Design and production of a novel antimicrobial fusion protein in Escherichia coli

Abstract

In recent years, antimicrobial peptides (AMPs) have attracted increasing attention. The microbial cells provide a simple, cost-effective platform to produce AMPs in industrial quantities. While AMP production as fusion proteins in microorganisms is commonly used, the recovery of AMPs necessitates the use of expensive proteases and extra purification steps. Here, we develop a novel fusion protein DAMP4-F-pexiganan comprising a carrier protein DAMP4 linked to the AMP, pexiganan, through a long, flexible linker. We show that this fusion protein can be purified using a non-chromatography approach and exhibits the same antimicrobial activity as the chemically synthesized pexiganan peptide without any cleavage step. Activity of the fusion protein is dependent on a long, flexible linker between the AMP and carrier domains, as well as on the expression conditions of the fusion protein, with low-temperature expression promoting better folding of the AMP domain. The production of DAMP4-F-pexiganan circumvents the time-consuming and costly steps of chromatography-based purification and enzymatic cleavages, therefore shows considerable advantages over traditional microbial production of AMPs. We expect this novel fusion protein, and the studies on the effect of linker and expression conditions on its antimicrobial activity, will broaden the rational design and production of antimicrobial products based on AMPs.

Evaluation of baiting fipronil-loaded silica nanocapsules against termite colonies in fields

“…In this way, D4S2 can be produced renewably and cost-effectively in industrially-relevant bacteria Escherichia coli ( E. coli ) [44, 45]. Furthermore, the modular design allows D4S2 to be isolated and purified from E. coli using a scalable, non-chromatographic approach, that is, selective thermochemical precipitation [45, 51, 52, 53] (Fig. 2A).…”

In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics

“…However, large-scale production of synthetic small peptides is challenging [31] and recombinant protein production in prokaryotic host is presented here as a promising alternative. Several experimental strategies have been explored to cope with the inherent toxicity of such peptides, when recombinantly produced, towards the prokaryotic host, including the fusion to partner proteins that seem to mask the antimicrobial activity [32][33][34], as well as the accumulation in the insoluble cell fraction of AMPs fused to scaffold proteins [35]. In any case, regardless of the mechanism of action of alpha-helical AMPs against microorganisms, the recombinant production of these peptides might be achieved by the neutralization of their net charge by fusion to solubility-enhancing proteins [36][37][38] or by enhancing formation of IBs in producing cells by the fusion to aggregation-prone proteins [39].…”

Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides

“…This protein conformation makes DAMP4 an excellent candidate for enhancing solubility of the fused AMP, as well as facilitating purification without the use of expensive chromatography tools (see "Purification of fusion proteins"). An additional sequence is required as a spacer between DAMP4 and pexiganan to provide sufficient flexibility for pexiganan to kill pathogenic bacteria while still linking to DAMP4, which would otherwise difficult to achieve (Sun et al 2018b). Setting the post-induction temperature to 25°C rather than 37°C is highly important to maintain the conformation of pexiganan, hence its antimicrobial activity.…”

“…A few fusion tags has made non-chromatography based purification of fusion protein possible, that is, by using a combined elastin-like polypeptide (ELP) and intein (Müller et al 2015;Sousa et al 2016), and four-helix bundle protein DAMP4 (Sun et al 2018a;Sun et al 2018b;Wibowo et al 2017;Wibowo et al 2015;Zhao et al 2015).…”

“…After re-solubilization in a simple buffer (low salt concentration), DAMP4-pexiganan with high purity can be obtained. Importantly, this fusion protein can be directly used as an antimicrobial agent without releasing the pexiganan from the inactive DAMP4, showing the same minimum inhibitory concentration (MIC) with the chemically-synthetized pexiganan (Sun et al 2018b). This was made possible through the use of a long and flexible linker coupled with engineered bioprocess (i.e., low post-induction temperature) which allowed for the pexiganan to maintain its active conformationality even when it is still in the fusion form.…”