Our current energy sources are largely non-renewable and actively being depleted. Furthermore, the greenhouse gases produced by fossil fuel consumption are harmful to the atmosphere. Hydrogenases are naturally-occurring enzymes that catalyze the reversible reduction of H2. However, natural hydrogenases are complex and produce low yields. Therefore, artificial hydrogenases are being engineered to produce enzymes with higher yields and that work under virtually any environmental condition. The Chakraborty group (University of Mississippi) converted a copper storage protein into a nickel binding protein (NBP), which functions as an artificial hydrogenase. To improve the yield and efficiency of the H2 reduction reaction, three mutants were designed: NBP_Tyr, NBP_L65R, and NBP_L58E. However, expression from the original recombinant vector, pET29α(+), yielded weak expression and failed purification. Therefore, we sought to improve expression, yield, and purity by using a different solubility tag. By adding an N-terminal Small Ubiquitin-like Modifier (SUMO) tag, all three mutant proteins were expressible in E. coli. The NBP_Tyr mutant was readily purified to &gt;90% purity at 87μM along with the NBP_L65R mutant, purified and concentrated to ~10 μM. The NBP_L58E mutant appears less stable and requires additional modifications to the purification strategy. Because the SUMO tag strategy was successful, it will enable subsequent generations of engineered hydrogenases to be purified and characterized.

Improved expression of engineered nickel binding proteins by attachment of a SUMO tag

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.