.Or
Person, far-red light fluorescin of the invention is as shown in SEQ ID NO:18.Alternatively, far-red light fluorescin such as SEQ of the invention
Shown in ID NO:19.Alternatively, far-red light fluorescin of the invention is as shown in SEQ ID NO:20.Alternatively, of the invention is remote red
Light fluorescin is as shown in SEQ ID NO:21.Alternatively, far-red light fluorescin of the invention such as SEQ ID NO:22 or,
Far-red light fluorescin of the invention is as shown in SEQ ID NO:23.Alternatively, far-red light fluorescin of the invention such as SEQ ID
Shown in NO:24.Alternatively, far-red light fluorescin of the invention is as shown in SEQ ID NO:25.Alternatively, far-red light of the invention
Fluorescin is as shown in SEQ ID NO:26.Alternatively, far-red light fluorescin of the invention is as shown in SEQ ID NO:27.Or
Person, far-red light fluorescin of the invention is as shown in SEQ ID NO:28.Alternatively, far-red light fluorescin such as SEQ of the invention
Shown in ID NO:29.
Preferably, the amino acid sequence of the far-red light fluorescin is as shown in SEQ ID NO:20.
In still yet another aspect of the present, a kind of fusion fluorescin is provided, the fusion fluorescin includes this hair
Bright above-mentioned far-red light fluorescin.
Further, the fusion fluorescin may further include another far-red light fluorescin.Preferably, described
Another far-red light fluorescin is BDFP far-red light fluorescin.It is highly preferred that another far-red light fluorescin is this hair
The bright far-red light fluorescin.
Alternatively, the fusion fluorescin may further include non-far-red light fluorescin, the non-far-red light fluorescence
Albumen is near infrared light fluorescin, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescence protein
And/or orange fluorescent protein.For example, the non-far-red light fluorescin can for EBFP, ECFP, mCerulean, TFP,
GFP, eGFP, EYFP, FRP, TagRFP or BDFP near infrared light fluorescin.The amino acid of the nearly feux rouges fluorescin of the BDFP
Sequence can be as shown in SED ID NO:2.
Further, it in fusion fluorescin of the invention, is connected between each fluorescin by connexon.It is preferred that
Ground, the connexon by 5~80 Amino acid profiles, such as by 5,6,7,8,9,10,11,12,13,14,15,16,17,
18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、40、45、50、 55、60、65、70、
75 or 80 Amino acid profiles.It is highly preferred that the connexon is by 5~70, preferably 5~60,5~55,5~50,5~45,
5~40,5~35 or 10~35 Amino acid profiles.
In still yet another aspect of the present, a kind of above-mentioned far-red light fluorescin of coding or above-mentioned fusion fluorescence egg are provided
White nucleic acid.
In still yet another aspect of the present, a kind of carrier including above-mentioned nucleic acid is provided.
As described above, modified BDFP fluorescin of the invention has lesser molecular weight, can covalently be tied with pigment
It closes, performance is stablized, and extreme environment is resistant to.In addition, modified BDFP line fluorescent albumen of the invention can also be used in the mankind
The active somatic cells such as cell line, elegans cell are marked, and are also applied for label Bacillus acidi lactici, plastid, mitochondria etc..
The maximum fluorescence emission peak of existing BDFP near infrared light fluorescin (near infrared light) near 710nm.This hair
Blue shift occurs for the fluorescence emission peak of the bright BDFP albumen by site mutation, obtained, and maximum emission wavelength is attached in 670nm
Closely (far-red light).Therefore, the far-red light BDFP fluorescin that the present invention is obtained by site mutation can with it is existing non-remote red
Light fluorescin (such as BDFP near infrared light fluorescin) is applied in combination, and dual biology mark is carried out to cell, tissue etc.
Note.
In addition, far-red light BDFP fluorescin of the invention can also be with another far-red light or non-far-red light fluorescin one
Building fusion fluorescin is played, to promote effective brightness when active somatic cell detection, or is combined fluorescent marker.Into one
The fused in tandem albumen of step ground, far-red light BDFP fluorescin of the invention and existing BDFP near infrared light fluorescin is not
The features such as only there is the effective brightness significantly improved, and be also equipped with large-scale Stokes displacement, it can be used for fluorescence resonance energy
Measure the repercussion study etc. of transfer.
Detailed description of the invention
Fig. 1 shows the homologous sequence comparison diagram of ApcB, ApcF2, BDFP1.1, BDFP1.2, BDFP1.6.
Fig. 2 shows mutant in HEK 293T intracellular fluorescence microscope image (a) and effective brightness contrast
Scheme (b).Each mutant is co-expressed with eGFP (FPs:IRES:eGFP) in HEK293T into the cell respectively.Fig. 2 a is respectively illustrated
The green fluorescence of eGFP and the red fluorescence (channel FR or NIR) of each mutant are observed under fluorescence microscope.In Fig. 2 b
In, the average far-red light fluorescent brightness of each mutant first takes ratio with its average eGFP fluorescent brightness, corrects expression；So
Afterwards again compared with the value of BDFP1.6 (v23), effective brightness final value is obtained.Cell imaging parameter: green channel (λex=470/
40nm, λem=510/40nm), far-red light channel (λex=630/20nm, λem=690/50nm), near infrared light channel (λex=
630/20nm, λem=690/50nm).The data acquisition time of BDFP1.1, v1, v2, v3, v4, v7, v8, v14 are 30s,
The data acquisition time of his mutant is 5s.
