Optimized Expression of Snake Fibrinolytic Enzyme Alfimeprase in Pichia pastoris and Its Activity Identification
- VernacularTitle:蛇毒纤溶酶Alfimeprase在毕赤酵母中的优化表达及活性鉴定
- Author:
Jing SHI
;
Shou-Tao ZHANG
;
Ya-Fei QI
;
Ai-Guang GUO
;
- Publication Type:Journal Article
- Keywords:
Pichia pastoris Alfimeprase rALF Fibrin plate method
- From:
China Biotechnology
2006;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Alfimeprase(ALF)is a recombinantly modified variant of non-hemorrhagic zinc metalloproteinase fibrolase.The target gene alf was obtained from the clone vector p43-alf and cloned into the Pichia pastoris expression vector pPICZ? A.Through high efficiency transformation and Zeocin selection,the recombinant strains of pPICZ?A-alf /GS115 were isolated.In order to achieve a high level expression of recombinant Alfimeprase(rALF),optimization of pH value,methanol daily addition concentration,cell density and methanol induction time points were carried out,and the production of rALF reached up to 425 mg/L.By His?Bind chromatography,the purity of secreted rALF was as high as 95 %.SDS-PAGE and Western blot analysis show that rALF has a molecular weight of about 24 kDa and is bound specifically to anti-His?tag monoclonal antibody.Activity identification results of the modified fibrin plate method demonstrate that the secreted rALF has high fibrinolytic activity.Thus sets up an optimized expression system for ALF,which will play an important role in its further studies and industrial production.
