Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase
Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.
- Research Organization:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22), Division of Chemical Sciences, Geosciences and Biosciences
- DOE Contract Number:
- AC36-08GO28308
- OSTI ID:
- 1050106
- Report Number(s):
- NREL/JA-2700-52560; TRN: US201218%%477
- Journal Information:
- PLoS One, Vol. 7, Issue 4; Related Information: Article No. e35886
- Country of Publication:
- United States
- Language:
- English
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