Fig. 3 shows mutant v13~v32 in HEK293T intracellular fluorescence microscope image (a) and effectively bright
It spends comparison diagram (b).Each mutant is co-expressed with eGFP in HEK293T into the cell respectively.Fig. 3 a respectively illustrates aobvious in fluorescence
The red fluorescence (channel FR) of the green fluorescence of the eGFP observed under micro mirror and each mutant.In Fig. 3 b figure, mutant
Average far-red light fluorescent brightness first takes ratio with its average eGFP fluorescent brightness, corrects expression；Then again with BDFP1.6
(v23) value (it is set to 1) compares, and obtains effective brightness final value.Cell imaging parameter, green channel (λex=470/
40nm, λem=510/40nm), far-red light channel (λex=630/20nm, λem=690/50nm), near infrared light channel (λex=
630/20nm, λem=690/50nm).Data acquisition time, v17 30s, other mutant are 5s.
Fig. 4 show BDFP1.2 (v6), BDFP1.6 (v23) albumen and known far-red light fluorescin iRFP670,
The curve graph of the stability contrast of miRFP670 and smURFP in vitro.Fig. 4 a shows the fluorescence intensity of each fluorescin in pH
Change in fluorescence in 9.5~2 ranges, wherein the medium of pH2~5 be 0.1M sodium acetate and 0.3M sodium chloride, pH5.5~10
Medium is 0.1M NaH2PO4With 0.3M sodium chloride.Fig. 4 b shows mixing hydrochloric acid of the fluorescence intensity in various concentration of fluorescin
Variation in guanidine (pH7.2) environment.Fig. 4 c shows variation of the fluorescence intensity of fluorescin after 80 DEG C of warm bath of solution.Fig. 4 d
Show that photobleaching is tested as a result, medium is kaliumphosphate buffer (pH7.2), the irradiation of 100W HBO 103W/2 light source is remote red
Light, which filters, is arranged (λex/bleach=630/20, λem=690/50nm), light amasthenic lens is 100 × C-Apochromat oil immersion object
Mirror, aperture 1.2, microscope are Zeiss Axioscope A1, and image camera is cool-snap HQ2CCD.
Fig. 5 shows the fusion protein that source of people albumen and monochromatic and double-colored BDFP albumen are expressed in living mammalian cells
Microphoto.(a) wide area microscope (wf) and structured lighting microscope (SIM, super-resolution microscope) image: HEK
293T expresses the fusion protein of each mutant and histone (H2B)；HeLa cell expresses each mutant and mitochondria imports sequence
(MTS), keratin (keratin), calcium connect the fusion protein of albumen (calnexin) or tubulin (α-tubulin)；U-
2OS cell expresses the fusion protein of each mutant and actin (β-actin)；Hamster CHO-K1 cell expresses mutant and flesh
The fusion protein of filamentous actin label (F-actinmarker) lifeact.(b) far-red light (BDFP1.6,1.2 or 1.3) He Jinhong
The dual colour imaging figure of outer light (BDFP1.1) fluorescin.(λ is arranged in far-red light image (left column) optical filterex=630/20nm,
λem=667/30nm), (λ is arranged near infrared light imaging figure (middle column) optical filterex=685/20, λem=740/40nm)；It is double
Color synthesizes coverage diagram (right column).Scale bar: 10 μm.
Fig. 6 shows the effective of BDFP1.2, BDFP1.6 and their same sequence fusion protein and existing fluorescin
Brightness contrast figure.It (a) is fluorescent microscopy images.Wherein, by each BDFP fluorescin and existing fluorescin
(iRFP670, miRFP670 and smURFP) is transiently transfected with eGFP building in identical carrier (BDFPs:IRES:eGFP)
Into HEK 293T cell.The fluorescent brightness of each BDFP fluorescin can be corrected based on the fluorescent brightness of eGFP.Observe channel
Setting is respectively green light (eGFP, λex=470/40nm, λem=510/40nm) and far-red light (λex=630/20nm, λem=
690/50 nm).Scale bar is set as 50 μm.(b) to the quantized result of the fluorescent brightness of (a).Data are soft using Origin 8.0
Part processing, the one-factor analysis of variance level of signifiance are α=0.05 (n=30).
Fig. 7 shows the same sequence of BDFP series albumen or the fluorescent brightness of different sequence series connection construct.(a) series connection building
The structural schematic diagram of body；(b) abosrption spectrogram for construct of connecting；(c) fluorescence spectra for construct of connecting；(d) row is on
The wide area dual colour imaging figure of Hela cell body internal labeling, lower row are SIM super-resolution imaging figure；(e) histogram of effective brightness,
Dark column indicates 630nm channel data (λex=630/20nm, λem=690/50nm), Bai Zhu indicates 660nm channel data (λex
=660/45nm, λem=710/50nm).Using 8.0 software data processing of Origin, the one-factor analysis of variance level of signifiance is
α=0.05 (n=30).
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art of the present technique,
Under the premise of not departing from general principles, several improvement can be made, these improvement are also considered as in the scope of the present invention
It is interior.Divide that the present invention is described in detail by specific embodiment below.It is understood, however, that the present invention is not limited to
Specific embodiment below.Protection scope of the present invention is defined by claims, within its scope, can be to this hair
Bright following embodiments carry out any change and combination.
Below in conjunction with specific embodiment, the present invention is explained further.
