Construction And Expression Of Tandem Multi-copy Gene Edcoding CA(1-8)ME(1-10) Hybrid Fused Acidic Peptide

Construction and Expression of tandem multi-copy gene edcodingCA(1-8)ME(1-10) hybrid fused acidic peptideTo improve antimicrobial peptide's expression, a novel mass-production method is proposed. It is based on the neutralization of the positive charges of antimicrobial peptide by fusing to an acidic peptide to avoid the lethal effect of the expressed peptide on the host cells. We designed and synthesized two DNA fragments encoding cecropin A(l-8)-melittin(l-10) hybrid and an acidic peptide, respectively. The two fragments were ligated to a whole fusion peptide gene, next was ligated with two adapters which have the same cohesive end (ATG/TAC), respectively. The whole gene was added every six hours instead of all in one time. In proper time, they were mixed and continued to ligate to multi-copy in the same direction. Proper multi-copy gene was selected and cloned into pUC19 vector. Restriction enzyme analysis and DNA sequencing confirmed that 5-copy gene was correctly inserted into the vector. The 5-copy gene was cloned into expression vector pBV220, pGEX-4T-l andpET21a digested with EcoR I and Sal I .After verified by sequencing, recombinant plasmids are transformed into several E.coli hosts. The 5-copy gene was expressed when induced in different hosts. SDS-PAGE analysis showed three recombinant plasimids all produced induced expression bands that accord with expected. Especially, the recombinant pET21a having 5 copy gene in E.coli BL21(DE3) got high expression and expression level was up to 25% of the total bacterial proteins. The analysis of product solubility revealed that 5-copy fusion protein mainly existed as inclusion body. The inclusion body was solulized in 8M Urea and purified by Poly-His protein purification kit. The product was further cut by CNBr and purified by CM ion-exchange chromatography and CA-ME hybrid was got. The purified CA-ME hybrid appears evident antibacterial activity with method of agar diffusion.