(620ag) 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.