Construction Of Prokaryotic Expression Of Bactofencin A

Bactofencin A,a type ?d bacteriocin,is a small unmodified cationic antimicrobial peptide originated from Lactobacillus salivarius DPC6502 which was isolated from pig intestines.It has strong inhibitory ability against a variety of Gram-positive bacteria,including Staphylococcus aureus and Listeria.In this study,the prokaryotic expression system for heterologous expressing of Bactofencin A was explored by soluble expression and secretory expression respectively.In the process of protein heterologous expression,exogenous genes usually face the problems of protein expression yield,solubility,purification,cytotoxicity and so on.In this study,the characteristics of peptide chain of Bactofencin A was analyzed,and the fusion expression strategy was established to protect the intact N-terminal and C-terminal structure while promoting dissolution and reducing host toxicity.Three Escherichia coli BL21(DE3)heterologous expression schemes were designed based on two aspects of soluble expression and one secreted expression.Among these different strategies,Bactofencin A gene was assembled with GST,SUMO,Yeb,rEK site,SP signal peptide,His-Tag and other molecular chaperone genes,and finally three independent fusion expression genes were designed.The soluble expression vectors p ET-His-SUMO-REK-BAC A,secretory expression vectors p ET-His-YEB-REK-BAC A and p ET-SP-GST-REK-BAC A were successfully constructed.The soluble expression vector p ET-His-SUMO-REK-BAC A was transfected and induced by IPTG,and the target fusion protein product was successfully expressed at about16 k Da.Subsequent detection results showed that His-SUMO-REK-BAC A fusion protein was mainly expressed in soluble form.After transfection and induction of the two secretory expression vectors,the expression products of the target fusion protein with about 17 k Da and 32 k Da were successfully detected in the periplasm of cells and the supernatant of bacterial fragmentation,respectively.After the subsequent fermentation conditions were optimized,the fusion protein yield of the three expression vectors was significantly increased.The purified protein products of His-SUMO-REK-BAC A and SP-GST-REK-BAC A showed weak inhibitory activity against S.aureus after digestion with enterokinase.The bacteriocin Bactofencin A was successfully expressed in Escherichia coli BL21(DE3)...