Expression,Purification and Characterization of Ricin A from E. coli

Abstract:

The target of this study is to achieve the cloning and expression of ricin A chain gene and produce the recombined ricin A chain protein(RTA) with high activity ane then assay it activity by RTB mediated by recombined adenovirus the RTA could enter cells and exert cytotoxicity.For this,the ricin A chain gene was amplified by PCR and cloned it into the fusion expression vector pET32a to construct plasmid(pET32a-His-RTA),the plasmid was transferred into E.coli BL21 and induced with low concentration of IPTG(1 mmol/L) at low temperature(20 ℃).Purified RTA by Ni-NTA column and the purified protein was identified by SDS-PAGE electrophoresis and Western-blot.It was found that the constructed recombinant plasmid(pET32a-His-RTA) was efficient expressed RTA fusion protein and about 50mg purified protein(Mr 47 000) was obtained from 1 liter culture broth.The cell death rate significantly increased and the highest being about 50-60 percentage point in the system containing RTB.