To obtain high efficiency of cleavage of thrombin in fusion protein containing a ANP fusetl to Re f pep-tides,the linker sequence deslgned as VIAGR which was dlfferent from GVRGPR formerly used was stud-ied. Plasmld p...To obtain high efficiency of cleavage of thrombin in fusion protein containing a ANP fusetl to Re f pep-tides,the linker sequence deslgned as VIAGR which was dlfferent from GVRGPR formerly used was stud-ied. Plasmld pHL carrying the fuslon gene Ref-NT-ANP downstream from PL Proruc)ter was derived fromexpression vector pLY1 by inserting the fragment of NT-ANP lnto lt. The exPresslon of fuslon gene waslnduced at 420,and the interested proteln Ref-NT-ANP accumlllated as inclusion bodies was lsolated bygradient centrifuge and then dissolved in 7 mol/L guanidinehydrochloride(Gdn-HCl). After dilution,renat-uration and dialyzation, the cleavage of thrombin was examined using samples with 1. 1 mol/1, (;dn-HC1and samples free of Gdn-HCl resI)ective1y. I)igestion result showed that the novel-ad()Pted cleavage sequencewas highly sensitive to thrombin when the substrate dissOlved in 1. 1 mol/I, tidn HCl. The time needed for87%cleavage (the ratio of substrate to thrombin was 5O pg/u)was less than 24 hOurs. This sequence described here which was sPecifically recognized by thrombin might be broadly applied in other fusion sys-tems.展开更多
文摘To obtain high efficiency of cleavage of thrombin in fusion protein containing a ANP fusetl to Re f pep-tides,the linker sequence deslgned as VIAGR which was dlfferent from GVRGPR formerly used was stud-ied. Plasmld pHL carrying the fuslon gene Ref-NT-ANP downstream from PL Proruc)ter was derived fromexpression vector pLY1 by inserting the fragment of NT-ANP lnto lt. The exPresslon of fuslon gene waslnduced at 420,and the interested proteln Ref-NT-ANP accumlllated as inclusion bodies was lsolated bygradient centrifuge and then dissolved in 7 mol/L guanidinehydrochloride(Gdn-HCl). After dilution,renat-uration and dialyzation, the cleavage of thrombin was examined using samples with 1. 1 mol/1, (;dn-HC1and samples free of Gdn-HCl resI)ective1y. I)igestion result showed that the novel-ad()Pted cleavage sequencewas highly sensitive to thrombin when the substrate dissOlved in 1. 1 mol/I, tidn HCl. The time needed for87%cleavage (the ratio of substrate to thrombin was 5O pg/u)was less than 24 hOurs. This sequence described here which was sPecifically recognized by thrombin might be broadly applied in other fusion sys-tems.
