Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Abstract

BackgroundRecombinant 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.Principal FindingsWe 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−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1).SignificanceThe [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.

Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii

“…Purification was carried out under strict anaerobic conditions in either a Coy Chamber or a MBRAUN glove box. Hydrogenase activity of pure HYDA1 was verified using the reduced methyl viologen assay as earlier reported (34).…”

“…For FNR1, the protein was released from the Talon metal affinity resin by overnight incubation at 4°C with 500 units of thrombin. HYDA1 (from C. reinhardtii) was heterologously expressed in E. coli and purified as described previously (34,35). Purification was carried out under strict anaerobic conditions in either a Coy Chamber or a MBRAUN glove box.…”

Hydrogen production at high Faradaic efficiency by a bio-electrode based on TiO2 adsorption of a new [FeFe]-hydrogenase from Clostridium perfringens

“…Recombinant expression of the hydrogenase CpHydA from Clostridium perfringens SM09 [22] was performed in E. coli 30 Rosetta2(DE3) as previously described [31] upon transformation of the competent cells with plasmid pECPF2655, obtained by ligating the entire coding sequence of CPF_2655 into the empty expression vector pECr1 [23,24] between NdeI and XhoI sites. After the induction, cells were incubated over night under pure argon flow to maintain anaerobic conditions in a water bath at 30°C.…”

Expression of different types of [FeFe]-hydrogenase genes in bacteria isolated from a population of a bio-hydrogen pilot-scale plant

“…[FeFe]-hydrogenases are the best performing hydrogen producing biocatalysts [16][17][18][19][20][21], with turnover frequency of up to 10 4 s -1 . These enzymes have a modular structure: in addition to the catalytically active domain (named H-domain), several other accessory domains may be present [17,22].…”