Development of a high-level expression system of an antimicrobial peptide histonin in Escherichia coli

Abstract

The increasing resistance of bacteria and fungi to currently available antibiotics is a major concern worldwide, leading to enormous efforts to develop new antibiotics with new modes of actions. One potential source of novel antibiotics is the antimicrobial peptides (AMPs). AMPs have received increasing attention as new antimicrobial substances because they possess antimicrobial activity against pathogenic microorganisms that are resistant to the conventional antibiotics with minimal inhibitory concentrations as low as $0.25 \mdash 4 \mu g/ml$. Numerous biological expression systems have been introduced for cost-effective production of AMPs because a large quantity of AMPs needs to be produced economically for pharmaceutical applications. However, direct expression of AMP in $Escherichia coli$ causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. Herein this study, an efficient method for the mass production of an intact histonin has been developed. Histonin (RAGLQFPVGKLLKKLLKRLKR) is a hybrid peptide $BUF II(5-13)[KLLK]_2RLKR$, which consists of the extended \alpha-helical region containing a proline hinge BUF II(5-13) and three \alpha-helical motifs $[KLLK]_2RLKR$. The hybrid peptide histonin showed stronger antimicrobial activity against a broad spectrum of pathogenic microorganisms than BUF II and also has antitumor activity against mouse xenograft models. In this study, the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C-terminus of histonin, Furin cleavage of the multimeric hist...