Recombinant Expression,Purification And Antimicrobial Activity Of Antimicrobial Peptide Mytichitin-CB

In today’s life,Antibiotics have been widely known and widely used for its rapidly effect and convenience.The heavy use of antibiotics led to a number of serious food safety problems and environmental pollution,such as drug resistance and antibiotic residues,the latter has become a national and world-wide public interest in food safety issues.Researching and developing alternative to antibiotics and effective bactericidal antimicrobial new drugs is impending.Antimicrobial peptides are recognized around the world as one of the most promising alternative to antibiotic products,their main advantages include:broad antibiogram,improving immunity,hot stability.This study used genetic engineering techniques to express Mytichitin-CB which was an antimicrobial peptide of defensing family in E.coli BL21 and pichia pastoris,it provided an effective way to express high purity and high activity of antimicrobial peptides.Mytichitin-CB,a novel antimicrobial peptide with 55 amino acid residues,was isolated from the hemolymph of Mytilus coruscus.This antimicrobial peptide,with molecular mass of 6.3 kDa,was characterized by 6 Cysteine residues engaged in three intra-molecular disulfide bridges.Mytichitin-CB gene was cloned into three prokaryotic expression vectors:pMAL-c2x vector,pGEX-2T vector,pET-28a(+)vector.Then following the procedure below:transforming the expression vectors to E.coli BL21 respectively,inducing hosts with Isopropyl-thio-galactoside(IPTG),purifying proteins successfully expressed using affinity column.The results show that two recombinant proteins,MBP-Mytichitin-CB and GST-Mytichitin-CB,were expressed,the first one was purified successfully.The recombinant protein MBP-Mytichitin-CB was degested by enterokinase to obtain the target protein Mytichitin-CB.The antimicrobial activity was determined for various indicator strains using well diffusion assay,the result revealed that Mytichitin-CB was no acrivity for indicator strains.Mytichitin-CB gene was cloned in eukaryotic expression vector pPIC-zaA(anti-zeocin),then the vector linearized with endonuclease SacI was transformed into pichia pastoris GS115 by electroporation.The recombinant protein was expressed successfully with molecular mass of 7.7 kDa.The results of well diffusion assay showed that the recombinant Myrichitin-CB had antimicrobial actitivity against Bacillus subtilis(Lzz-133,151-1,L300-1).The MIC result showed that the recombinant mytichitin-CB had significant antimicrobial activity against B.subtilis Lzz-133(66.7 μg/mL),151-1(66.7μg/mL),L300-1(50 μg/mL).The optimal fermentation conditions were that the fermentation time chose 96 h,methanol concentration was 1%(v/v),the cell density was 30 g/L,the volume of 100 mL flask chose 15 mL.The quality of Mytichitin-CB increased 19.3%from 123.9 mg/L to 147.8 mg/L.