Development of an intein-based bioprocessing platform for recombinant production of a potent antimicrobial peptide: Lasioglossin-III

Abstract

The rapid emergence of antimicrobial resistance has intensified the demand for novel anti-infective agents, positioning antimicrobial peptides (AMPs) as promising therapeutic alternatives. However, large scale production of AMPs remains challenging due to their small size, cationic nature, host toxicity, and high cost of chemical synthesis. In this study, we report an efficient and economically viable intein-mediated recombinant bioprocess for the production of the short antimicrobial peptide Lasioglossin-III (LL-III) in its C-terminally amidated form using Escherichia coli-based expression platform. A codon-optimized LL-III gene was fused to the Mxe GyrA intein along with a chitin binding domain (CBD) using the pTWIN1 expression system, enabling stable expression, affinity purification, and controlled auto-cleavage. The fusion protein was expressed in soluble form at low temperature and purified using chitin resin under optimized binding conditions. Notably, the chitin resin retained its binding capacity across multiple reuse cycles, improving the overall process-economics; auto-cleavage under mild reducing conditions released the mature peptide, which was confirmed by RP-HPLC and LC/MS to be identical to chemically synthesized standard peptide. The optimized process yielded ∼4.4 mg L⁻¹ of recombinant peptide leading to significant reduction in production costs at the current cleavage efficiency. Overall, this study establishes a scalable, environmentally sustainable, and cost-effective bioprocessing platform not only for short AMPs, but also for other peptide-based therapeutics as well.