Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production
Figure 7
Plasmid pEA2-SHI and pET-A-SHI were used to express rSHI in E. coli, and pET-A-SHI was used to generate SHI with a Gateway sequence and TEV recognition site attached to the N-terminal of PF0891 subunit. The plasmid pC11A2-CDABI contains the operons hypCD and hypAB, hycI from P. furiosus. The operons hypCD and hypAB, hycI of P. furiosus were linked by an intergenic region shown in Figure 3c. The Gateway sequence is located at the N-terminus of the HypC protein. The plasmid pC3AR2-SdFa has slyD and frxA of P. furiosus cloned behind Phya, the recombinant SlyD has the N-terminal Gateway sequence/TEV site. Plasmid pRA2-EF was constructed to co-express HypE/HypF, the same linker as in the plasmid pC11A2-CDABI or pC3AR2-SdFa, is inserted between hypE and hypF gene, and HypE contains the N-terminal Gateway sequence.