(A) study on the production of antimicrobial peptides with a novel genetic engineering approach in Escherichia coli

Abstract

The emergence and rapid horizontal spread of antibiotic-resistant traits in bacteria of human and veterinary clinical significance has been a driving force in the search for new classes of antibiotics. Antimicrobial peptides (AMPs) have received increasing attention as new antimicrobial substances because these peptides show a direct killing activity against a wide range of microorganisms and they possess antimicrobial activity against pathogenic microorganisms that are resistant to the conventional antibiotics with minimal inhibitory concentration as low as 0.25-4 μg/ml. Numerous biological expression systems have been introduced for cost-effective production of AMPs for pharmaceutical applications. Among them, expression of AMPs 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. Here, we describe a novel prokaryotic expression system for the production of cationic AMPs. The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human interferon-γ) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the co-expressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, about 100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from a 1 L Escherichia coli culture. In addition, fed-batch fermentations were carried out fo...