Coprinolysin pore-forming protein for targeted insect cell receptor identification

Overview

This research program investigates Coprinolysin, a pore-forming protein natively found in the fungus Coprinopsis cinerea, as a model system for understanding how protein-based pesticides recognize and kill specific insect cells. Preliminary in vitro studies have shown that Coprinolysin substantially reduces the viability of Spodoptera cell lines while leaving Drosophila cell lines completely unaffected, demonstrating species-specific targeting. The program aims to characterize the receptor binding domain of Coprinolysin to enable rational design of protein pesticides with customizable insect specificity, addressing an under-reported area of how pore-forming proteins interact with their target receptors.

Technical specifications

Key features:

• Species-specific activity: Demonstrated selective toxicity against Spodoptera insect cell lines with no effect on Drosophila cells, confirming receptor-mediated specificity
• Aerolysin-like pore-forming mechanism: Coprinolysin utilizes a pore-forming mechanism previously unobserved in this protein family
• Chimeric protein engineering: A designed chimera combining the receptor binding domain of a related family member with the pore-forming region of Coprinolysin successfully redirected targeting to a different cell type in vitro
• Proximity labelling approach: Construction of a miniTurbo biotinylation tag fusion with Coprinolysin enables identification of target receptors within approximately 10 minutes of labelling
• Mass spectrometry identification: Labelled target receptors can be enriched and identified using standard mass spectrometry workflows
• Generalizable methodology: The proximity labelling approach may serve as a general strategy for identifying pore-forming protein–target interactions across related family members

Technology readiness level

This research is at an early experimental stage. The proof-of-concept has been established through in vitro assays demonstrating selective insect cell killing and successful chimeric protein retargeting. The receptor binding domain of Coprinolysin has not yet been characterized, which represents the core of the proposed future validation work. The next phase involves constructing and testing a miniTurbo-Coprinolysin fusion protein against insect cell lines including Sf9 and Hi5 to identify target receptors via mass spectrometry. The methodology is positioned as a foundational research tool with potential downstream applications in protein pesticide design, though commercial product development would require further validation and optimization beyond the current scope.