[Fermentation and purification of Echistatin fusion protein expressed in Escherichia coli].
Lots of studies of Echistatin (Ecs) have proved its wide use in many aspects. However, the low yield of Ecs has impeded the relative researches of the protein. To establish the high-level expression system of Ecs, the fermentation and purification process of Ecs fusion protein expressed in E. coli were optimized. The Ecs gene was introduced into vector pTXB1 and placed under the control of highly efficient T7 promoter system. The cloned Ecs gene was expressed in E. coli BL21 (DE3) as soluble form. The Ecs production and biomass accumulation were optimized by examining medium composition, point of induction and induction time in fed-batch fermentation. Biomass accumulation was greatly affected by medium gradient, reaching 50.3g/L in 2 x YT medium. Ecs production was found to increase to 35% of total protein with 75g/L biomass accumulation after induced for 4h. Purification of Ecs from supernatant of sonication was done using one-step chromatographic procedure with chitin affinity chromatography and DTF cleavage, resulting in yields of 28mg/L and > 90% purity. The bioactivity of purified Ecs was determined and the result showed that purified Ecs could inhibit the aggregation of platelet in vitro with similar bioactivity to wild Ecs. This optimized method is readily scaled up for the expression and purification of Ecs in sufficient quantities for further structural and biological studies and applications.
Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China. yanglijunmm@126.com
Li-jun
LJ
Yang
Tao
T
Yang
Niu-liang
NL
Cheng
Jun
J
Xie
Bo
B
Niu
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014757:Viper Venoms / Q000096:biosynthesis* / Q000302:isolation & purification
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C057986:echistatin