Cloning and Overexpression of Staphylococcal Enterotoxin B gene synthetic (SEBsyn) in pET-28a: Pre-development bacterial-toxin therapy for cancer

Abstract

Staphylococcal enterotoxin B (SEB) from Staphylococcus aureus is a potential therapeutic agent for cancer. SEB can activate the immune response, which could induce apoptosis of various cancer cells. This study aimed to design and produce a recombinant SEB protein using Escherichia coli BL21(DE3) expression system. We designed and optimized the codon of the inserted gene for E. coli and then transformed it into E. coli BL21(DE3). The transformants were verified by selective media containing kanamycin followed by colony PCR with T7 promoter and T7 terminator primers and DNA sequencing. The results showed that the SEBsyn encoding gene could be synthesized and cloned into pET-28a(+) expression plasmid. This recombinant plasmid that carried the SEB encoding gene (pET-28a_SEBsyn) was successfully transformed into E. coli BL21(DE3). The amplicons of colony PCR visualized in 2% agarose gel showed that transformants carrying the recombinant plasmid had an inserted gene length of about 1 kb. Gene inserts sequence verification by DNA sequencing resulted in 1,148 bp sequence consensus. The blastx analysis showed that it had the best hit with enterotoxin B in the NCBI database accession id CAC6284025.1. The recombinant SEB protein was also successfully overexpressed in E.coli BL21(DE3) with 0.1 mM IPTG. Our recombinant SEB protein was dominantly insoluble in the inclusion body; however, some SEB protein was expressed as soluble. The molecular weight of the target protein is about 29 kDa.