Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

Figure 1

Models of tetrameric (a), Ni-free trimeric (b), and dimeric (c) forms of SHI. These are modified from [10] and are based on the cofactor and iron-sulfur cluster contents in sequence analysis. The proposed electron flow from NADPH oxidation to hydrogen evolution is also shown. Four different strains of P. furiosus were constructed to obtain the various forms of SHI. They are designated as MW400, MW430, MW434, and MW402 and their properties are listed in Table 2. These were used to prepare PF0891 Strep-tag II SHI [11], PF0891 9x-His tag SHI [12], PF0894 9x-His tag SHI [12], and PF0893 9x-His tag dimeric SHI [13], respectively. SDS PAGE gels show the purity of the different forms: (d) lane 1, Strep-tag II tetrameric SHI; (e) lane 2, 9x-His tag tetrameric SHI; lane 3, 9x-His tag Ni-free trimeric SHI; (f) lane 4, native SHI; lane 5, 9x-His tag dimeric SHI (PF1542 is an unrelated protein that is a persistent contaminant that copurified with dimeric SHI). The SDS PAGE gel data were modified from [11–13].

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