One-step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of Strep-tag affinity chromatography.
The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron-sulfur clusters. Here, we report the one-step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep-tag II peptide to the C-terminus of NuoF or NuoG. Fusion of this peptide to the N-terminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment.
Bungert
S
S
Heinrich-Heine-Universität Düsseldorf, Institut für Biochemie, Universitätsstr. 1, D-40225, Düsseldorf, Germany.
Krafft
B
B
Schlesinger
R
R
Friedrich
T
T
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification*; D009245:NADH Dehydrogenase / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D016660:NAD(P)H Dehydrogenase (Quinone); D009245:NADH Dehydrogenase