Expression Of Antibacterial Peptide BSN-37 By SUMO Technique In E.coli

At present,the irrational use of antibiotics is a serious social problem.The emergence of drug resistance strain and antibiotic residue in animal products highlight the need for more safe and effective therapy.Antimicrobial peptides are small defensive peptides produced by organisms to resist the invasion of foreign microorganisms,which characterize as potential substituting for antibiotics.The traditional extraction method of natural antimicrobial peptides such as extraction from organisms and chemical synthesis is too complex and long operation time.Therefore,expression it in vitro through genetic engineering technique will become the major direction in future.State-controlled bharat sanchar nigam 37(3.3 k Da)is one of the antibacterial peptides in our laboratory with independent intellectual property rights which consist of 37 amino acids(sequence is: FRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPP)and appeared good antibacterial activity.In order to settle the deficiencies in traditional extraction methods,a BSN-37 fusion expression plasmid was successfully constructed in E.Coli in this study.The 126 bp BSN-37 genes which reverse translated according to the amino acid sequence and the codon preference characteristics of E.Coli was cloned into PUC 57 vector and named PUC-BSN-37.After the expression vector p GEX-6p-1 and the target gene were digested by Bam H I and Xho I respectively,T4 DNA ligase was employed to ligated and transformed into E.Coli DH 5? and constructed new recombinant plasmid p GEX-6p-1-BSN-37.The recombinant plasmid was further identified by PCR,plasmid digestion and sequencing.The constructed positive plasmid was transformed into E.Coli BL21(DE3)for expression,and SDS-PAGE andWestern Blot analysis showed that an obvious expression of GST –tag in the empty carrier engineering bacteria at 26 k Da,while the fusion protein containing BSN-37 had low expression at30 k Da.In order to increase the expression level of the antimicrobial peptide BSN-37,we selected the molecular chaperone SUMO as fusion expression system expressed the antimicrobial peptides in E.Coli.Similar to plasmid p GEX-6p-1-BSN-37,we constructed the recombinant plasmid p Cold-SUMO-BSN-37.The recombinant plasmid was identified by PCR,plasmid digestion and sequencing.To exploreed the best expression condition,the constructed positive plasmid was transformed into E.Coli BL21(DE3),and the expression conditions were optimized.It was demonstrated the fusion protein expressed perfect that induced by 0.4 ?M IPTG for 6h in 37 ?and p H 7.2,The SDS-PAGE assay further indicated the fusion protein expressed in the soluble form.To remove the His tag in N-terminal of BSN-37,the target protein was purified by affinity chromatography and detected by Western Blot.The purified protein was incubated with the SUMO protease at 4 ?for 12 h,and then obtained the target protein after concentrated and desalted.The activity of the target protein was detected by Double layer AGAR diffusion test.The results showed that the target protein was about 25 k Da,while the empty carrier was about 19 k Da,which was consistent with the expected results.The target protein has good antibacterial activity against Salmonella CVCC541.Above all,we constructed a fusion expression system of antimicrobial peptide in E.coli.From it,we obtained high purity BSN-37 that exhibited antimicrobial activity.This researchestablished a foundation for further study on the expression of bovine antimicrobial peptide and the development of new antimicrobial drugs.