Fusion Expression Of Antimicrobial Peptides And Their Activity Analysis

Antibiotic resistance has become a globle problem, thus developing newantibiotics has been a research hotspot of this area. AMPs found among majority ofliving organisms are usually positively charged and amphipathic molecules of lowmolecular weight, with broad-spectrum antimicrobial activity. Compared withantibiotics, AMPs don’t have the problem of antibiotic resistance. It is believed thatAMPs could serve as new drug candidates for pharmaceutical application instead ofantibiotic, which have tremendous prospect and pratical value. The principallimitation for promoting the clinical use of antibacterial peptides is the highproduction cost. Nowadays AMPs production by genetic engineering is widely used.In this research, GST was chosen as fusion tag to produce nine kinds of commonAMPs in E.coli expression system.Nine kinds of AMPs were screened from NCBI by bioinformatic method,named GK10, LKW, Fowlicidin-3, Hclocidin18, KSLS, RM12, RM13, Magainin IIand SPFK. Based on their amino acid sequences and according to the codonpreference of E.coli, nine pairs of primers with reversed complementary nucleotidesequences were designed and synthesised. The sequence of hydroxylaminehydrochloride cleavage site (Asn-Gly) was introduced at the5’-terminal of eachupstream primer. Nine target fragments of AMPs were amplified by PCR, whichthen were inserted into express vector pGEX-4T-1to construct recombinant plasmidspGEX-4T-1-AMPs. The identified recombinant plasmids were transformed intoE.coli BL21(DE3) and induced by IPTG. The analysis of12%SDS-PAGE andWestern blotting showed that the nine fusion protein GST-AMPs were allsuccessfully expressed and mainly in the supernatant, with molecular weightscorresponding with their theory values. Nine fusion proteins purified by GlutathioneSepharose4B affinity chromatography were then cleavaged in hydroxylaminecleavage buffer to release antimicrobial peptides. The antibiotic activity against bothEscherichia coli and micrococcus lysodeikicus was tested by inhibition experiment,with PBS buffer and uncleavaged fusion protein as negative control. There were six peptides (F3, GK10, Hc18, LKW, M2and RM13) showing antibacterial activitityagainst E.coli and micrococcus lysodeikicus contrast with negative control by agardiffusion method. The results of microdilution antifungal susceptibility test showedthat GK10has the highest antibacterial activity, then F3, Hc18, LKW, M2and RM13were of gradually decrease. This study would provide reference for the feasibility ofusing E.coli expression system to produce AMPs，and lay a good foundation forscreening new antimicrobial drugs and antimicrobial peptides industrialization.