Material and method
1. carrier
PET28 (or pET30) and pACYCDuet (Novagen) is T7 promoter expression vector.PACYCDuet is through setting
Meter can be used in double objective gene sequences in the interior cotransformation expression of Escherichia coli (E.coli).Expression vector pcDNA3.1
It (Invitrogen) is the mammal expression vector with CMV promoter.
When HEK 293T is screened into the cell, using expression vector pcDNA3.1, amalgamation and expression sequence is BDFP:
IRES:eGFP.When making brightness contrast, the fluorescent brightness of BDFP albumen can be modified based on the fluorescent brightness of eGFP.
2. mutation
Mutation initiation template be BDFP1.1 (as shown in SEQ ID NO.2, i.e. ApcF2 (20-169)-S46T/I51V/
N72C/Y82C/Y92M/N136K/V160I/V161A)。
Locus specificity and site saturation mutagenesis (20 amino acid all use primer mixture to encode) use Zheng
Et al. (L.Zheng, U.Baumann, J.L.Reymond, An efficient one-step site-directed and
Site-saturation mutagenesis protocol, Nucleic AcidRes.32 (2004) e115) an effective step
Formula scheme carries out.Random mutagenesis is carried out by fallibility PCR, and used condition causes every 1000 base-pairs (bp) to produce
Raw up to 16 mutation frequencies (O.Griesbeck, G.S.Baird, R.E.Campbell, D.A.Zacharias,
R.Y.Tsien, Reducing theenvironmental sensitivity of yellow fluorescent
Protein.Mechanism and applications, J.Biol.Chem.276 (2001) 29188-29194).
The gene library obtained by random mutagenesis is cloned on pET28 vector plasmid.Then the plasmid will be with generation
The plasmid pACYC-ho1 of BILE PIGMENTS (BV), conversion enters e. coli bl21 (DE3) together.Cell is first under the conditions of 37 DEG C
Then agar plate culture 10h cultivates 48h under the conditions of 17 DEG C.Inducer uses isopropyl-β-D-thiogalactoside
(IPTG), concentration 0.05mM.Hyperfluorescence clone on plate, is the bacterium colony selection system using model Qpix 420
628/65nm/ is arranged in (Molecular Devices), feux rouges excitation channel, and 692/65nm is arranged in far-red light fluorescence channel.Often
The clone with high brightness fluorescent that a library is selected, it will the fallibility pcr template as next round.
3. the expression of the recombinant protein in Escherichia coli
PET expression vector with encoding fluorescent protein is transformed into coli strain BL21 (DE3) (Novagen)
Afterwards, then by carrier pACYC-ho1 it is transformed into same bacterial strain.By transformed BL21 cell at 18 DEG C, culture is being supplemented with card
In that mycin (20 μ g/ml) and chloramphenicol (17 μ g/ml) LB culture medium.When O.D value reaches 0.4~0.6,1mM isopropyl is used
Base-β-D- thiogalactoside (IPTG) inducing expression 5~16 hours, then at 4 DEG C, 12,000 × g is centrifuged 3min, collects
Cell rinses 2 times through water, and short-term preservation is at 4 DEG C or is placed in -20 DEG C of long-term preservations.
4. the purifying of albumen and quantitative
Wet thallus is suspended in start buffer [kaliumphosphate buffer (KPB, 20mM, pH7.2), the sodium chloride of ice pre-cooling
(NaCl, 0.5M)] in.It is broken through 50W power ultrasound (JY92-II, NingBo XinZhi Biology Science Co., Ltd, China),
5 minutes.At 4 DEG C, 12000 × g is centrifuged 60 minutes suspension.Obtained supernatant is through Ni2+Affinity column (Amersham
Biosciences it) purifies, wherein using start buffer [potassium phosphate (KPB, 20mM, pH 7.2) loading, using additionally containing
The buffer of 0.5M imidazoles is eluted.The sample being collected into, at least twice with start buffer (pH 7.2) dialysis.Pass through
Bradford method measures protein concentration, and bovine serum albumin(BSA) is used to be corrected as standard items.
5. the culture of mammalian cell
By HEK 293T, HeLa or U-2 OS cell culture in the DMEM culture medium for containing 10% fetal calf serum
(Invitrogen) in, by CHO-K1 cell culture in the F-12K culture medium containing 10% fetal calf serum.It uses2000 (Invitrogen) are transfected.When transfection,2000 and DNA is with 3: 1 (μ
L: μ g) ratio is in serum free mediumMiddle mixing after ten minutes, is added in the cell to be transfected immediately.
After 5~6h, fresh DMEM culture medium is replaced.
6. (in-vivo imaging) is imaged in active somatic cell
It completes to transiently transfect in 20mm glass Tissue Culture Dish (Nest), starts to carry out active somatic cell imaging for 24 hours afterwards.At
It is first rinsed twice with 1mL PBS as before, is then rinsed once with DEME culture medium is (no phenol red).Imaging device is that inversion is micro-
Mirror Nikon Ti is furnished with 20 0.45-DIC L-WD of cool-snap HQ2 CCD camera and Nikon Plan Fluor ELWD
Object lens.Excitation and transmitting are provided that eGFP is green channel, λex=470/40, λem=510/40nm；Far-red light fluorescence egg
White is far-red light channel, λex=630/20, λem=690/50nm.Picture uses ImageJ software (National
Institutes of Health) it is analyzed and is handled.
