Prokaryotic expression and purification of the efaA protein from Enterococus faecalis antigens / 中国人兽共患病学报

ABSTRACT

To prokaryotic express prokaryotically and to purify the efaA protein from Enterococus faecalis so as to provide the basis for the further study on the pathogenesis and clinical sero-diagnosis of endocarditis caused by E.faecalis, efaA gene of E.faecalis was amplified by PCR, the PCR-amplified product was digested with restriction enzymes and cloned into prokaryotic vector pET32a to construct the recombinant plasmid pET30a/efaA. This recombinant plasmid was confirmed by double enzyme digestion with BamhI and Xhol and then subjected to sequencing, and transformed to E.coli BL21 (DE3). Expression of the fusion protein was induced by IPTG, and analyzed by SDS-PAGE and Western blotting. The recombinant fusion protein was purified by His-binding affinity chromatography.It was shown that efaA gene of 943 bp in size was amplified from Enterococus faecalis and the recombinant plasmid pET30a,/ efaA was successfully constructed and expressed in E.coli BL21. The purified product was found to be 34 kDa in molecular weight as demonstrated by SDS-PAGE and Western blotting. It is evident that the efaA protein of E.faecalis can be successfully expressed and purified.