Converting a Thermostable Enzyme into a Binder of Explosive Molecules (Rapid Fire)

Converting a Thermostable Enzyme into a Binder of Explosive Molecules

Jennifer Haghpanah¹ and Scott Banta¹

¹Department of Chemical Engineering, Columbia University

The search for proteins that bind to small molecules is driven by the potential to develop sensors applications such environmental monitoring. Antibodies are generally utilized for molecular recognition; however, they can be difficult to generate and are generally unstable. By evolving a thermostable enzyme, (alcohol dehydrogenase (AdhD) from Pyrococcus furiosus) into a selective binding protein, we aim to overcome the problems associated with yield, purity, stability, and cost of antibodies. Cyclotrimethylenetrinitramine (Royal Demolition Explosive, RDX) is a harmful chemical and a common military-grade explosive that contaminates many environments around the world. The current detection methods for this explosive are expensive and time consuming. We are evolving the AdhD enzyme into a binder with affinity for RDX. Multiple AdhD libraries were generated by randomizing sites within and outside the cofactor binding pocket. Ribosome display techniques were employed to identify library candidates demonstrating an affinity towards RDX. To further investigate where the binding was taking place, we truncated the enzyme and confirmed the importance of the cofactor pocket for the binding event. After multiple rounds of selection, clones have been identified from several libraries and we are currently introducing diversity to our converged sequences to obtain a high-affinity binder. Promising candidates are being screened via enzyme-linked immunosorbent assay (ELISA) and isothermal calorimetry (ITC). The assay method ensures the mutants retain their thermal stability which will make them superior for the development of new biosensors for RDX detection.