Improved production of holotoxin Stx2 with biological activities by using a single-promoter vector and an auto-induction expression system

Abstract

The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinant Shiga toxin 2 (rStx2). rStx2 was expressed in the E. coli periplasm in a completely soluble, biologically active form. The final yield of purified rStx2 from each liter of culture in LB medium and ZYM-5052 medium was approximately 2.3 mg and 3.5 mg, respectively. The highest amount of rStx2 accounted for 27.8% of total bacteria protein in ZYM-5052 medium. rStx2A and rStx2B isolated from rStx2 by electroelution were, respectively, identified by N-terminal protein sequencing. Signal peptides with the sequence MKCILFKWVLCLLLGFSSVSYS and MKKMFMAVLFALASVNAMA were identified at the N terminus of rStx2A and rStx2B, respectively. Our rStx2 possessed Vero cell CD 50 value about 500 pg and LD 50 value approximately 6 ng. rStx2 can be substitute for natural toxin Stx2, which can be used for animal models, drug screening, vaccine research, and so on.