Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag inEscherichia coli

Abstract

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human β-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human β-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

Antimicrobial Peptide Structures: From Model Membranes to Live Cells

“…In particular, how lipid composition modulates the mechanism of AMP self‐assembly is a crucial piece of the puzzle still to be elucidated. This challenge may well be addressed by in‐cell DNP‐NMR studies using recombinant expression of labelled AMPs and bacteria that have been genetically engineered to have different membrane lipid compositions …”

Cathelicidins: Immunomodulatory Antimicrobials

“…Given their antimicrobial potential, recombinant expression in bacterial species is a challenge. Some methods like bacterial production of fusion proteins or inteins do exist, for example in E. coli [ 140 ], Bacillus subtilis [ 141 ], or the yeast Pichia pastoris [ 142 ]. The purified peptides often retain their antibiotic activity but are (in these examples) not tested for endotoxins or immunomodulatory activity.…”

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

“…IBs with high specific enzyme activity have been verified as potentially useful biocatalysts [36]. Target proteins were successfully released from active IBs upon cleavage of the intein between the peptide tag and the target protein [27]. It has become a promising method to develop a quick technique to enable protein expression and purification in bacteria.…”