7. wide area and super-resolution microscope inspection
In room temperature, by stand-alone mode, Nikon is inverted using the ECLIPSE Ti-E equipped with 100 × 1.49NA oil immersion objective
Nikon structured lighting system on microscope obtains wide area and structured lighting microscope (SIM) photo.Use 640nm's
Semiconductor laser (100mW, CUBE 640-100C, COHERENT) excites FR fluorescence.Using soft by NIS-Elements AR
The electron multiplication CCD camera (Andor iXon3 DU897) of part (Nikon) control carries out data acquisition.Use NIS-
Elements AR handles image.
8. the building of fused in tandem albumen
The fluorescin of BDFP series can pass through following connexon (1inker) construction of fusion protein:
The connexon of 11 amino acid residues: GHGTGSTGSGS；
The connexon of 23 amino acid residues: GHGTGSTGSGSSGTASSEDNNMA；
The connexon of 31 amino acid residues: GHGTGSTGSGSHGTGSTGSSGTASSEDNNMA.
Experiment discovery, the connexon of above-mentioned 3 kinds of different lengths have not significant impact that (result is not shown to the fluorescence of fusion protein
Out).
9. homologous sequence compares
BDFP fluorescin structure alignment, is completed on SWISS-MODEL remote server.The template sequence used is blunt
The phycobniliprotein ApcB (pdb code:1ALL) of top spirulina (Spirulina platensis), comparison software Swiss-
PDBViewer, edition 4 .1.Protein structure figure is created using PyMOL (http://www.pymol.org/).Using Clustal
Protein sequence comparison chart is completed in (http://www.clustal.org/) creation.
10. spectrum analysis
The ultraviolet-ray visible absorbing light of chromoprotein is detected by spectrophotometer (DU800, Beckman-Coulter)
Spectrum.The extinction coefficient of fluorescin is absorption coefficient ε=39,900M according to BILE PIGMENTS BV at 390nm-1cm-1, joined
Than conversion.
Fluorescence spectrum is detected by sepectrophotofluorometer (F320, Tianjin Gangdong Technology Development Co., Ltd.).
With reference to known iRFP670 fluorescin (ΦF=0.122) the sub- yield Φ of amount of fluorescence detectedF, sample detection environment is potassium phosphate
Salting liquid (20mM, pH 7.2, KPB).
11. the Detection of Stability of fluorescin
Detect the stability experiment under condition of different pH.The BDFP fluorescin solution of detection after purification, it is slow in different pH
Fluorescence intensity change under fliud flushing environment.It is 2~10 that range, which is arranged, in pH, and wherein pH2~5 uses 0.1M sodium acetate, 0.3M chlorination
The buffer system of sodium；PH5.5~10 use 0.1MNaH2PO4, the buffer system of 0.3M sodium chloride.
Detect the stability experiment in strong denaturant environment.The BDFP fluorescin solution of detection after purification, different dense
Fluorescence intensity change under the guanidine hydrochloride solution environment of degree, pH7.2.
Detect stability experiment in a high temperauture environment.The BDFP fluorescin solution of detection after purification, in 80 DEG C of water-baths
In pot, fluorescence intensity changes with time.
Photobleaching experiment.Fluorescin solution (pH7.2,20mM KPB) is instilled in mineral oil, with 100W HBO 103
100% power illumination of W/2 light source, far-red light, which filters, is arranged (λex=630/20, λem=690/50nm), light amasthenic lens is 100
× oil mirror (C-Apochromat oil-immersion lens), aperture 1.2, using microscope Zeiss Axioscope
A1, image camera are cool-snap HQ2 CCD.With software I mageJ (National Institutes of Health)
Analyze data.
It will be understood by those skilled in the art that for the experimental methods of molecular biology for not making to illustrate herein, it can
Referring to specific method listed in " Molecular Cloning:A Laboratory guide " (fourth edition, M.R. Green and J. Pehanorm Brooker write) book
It carries out, or is carried out according to kit and product description.Used kit biomaterial unless otherwise specified can be from quotient
Industry approach obtains.
Embodiment
1. homologous sequence of embodiment compares analysis
Fig. 1 shows the comparison of several homologous sequences, and wherein ApcF2 and its derived sequence BDFP1.1 are all from
Chroococcidiopsis thermalisPCC 7203；ApcB comes from blunt top spirulina Spirulina platensis, should
Algae is not suitable with far-red light and near infrared light growing environment.It will be seen from figure 1 that L113 ApcB, ApcF2 and BDFP1.1 it
Between be highly conserved.
On the basis of BDFP1.1 sequence (SEQ ID NO.:2), fixed point saturation mutagenesis is carried out to L113；Screening obtains prominent
Variant is reacted by fallibility PCR, is carried out lasting random mutation, is established mutant library.Using external spectral property detection and
The method of active somatic cell imaging screens the mutant in mutant library according to following screening criteria: with BDFP1.1
It compares, obvious blue shift occurs for targeted mutagenesis body maximum emission wavelength, but is still greater than 660nm；Simultaneously with known fluorescin
Mark iRFP670 (ε Φfl=10.2mM-1cm-1) compare, molecule brightness is suitable.
By screening, 7 mutant are obtained first.This 7 mutant are sequenced, sequencing result finds that they distinguish
With amino acid mutation as shown in Table 1 below.
In this 7 mutant of expression in escherichia coli, then pass through Ni2+Each mutant of affinitive layer purification expression is gone forward side by side
Row protein quantification.The mutant protein of equivalent is placed in detection solution kaliumphosphate buffer (KPB, 20mM, pH 7.0) and 0.5M
In NaCl.The a length of 620nm of the excitation light wave of fluorescence emission spectrum；Molecule brightness contrast object of reference is iRFP670 (ε Φfl=
10.2 mM-1cm-1).The spectral property of 7 mutant is detected, the results are shown in Table 2.
