High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes in Escherichia coli.
A novel strategy for constructing multiple joined genes of acidic partner-mediated antimicrobial peptide is described. This strategy allows the expression of antimicrobial peptide by Escherichia coli in a stable form and with high yield. Cecropin A (1-8)-melittin (1-10) (CAME) hybrid peptide was selected as a model of antimicrobial peptide. An acidic fragment from magainin intervening sequence was fused to the antimicrobial peptide as a partner to neutralize the lethal effects on the host cells. Multiple copies of the fusion peptide gene were tandemly linked and cloned into the expression vector pET21a. Multimers were expressed at high levels, reaching up to 36% of total cell proteins, and expression levels were proportional to the degree of multimerization. The fusion proteins were mainly expressed as inclusion bodies, probably owing to cysteine residues in the multimers. The target CAME peptide was obtained by cleaving the multimers with cyanogen bromide and purified by cation-exchange chromatography. Recombinant CAME peptide showed strong antimicrobial activities against both Gram-negative and -positive bacteria. These results might provide an efficient solution for high-level expression of various kinds of antimicrobial peptides that are toxic to the host.
Wang
Yun-Qi
YQ
Department of Chemistry, Jinan University, Guangzhou, 510632, The People's Republic of China.
Cai
Ji-Ye
JY
D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D023181:Antimicrobial Cationic Peptides; D002412:Cations; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003545:Cysteine