Recombinant Expression of Hydrogenases

The C. reinhardtii hydA1 and C. pasteurianum hydA genes were used for expression of the HydA1 and CpI [FeFe] hydrogenases, respectively. Both genes were previously codon-optimized for expression in E. coli[40] (link). The coding sequencing for a C-terminal Strep-tag II® extension (IBA GmbH) with a two residue linker (5′-SAWSHPQFEK-3′) was added by PCR amplifying the hydrogenase genes from the plasmids pY71 shydA1*[8] (link) and pK7 shydA[40] (link). PCR products were then cloned into the pET-21(b) expression vector (Novagen). The plasmid pACYCDuet-1–hydGX–hydEF[8] (link) was used for expression of the S. oneidensis [FeFe] hydrogenase maturases HydE, HydF, and HydG. Multiple cloning sites I and II contain the hydGX and hydEF nucleotide sequences, respectively. The hydX sequence (Accession code AAN56899) is a part of the S. oneidensis [FeFe] hydrogenase operon and encodes a soluble protein with no identified functions. The petF gene from Synechocystis sp. PCC 6803 was PCR amplified from the pK7 expression vector and cloned into the pET-21(b) plasmid [41] (link). All expression constructs were confirmed by DNA sequencing and transformed into the E. coli strains BL21(DE3) (Novagen) and BL21(DE3) ΔiscR by selection with the appropriate antibiotics.