The mutational site of each mutant and the spectral results of table 2 listed in conjunction with table 1 can be seen that BDFP1.1 most
Big launch wavelength is about 707nm；Mutant v1 and v2 mutate at L113, their maximum emission wavelength is blue shifted to
About 700nm；Mutant v4 mutates at L113 and G125, and maximum emission wavelength is further blue shifted to about 670nm；It is prominent
Variant v6, v7 and v8 at L113 and G125 other than mutating, and also with the mutation in other sites, but they are most
Big launch wavelength is in 670nm or so.
The above results show the mutation at L113 so that the blue spectral shift of fluorescin (is blue shifted to about from about 707nm
700nm), and L113 and G125 combinatorial mutagenesis the fluorescin blue spectral shift effect that is more significantly (are blue shifted to about
670nm).Other sites, the mutation at F30, Q39, N47, M81, D101, V143 or T151 not will cause fluorescin spectrum
Further blue shift.
In addition, compared with BDFP1.1, it can be seen that L113, which is mutated, improves the molecule brightness of fluorescin, and L113 with
G125 combinatorial mutagenesis further improves the molecule brightness of fluorescin.Specifically, the molecule brightness of BDFP1.1 is 39, mutation
The molecule brightness of body v1 and v2 (being mutated with L113) are increased to 70 and 57 respectively；Mutant v4 (is combined with L113 and G125
Mutation) molecule brightness be further increased to 157；Point of other mutant v6~v8 with L113 and G125 combinatorial mutagenesis
Sub- brightness is above 150.
Embodiment 2. detects the effective brightness intracellular in HEK 293T of each BDFP fluorescin
The nucleic acid (FPs:IRES:eGFP) for expressing each mutant is constructed in expression vector pcDNA3.1, then instantaneously
It is intracellular to be transfected into HEK 293T, observes fluorescent brightness using inverted fluorescence microscope.As a result shown in Figure 2.
Fig. 2 a shows the fluorescent image observed under green channel and the channel FR/NIR respectively.Because BDFP1.1,
V1, v2, v3, v4, v7, v8, v14 are under the channel FR/NIR, and the 5s time for exposure does not collect the image of effective brightness, therefore
Take the 30s time for exposure.Other mutant take the 5s time for exposure.Mutant v6 (BDFP1.2), v7 are shown in Fig. 2 b
With the quantized result of the effective brightness of v8.
Can be seen that mutant v1, v2, v4, v6 (BDFP1.2), v7 and V8 from result above can lead in FR/NIR
Fluorescence is observed under road.In addition, though the molecule brightness of mutant v4, v7 and V8 are significantly improved, but thin in HEK293T
Effective brightness intracellular is not high.In contrast, effective brightness of mutant v6 (BDFP1.2) is higher.
The screening of the further mutant of embodiment 3.
In order to further find the mutant of excellent, inventor has carried out another wheel mutation.With same as Example 1
Screening criteria, filter out 20 mutant v13~v32, as shown in table 3 below.
Meanwhile the spectral property of mutant v13~v32 is had detected, as a result shown in table 4 as above.
It can be seen that the maximum emission wavelength of v13~v32 is similar with v6 (BDFP1.2) from the result in table 4, exist
670nm or so, and molecule brightness is also higher.
Embodiment 4. detects mutant v13~v32 effective brightness intracellular in HEK 293T
In method same as Example 2, the expression vector for co-expressing each mutant and eGFP is transiently transfected to HEK
293T is intracellular, observes fluorescent brightness using inverted fluorescence microscope.As a result it is shown in Fig. 2 (v13~v15) and Fig. 3.
Fig. 2 and 3a shows the fluorescent image observed under green channel and the channel FR respectively.Wherein, due to mutant
V17 does not collect effective luminance picture in the 5s time for exposure, thus take the 30s time for exposure to acquire image (v14 is in 30s
Effective luminance picture is not collected when the time for exposure, therefore is being not shown in the figure) yet.When other mutant take 5s to expose
Between.Fig. 2 a and Fig. 3 b respectively quantify fluorescent brightness.
It can be seen from the figure that other mutant are equal in the intracellular effective brightness of HEK293T other than v14 and v17
It is very high.
Embodiment 5. detects the stability of each fluorescin under in vitro conditions
The present embodiment has detected fluorescin to conditions in vitro, such as the tolerance of soda acid, Denaturing, high temperature, photobleaching
Property.As a result shown in Figure 4.
Fig. 4 a shows fluorescence intensity of each fluorescin at different pH.It can be seen from the figure that BDFP1.2 (v6) and
The fluorescence intensity of BDFP1.6 (v23) fluorescin fluorescence in pH2 also retains 50% or more (compared with when pH7), and
IRFP670 and miRFP670 fluorescence in pH3.5 is quenched completely.It is relatively stable that this, which illustrates BDFP1.2 at a low ph,
's.
Fig. 4 b shows fluorescence intensity of each fluorescin in the guanidine hydrochloride solution of various concentration.It can from figure
Out, BDFP1.2 (v6) still remains with 70% or more fluorescence under the conditions of 3M guanidine hydrochloride (pH7.2).BDFP1.6 (v23) is glimmering
The tolerance of photoprotein is higher, under the conditions of 3.5M guanidine hydrochloride, moreover it is possible to retain about 60% fluorescence.
