Engineering a Penetratin-Conjugated Soybean Trypsin Inhibitor for Improved Insecticidal Activity Through Oral and Topical Delivery

Soybean trypsin inhibitor (STI) is a plant defense protein with established activity against insect digestive proteases. In the present study, a recombinant STI-penetratin fusion protein (rSTIP) was developed to evaluate its insecticidal activity following oral and topical exposure. The codon-optimized rSTIP construct was cloned into pET-28a(+) and expressed in E. coli BL21(DE3). The recombinant protein was predominantly recovered as inclusion bodies and subsequently solubilized, refolded, and purified using Ni-NTA affinity chromatography. Two refolding strategies were evaluated: on-column refolding and glutathione-assisted dialysis. Glutathione-assisted refolding followed by dialysis resulted in the highest recovery of trypsin inhibitory activity (65.5%). LC-MS/MS analysis provided sequence-level confirmation of the STI component. In Drosophila melanogaster larvae, rSTIP exhibited stronger insecticidal activity than the native STI following both oral and topical exposure, with topical application resulting in complete larval mortality within 36 h. Although direct protein penetration or cellular uptake was not evaluated in this investigation, the increased insecticidal activity of STI after conjugation with penetratin suggests improved delivery as a possible mechanism. In the present study, a recombinant STI-penetratin (rSTIP) fusion protein was developed to investigate whether fusion with penetratin could alter the insecticidal activity of STI.