Expression of a recombinant peptide with bioinsecticidal potential for the control of agricultural pests

Soybean production faces persistent pressure from the defoliating pest Anticarsia gemmatalis. Here, we report the design, production, and characterization of a recombinant chimeric peptide (GORE 1-2 T) with bioinsecticidal potential targeting digestive trypsins from Anticarsia gemmatalis (A. gemmatalis). A synthetic gene encoding GORE 1-2 T-based on trypsin-binding tripeptides-was cloned into pET-41(a)+ and expressed in Escherichia coli; among the strains tested, BL21(DE3)pLysS yielded the most consistent production as a GST fusion, predominantly in the insoluble fraction. The peptide was purified after GST cleavage, and its identity was confirmed by Tris-tricine SDS-PAGE with silver staining and LC-MS (≈4.6 kDa). Enzyme kinetics using midgut trypsin-like fractions demonstrated competitive inhibition by GORE 1-2 T with an apparent Kᵢ ≈ 100 μM, consistent with prior tripeptide data and indicative of engagement of multiple subsites (S1-S3). In silico analyses supported the experimental findings: docking located GORE 1-2 T at/near the catalytic cleft, and 100-ns molecular dynamics simulations showed a stable backbone RMSD, a compact radius of gyration, and persistent hydrogen bonds, consistent with a well-structured, inhibitory complex. Collectively, these results validate GORE 1-2 T as a selective inhibitor of lepidopteran digestive serine proteases and establish a reproducible framework that integrates molecular modeling with heterologous expression for the development of next-generation bioinsecticidal peptides. This approach offers a more sustainable alternative to conventional insecticides and a plausible route toward crop protection applications.