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.