Soluble Expression and Purification of Snake Venoms Fihrino(geno)lytic Emzyme Alfimeprase in E.coli
- VernacularTitle:蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化
- Author:
Shou-Tao ZHANG
;
Yan-Sheng ZHOU
;
Xue-Hua LAI
;
Xing-Feng BAO
;
Ai-Guang GUO
;
- Publication Type:Journal Article
- Keywords:
Alfimeprase Chaperone Soluble expression Purification
- From:
China Biotechnology
2006;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.
