Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli.
Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.
D004251:DNA Transposable Elements; D003901:Desulfovibrio / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D006864:Hydrogenase / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D006864:Hydrogenase