Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant.
The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the Spinacia oleracea fused to MalE. Overexpressed fusion proteins were predominantly found (from 55 to 70%) in cytoplasm in a soluble form. The single affinity chromatography step (amylose resine) was used to purify about 15mg of protein from 1 liter of E. coli culture. The isolated proteins were electrophoretically pure and could be used for further experiments. The NifS-like protein IscS from the cyanobacterium Synechocystis PCC 6803 mediates the incorporation of 2Fe-2S clusters into apoferredoxin and cyanobacterial Rieske apoprotein in vitro. Here, we used the recombinant IscS protein for the enzymatic reconstitution of the iron-sulfur cluster into full-length Rieske fusion and truncated Rieske fused proteins. Characterization by EPR spectroscopy of the reconstituted proteins demonstrated the presence of a 2Fe-2S cluster in both full-length and truncated Rieske fusion proteins.
Institute of Biochemistry and Molecular Biology, Wrocław University, Tamka 2, 50-137 Wrocław, Poland.
Beata
B
Gubernator
Andreas
A
Seidler
Matthias
M
Rögner
Andrzej
A
Szczepaniak
D000688:Amylose / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D002845:Chromatography / Q000379:methods; D000458:Cyanobacteria / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D008280:Magnetics; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018724:Spinacia oleracea / Q000378:metabolism*; D013347:Subcellular Fractions
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C037989:Rieske iron-sulfur protein; D000688:Amylose; D014450:Electron Transport Complex III