Developement of a biotechnological process suitable for the large-scale production of antimicrobial peptides

Abstract

The drug pollution of aquatic enviroments including surface water, groundwater and even drinking water is a growing concern. Antibiotics, which belong to the most useful family of pharmaceuticals, pose another threat aside from drug pollution, since their misuse and overuse can lead to the emergence of drug-resistant microbes. The ultimate goal of the interdisclipinary project called PharmCycle is the reduction of the drug pollution of aquatic environments. One approach for achieving this goal is the development of sustainable drugs. Antimicrobial peptides (AMPs) are regarded as a promising candidate for the developement of sustainable antimicrobial/antibacterial agents, since it is harder for microbes to develope resistance against them. AMPs mainly exert their antimicrobial effect by disrupting the cell envelope of the target cell. In order to establish AMPs as an alternative to antibiotics, it has to be produced in a sufficient quantity. Developing a biotechnological process aided by recombinant DNA technology has the potential of producing high yields of AMPs. This study, which is part of PharmCycle, focuses on developing a biotechnological process, that can be scaled up in order to enable production of greater amounts of the AMP NK2-ALK, which is derived from the porcine NK-Lysin. In the upstream segment of the process, a high cell density culture (HCDC) was performed using E. coli BL21 (DE3). NK2-ALK is linked to a carrier protein called Onconase, which leads to the expression of the fusion protein in an insoluble state (formation of inclusion bodies). Thus, the first step of downstream process was the disruption of the cells via high pressure homogenization followed by the solubilization of the inclusion bodies. The fusion protein had a relatively high purity ( 80%) after just one Immobilized metal ion affinity chromatography (IMAC) step. Due to the linker containing an acid susceptible motif, acetic acid was sufficient to seperate NK2-ALK from its fusion partner. The recombinant NK2-ALK retained its antimicrobial activity as verified by radial diffusion assay. The yield was determined to be up to 2:6mg of fusion protein per 1 g of wet biomass. Thus, the theoretical maximum amount of AMPs, which can be released from the fusion protein, is 0:51mg per 1 g of wet biomass