Expression of anthrax protective antigen in E.coli and purification and analysis of biological activity of expression products

Objective:To construct pET-32a(+)-PA recombinant plasmid,express the Trx-PA fusion protein in Escherichia coli BL21(DE3), and obtain recombinant PA protein with biological activity.Methods:The coding sequence of PA was ampilified by PCR and fused in frame with the coding region of Trx in the pET-32a(+) vector to generate the pET-32a(+)-PA recombinant plasmid which was transformed into competent E.coli BL21(DE3).Induced by IPTG,the pET-32a(+) fusion protein was expressed in E.coli BL21(DE3) and was characterized by SDS-PAGE and Western blot.Then,the expressed product was purified by Ni2+ affinity chromatography and renatured by dialysis.The biological activity of Trx-PA was detected by proteolytical cleavage and cytotoxicity assay.Results:The pET-32a(+) recombinant plasmid was successfully constructed and the Trx-PA fusion protein was purified.SDS-PAGE analysis indicated that the relative molecular weight of fusion protein was right.After purification and renaturation,the fusion protein reached a purity of 76%.The Trx-PA could be cleaved by furin in vitro and in vivo correctly.The cytotoxicity of LF for Raw264.7 cell was dependent on the digestion of Trx-PA.Conclusion: Recombinant Trx-PA fusion protein is efficiently expressed in E.coli BL21(DE3) and showed natural biological activities.