Fig. 4 c shows each fluorescin time that fluorescence is kept under high temperature (80 DEG C).It can be seen from the figure that
BDFP1.2 (v6) and BDFP1.6 (v23) still remain with 40% or more fluorescence after 80 DEG C of warm bath 2h.And iRFP670's is glimmering
Light is quenched completely after 80 DEG C of warm bath 2h, and the fluorescence of smURFP is also reduced to 25% or less.
Fig. 4 d shows the retention time of each fluorescin fluorescence in photobleaching processing.It can be seen from the figure that
BDFP1.2 (v6) and BDFP1.6 (v23) are similar with iRFP670, miRFP670 and smURFP in photobleaching.
Based on the above results, it can be deduced that BDFP1.2 (v6) and BDFP1.6 (v23) is in low pH, concentrated hydrochloric acid guanidine solution
Or it in the environment of high temperature, all has excellent stability, and be resistant to photobleaching.
Embodiment 6. detects effective brightness and its polymerization state of each fluorescin in cell
Sample needs to pass through Ni before detecting spectrum2+Affinitive layer purification, detection solution environmental are KPB (20mM, pH
And 0.5M NaCl 7.0)；The a length of 660nm of the excitation light wave of fluorescence emission spectrum.The comparison object of reference of molecule brightness is
iRFP670 (ε·Φfl=10.2mM-1cm-1).In intracellular effective fluorescence (far-red light) brightness of HEK293T, transfecting
Detection obtains pcDNA3.1- FPs:IRES:eGFP plasmid afterwards for 24 hours, without adding external source BILE PIGMENTS BV or coexpression ferroheme
Oxidizing ferment HO-1.As a result it is shown in the following table 5.
It is similar with result above, the maximum emission wavelength of BDFP1.1 be about 707nm, BDFP1.2 and BDFP1.6 most
Obvious blue shift, about 670nm occur for big launch wavelength.Therefore, BDFP1.2 and BDFP1.6 can be applied in combination with BDFP1.1,
Carry out double fluorescent label.
In addition, BDFP1.2 effective brightness in HEK 293T, every BV is 10.5, and every kDa is 21.8；BDFP1.6 is in HEK
Effective brightness in 293T, every BV are 61.6, and every kDa is 128.This shows that effective brightness of BDFP1.2 and BDFP1.6 is aobvious
It writes and is higher than BDFP1.1, and effective brightness of BDFP1.6 has been got back compared with BDFP1.2 and further increased.
Embodiment 7. detects the single expression of fluorescin in mammalian cells and its combinational expression with BDFP1.1
In the present embodiment, has detected BDFP1.2 and individually import sequence (MTS), keratin with histone (H2B), mitochondria
(keratin) or calcium connects the expression of the fusion protein of albumen (calnexin) in mammalian cells, has detected simultaneously
BDFP1.2, BDFP1.6 effect with BDFP1.1 composite marking respectively.As a result shown in Figure 5.
Fig. 5 a shows the result of BDFP1.2 separate marking.BDFP1.2 and histone are expressed in HEK293T cell
(H2B) fusion protein；BDFP1.2 is expressed in HeLab cell individually imports sequence (MTS), keratin with mitochondria
(keratin) or calcium connects the fusion protein of albumen (calnexin).It can be seen from the figure that micro- using wide area
(Widefield, wf) and structured lighting micro- (SIM, super-resolution microscope) can be clearly observed fusion protein
Fluorescence.
Figure 5b shows that the results that BDFP1.1 and BDFP1.2 or BDFP1.6 are combined label.By fusion protein
BDFP1.2- MTS is with BDFP1.1-NLS cotransfection with combinational expression in HeLa cell.Then it is detected under fluorescence microscope.
The optical filter of left column is set as λex=630/20nm, λem=667/30nm；The optical filter of middle column is set as λex=685/
20, λem=740/40nm；Right column is overlapping image.Similarly, fusion protein BDFP1.6-H2B and BDFP1.1-MTS is total to
Transfection is in HeLa cell with combinational expression.It can be seen from the figure that the fusion protein of BDFP1.2 and BDFP1.6 was issued
Far-red light fluorescence can be distinguished with the near infrared light fluorescence of BDFP1.1, so as to carry out double labeling to cell simultaneously.
Embodiment 8. detects the Absorption and emission spectra property of BDFP series albumen series connection construct
The present embodiment use 23 amino acid connexon, further construct series connection construct BDFP1.1:1.2,
BDFP1.1:1.6 (as shown in Figure 7a).
Series connection construct BDFP1.1:1.2, BDFP1.1:1.6, BDFP1.2:1.2 and BDFP1.6:1.6 of building are turned
Change into Escherichia coli, and inducing expression.Then the fusion fluorescent protein B DFP1.1 expressed by affinitive layer purification:
1.2, BDFP1.1:1.6, BDFP1.2:1.2 and BDFP1.6:1.6.Using ultraviolet-visible absorption spectroscopy instrument and Fluorescence Spectrometer
The spectral property for detecting these fusion fluorescins, is as a result shown in the following table 6.
It can be seen that the transmitting of same sequence series connection construct BDFP1.2:1.2 and BDFP1.6:1.6 from table 6 and Fig. 6 b, c
Spike length is each about 670nm, and significant change does not occur.And different sequence series connection construct BDFP1.1:1.2 and BDFP1.1:1.6,
Absorption peak is blue shifted near 640nm, and near fluorescence peak red shift to 710nm, this shows to increase Stokes displacement.
In addition, fluorescin becomes free state after fusion, it is not susceptible to aggregate and precipitate phenomenon (data are not shown).
