Abstract
Antimicrobial peptides (AMPs) have significant potential as alternatives to classical antibiotics. However, AMPs are currently prepared using processes which are often laborious, expensive and of low-yield, thus hindering their research and application. Large-scale methods for production of AMPs using a cost-effective approach is urgently required. In this study, we report a scalable, chromatography-free downstream processing method for producing an antimicrobial peptide, pexiganan, using recombinant Escherichia coli (E. coli). The four helix bundle structure of the unique carrier protein DAMP4 was used to facilitate a simple and cheap purification process based on a selective thermochemical precipitation. Highly pure fusion protein DAMP4var-pexiganan was obtained at high yield (around 24 mg per 800 mL cell culture with a final cultivation OD600 ~ 2). The purification yield of DAMP4var-pexiganan protein is increased twofold with a 72.9% of the protein recovery in this study as compared to the previous purification processes (Dwyer in Chem Eng Sci 105:12–21, 2014). The antimicrobial peptide pexiganan was released and activated from the fusion protein by a simple acid-cleavage. Isoelectric precipitation was then applied to separate the pexiganan peptide from the DAMP4var protein carrier. The final yield of pure bio-produced pexiganan was 1.6 mg from 800 mL of bacterial cell culture (final cultivation OD600 ~ 2). The minimum bactericidal concentration (MBC) test demonstrated that the bio-produced pexiganan has the same antimicrobial activity as chemically synthesized counterpart. This novel downstream process provides a new strategy for simple and probable economic production of antimicrobial peptides.
Highlights
Over the past few decades, the vast number of studies on antibiotics has greatly stimulated the discovery and development of modern medicines
In order to obtain D AMP4var-pexiganan protein from E. coli cells, protein was recovered from cells by sonication, and the released protein was purified by three main consecutive steps: (1) precipitation of DNA; (2) precipitation of protein contaminants; and (3) isolation of DAMP4var-pexiganan protein
This paper presents a new downstream processing method to produce antimicrobial peptides from recombinant E. coli based on selective thermochemical lysis (Dwyer et al 2014) and precipitation (Wibowo et al 2017) methods
Summary
Over the past few decades, the vast number of studies on antibiotics has greatly stimulated the discovery and development of modern medicines. The utility of antibiotic drugs has been increasingly viewed as limited, and potentially harmful long-term due to their tendency to cause bacterial resistance (Andersson et al 2000). Antimicrobial peptides (AMPs) has received significant attention as an ideal alternative to classical antibiotics because of their unique antimicrobial properties (Fox 2013). The antimicrobial properties of AMPs as well as their mechanisms of action have been documented against a broad spectrum of pathogenic microorganisms (Brogden 2005). AMPs can effectively disrupt bacterial cell membranes causing a quick leakage of the intracellular materials (Matsuzaki et al 1995a). The rapid antimicrobial activity and quick killing effect of AMPs make it difficult for microorganisms to develop resistance against AMPs (Guaní-Guerra et al 2010). Many AMPs exhibit toxicity against prokaryotic cells but not against eukaryotic cells such as human cells due to different components of their
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