Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin.
A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.
Davasse
V
V
Département de Biologie Moléculaire et Structurale, Laboratorie Métalloprotéines, C.E.N.G., Grenoble, France.
Moulis
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003013:Clostridium / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D005813:Genes, Synthetic / Q000235:genetics*; D006864:Hydrogenase / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013053:Spectrophotometry
D005288:Ferredoxins; D011994:Recombinant Proteins; D006864:Hydrogenase; D015252:Deoxyribonucleases, Type II Site-Specific