Effective brightness of the detection series connection building of embodiment 10. and its polymerization state
In the present embodiment, by connect construct BDFP1.1:1.2, BDFP1.1:1.6, BDFP1.2:1.2 and BDFP1.6:
1.6 transiently transfect into HEK 293T cell, in fluorescence microscopy its effective brightness under the microscope, are as a result shown in table 6, Fig. 6 and figure
In 7e.
It can see from table 6 and Fig. 6 b, with the effective bright of sequence series connection construct BDFP1.2:1.2 and BDFP1.6:1.6
Degree is significantly improved, and is higher than the summation of effective brightness when two BDFP fluorescins are used alone.Fig. 7 e is also obtained
Similar results.This result shows that, connect construct in two BDFP fluorescin cooperating types improve series connection construct
Effective brightness.
It is arrived in addition, fusion protein BDFP1.2:1.2- histone and BDFP1.1:1.6- mitochondria are imported sequence cotransfection
Hela is intracellular, is observed under fluorescence microscope, is as a result shown in Fig. 7 d.Wherein, BDFP1.1:1.6- mitochondria imports
Sequence is observed using near infrared channels, and BDFP1.2:1.2- histone is observed using far infrared channel.It can from Fig. 7 d
The fluorescence of BDFP1.2:1.2 and BDFP1.1:1.6 can distinguish out, so as to carry out double labeling to cell.
SEQUENCE LISTING
<110>Guangzhou Tianbao Songyuan Biology Science & Technology Development Co., Ltd.
<120>novel B DFP fluorescin and its fusion protein through genetic modification
<130> P18GZ1NN03250CN
<160> 29
<170> PatentIn version 3.5
<210> 1
<211> 169
<212> PRT
<213> Chroococcidiopsis thermalis
<400> 1
Met Gln Asp Lys Leu Thr Ser Val Ala Lys Asn Cys Asp Leu Thr Gly
1 5 10 15
Ser Ser Leu Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala
20 25 30
Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Ser Asn Ala
35 40 45
Ala Glu Ile Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro
50 55 60
Glu Leu Val Ser Pro Gly Gly Asn Ala Tyr Thr Thr Arg Arg Tyr Asn
65 70 75 80
Met Tyr Val Arg Asp Met Asn Tyr Phe Leu Arg Tyr Cys Ser Tyr Ala
85 90 95
Ile Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly
100 105 110
Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala
115 120 125
Arg Ser Ile Gln Leu Met Lys Asn Ile Val Lys Glu Lys Leu Val Thr
130 135 140
Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Val
145 150 155 160
Val Arg Glu Ile Ser Glu Thr Glu Ile
165
<210> 2
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> BDFP1.1
<400> 2
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 3
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v1
<400> 3
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 4
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v2
<400> 4
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Met
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 5
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v4
<400> 5
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 6
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v5
<400> 6
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asp Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 7
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v6
<400> 7
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 8
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v7
<400> 8
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Ser Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Ser Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Thr
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 9
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v8
<400> 9
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Arg Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Thr
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 10
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v13
<400> 10
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 11
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v14
<400> 11
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Cys Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 12
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v15
<400> 12
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Cys Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 13
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v16
<400> 13
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 14
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v17
<400> 14
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Gly Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 15
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v18
<400> 15
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Ala Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 16
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v19
<400> 16
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 17
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v20
<400> 17
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 18
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v21
<400> 18
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 19
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v22
<400> 19
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Ala Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 20
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223>v23(EDFP1.6)
<400> 20
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 21
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v24
<400> 21
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 22
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v25
<400> 22
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Ala Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 23
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v26
<400> 23
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 24
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v27
<400> 24
Met Ala Asn Arg Glu Val Phe Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Ala Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 25
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v28
<400> 25
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 26
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v29
<400> 26
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 27
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v30
<400> 27
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 28
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v31
<400> 28
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Ala Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 29
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v32
<400> 29
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Ala Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
Claims (10)
1. a kind of far-red light fluorescin, which is characterized in that the far-red light fluorescin includes BDFP near infrared light fluorescence egg
White amino acid sequence, and including at the 30th, 101,107,109,113,130,143,151 and 163 amino acid
Mutation,
The far-red light fluorescin does not include the mutation at the amino acids of the 35th, 120 and 122,
The amino acid sequence of the BDFP near infrared light fluorescin is as shown in SED ID NO:2.
2. far-red light fluorescin according to claim 1, which is characterized in that the far-red light fluorescin further wraps
Include the mutation at the 24th, 29,54,57,75,76,127,144 and/or 148 amino acid.
3. far-red light fluorescin according to claim 1 or 2, which is characterized in that the 30th phenylalanine sports bright
Propylhomoserin；101st Aspartic acid mutations are glycine；107th glutamic acid mutation is glycine；109th leucine mutation
For methionine；113rd leucine sports methionine or phenylalanine；130th mutant serine is glycine；The
143 valine mutations are alanine；151st threonine sports alanine；Alternatively, the 163rd glutamic acid mutation is figured silk fabrics
Propylhomoserin.
4. far-red light fluorescin according to claim 1 or 2, which is characterized in that the ammonia of the far-red light fluorescin
The base acid sequence such as institute of SEQ ID NO:10,12,13,15,16,17,18,19,20,21,22,23,24,25,26,27,28 or 29
Show, it is preferable that the amino acid sequence of the far-red light fluorescin is as shown in SEQ ID NO:20.
