Background: Clostridium thermocellum is a thermophilic and cellulolytic anaerobe that produces hydrogen during cellulose fermentation. Hydrogenases catalyze the oxidation or evolution of H2. Clostridium thermocellum contains three putative FeFe-hydrogenases (CtHydA1, CtHydA2 and CtHydA3) yet little is known about the physiological functions of these hydrogenases. Due to the novelty of CtHydA3, it was selected for the initial study. Methods: Genes encoding CtHydA3, a ferredoxin-like protein (Ct_3004), and three FeFe-hydrogenase maturation proteins (CtHydE, CtHydF, and CtHydG) were cloned into three plasmids and co-transformed into E. coli strains Rosetta (DE3) and BL21 (DE3) for heterologous expression. In addition, a 6X His-tag sequence was fused to a C-terminus of CtHydA3. Protein expression was monitored by SDS-PAGE and western-blotting. The clarified crude extracts were passed over a TALON metal affinity column for the purification of His-tagged protein. Results: Recombinant plasmids harboring C. thermocellum hydA3, three maturation genes (HydE, HydF, and HydG), and ferredoxin were successfully constructed for the expression. Based on western blots, HydA3 hydrogenase protein was expressed in E. coli Rosetta (DE3), but not in E. coli BL21 (DE3) likely due to the differences in codon usage, especially arginine, glycine, isoleucine, between C. thermocellum and E. coli. The expression is further corroborated by a two-fold increase in in vitro hydrogenase activity in the soluble cell extract of the recombinant Rosetta strain, mediated by reduced methyl viologen. However, no difference in in vivo hydrogen production was detected in the recombinant Rosetta strain, suggesting an inability of the recombinant hydrogenase to contribute to the host’s hydrogen metabolism. The C-terminal His-tagged protein was bound to a metal affinity column to facilitate its purification, yet it yielded low levels of hydrogenase activity. Conclusion: we have developed a heterologous expression system for biochemical characterization.
Full conference title:
110th General Meeting American Society for Microbiology
- ASM 110th (2010)