In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli.
We have developed a strategy for overproduction of spinach apocytochrome b6 as a fusion protein to maltose-binding protein (MBP) in Escherichia coli, using the expression vector pMal-c2. The fusion protein was purified to virtual homogeneity by gel filtration chromatography and the method of insertion of hemes into fusion protein was elaborated. The ambient and low-temperature absorption spectra of the reconstituted cytochrome b6 were similar to those of cytochrome b6 spectra in isolated proteins or cytochrome b6f complexes and are typical for bis-histidine ligated b-type cytochromes. Optical circular dichroism (CD) spectra of the visible region further confirmed the appropriate binding of hemes by the apocytochrome b6 protein. We found that the incorporation of hemes was required for the refolding of the cytochrome b6 protein into the more compact structure found in the native cytochrome protein. Heme staining experiments suggested that the two hemes in the reconstituted cytochrome b6 protein are bound with different affinities. The reconstituted cytochrome b6 protein was cleaved by Xa factor proteolysis from fusion protein and separated for characterization. The procedure presented in this work for reconstitution of hemes into the cytochrome b6 protein should provide an important tool for structure/function studies of membrane-bound cytochrome proteins.
Króliczewski
Jarosław
J
The Institute of Biochemistry and Molecular Biology, University of Wrocław, Tamka 2, Wrocław, Poland.
Szczepaniak
Andrzej
A
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000378:metabolism*; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D015951:Factor Xa