5. a kind of fusion fluorescin, which is characterized in that the fusion fluorescin includes such as any one of Claims 1 to 4
The far-red light fluorescin.
6. fusion fluorescin according to claim 5, which is characterized in that the fusion fluorescin further comprises another
One far-red light fluorescin；
Preferably, another far-red light fluorescin is BDFP far-red light fluorescin；
It is highly preferred that another far-red light fluorescin is far-red light fluorescence egg as described in any one of claims 1 to 4
It is white.
7. fusion fluorescin according to claim 5, which is characterized in that the fusion fluorescin further comprises non-
Far-red light fluorescin, the non-far-red light fluorescin are near infrared light fluorescin, green fluorescent protein, blue-fluorescence egg
White, cyan fluorescent protein, yellow fluorescence protein and/or orange fluorescent protein.
8. the fusion fluorescin according to any one of claim 5~7, which is characterized in that lead between each fluorescin
Cross connexon connection, it is preferable that the connexon is by 5~80 Amino acid profiles, more preferably by 5~70 Amino acid profiles.
9. a kind of encode far-red light fluorescin as described in any one of claims 1 to 4 or as in claim 5~8
The nucleic acid of described in any item fusion fluorescins.
10. a kind of carrier including nucleic acid as claimed in claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811009726.6A CN109517057B (en) | 2018-08-30 | 2018-08-30 | Novel BDFP fluorescent protein modified by gene and fusion protein thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811009726.6A CN109517057B (en) | 2018-08-30 | 2018-08-30 | Novel BDFP fluorescent protein modified by gene and fusion protein thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109517057A true CN109517057A (en) | 2019-03-26 |
| CN109517057B CN109517057B (en) | 2022-03-29 |
Family
ID=65770836
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811009726.6A Active CN109517057B (en) | 2018-08-30 | 2018-08-30 | Novel BDFP fluorescent protein modified by gene and fusion protein thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109517057B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386977A (en) * | 2019-07-01 | 2019-10-29 | 广州天宝颂原生物科技开发有限公司 | A kind of near infrared light fluorescin and its fusion protein |
| CN110577593A (en) * | 2019-07-01 | 2019-12-17 | 华中农业大学 | A small molecule near-infrared fluorescent protein and its fusion protein |
-
2018
- 2018-08-30 CN CN201811009726.6A patent/CN109517057B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| DING,W等: "ASN64561.1", 《GENBANK》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386977A (en) * | 2019-07-01 | 2019-10-29 | 广州天宝颂原生物科技开发有限公司 | A kind of near infrared light fluorescin and its fusion protein |
| CN110577593A (en) * | 2019-07-01 | 2019-12-17 | 华中农业大学 | A small molecule near-infrared fluorescent protein and its fusion protein |
| CN110577593B (en) * | 2019-07-01 | 2021-09-14 | 华中农业大学 | Small-molecule near-infrared light fluorescent protein and fusion protein thereof |
| CN110386977B (en) * | 2019-07-01 | 2022-12-13 | 广州天宝颂原生物科技开发有限公司 | Near-infrared light fluorescent protein and fusion protein thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109517057B (en) | 2022-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2399563T3 (en) | Modified fluorescent green proteins and method for their use | |
| CN109134644A (en) | Far-red light fluorescin and its fusion protein | |
| RU2395581C2 (en) | Novel fluorescent proteins from entacmaea quadricolor and method of obtaining said proteins | |
| US20240027344A1 (en) | Fluorescent Probe for Branched Chain Amino Acids and Use Thereof | |
| JP5570803B2 (en) | Fluorescent protein and pH measurement method | |
| CN110386977B (en) | Near-infrared light fluorescent protein and fusion protein thereof | |
| JP6707472B2 (en) | Tag that activates and shifts fluorophores (FAST) | |
| US20130344591A1 (en) | Modified Fluorescent Proteins and Methods for Using Same | |
| WO2022215532A1 (en) | Novel polypeptide exhibiting fluorescent properties and use for same | |
| CN109517057A (en) | Novel B DFP fluorescin and its fusion protein through genetic modification | |
| CN109265523A (en) | Novel fluorescence marker and its fusion protein from BDFP near infrared light fluorescin | |
| US7972834B2 (en) | Modified fluorescent proteins and methods for using same | |
| CN109553689A (en) | A kind of fusion protein containing ApcE2 mutant and its application | |
| CN110577593B (en) | Small-molecule near-infrared light fluorescent protein and fusion protein thereof | |
| ES2375610T3 (en) | PHOTOPROTEIN MTCICLINA ISOLATED AND ITS USE. | |
| CN109748970B (en) | Alpha-ketoglutaric acid optical probe and preparation method and application thereof | |
| US8206978B2 (en) | Green fluorescent protein optimized for expression with self-cleaving polypeptides | |
| CN109553661A (en) | ApcE2 protein mutant and its application | |
| US9383366B2 (en) | Blue fluorescent protein and methods of use thereof | |
| CN112961225B (en) | Near-infrared fluorescent protein, recombinant vector, recombinant cell and application thereof | |
| JP2011212008A (en) | FLUORESCENT PROTEIN AND METHOD FOR MEASURING pH | |
| CN108834418A (en) | fluorescent protein | |
| RU2515903C2 (en) | Isolated nucleic acid coding fluorescent biosensor, expression cassette, cell, producing fluorescent biosensor, isolated fluorescent biosensor | |
| CN115806602A (en) | BDFPs muteins, fusion proteins and applications | |
| JP5796949B2 (en) | Fluorescent protein |
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 |