Purification of the membrane binding domain of cytochrome b5 by immobilised nickel chelate chromatography.
The purification of a eukaryotic membrane protein has been achieved using a prokaryotic expression system. Bovine cytochrome b5 is an integral membrane protein (Mr approximately 16500). It comprises of a globular haem containing catalytic domain positioned at the N-terminus of the protein and a hydrophobic membrane binding segment at the C-terminus. The membrane binding domain (MBD) is resistant to purification using conventional strategies that have proved successful in isolating the soluble haem containing fragment. We report here a versatile purification method for the isolation of the MBD involving a gene fusion system. The fusion protein incorporates thioredoxin at the amino terminus and six histidines as the metal affinity binding site followed by cytochrome b5 in a pET expression system. This supports high level expression of cytochrome b5 in E. coli C43(DE3) cells. The fusion protein is effectively solubilised from lysed cells with Triton X-100. A step gradient elution with imidazole under non-denaturing conditions on a His-Bind nickel chelate affinity column, saturated with proteins as a crude cell extract, purified the protein in a single step. Proteolytic digestion of pure fusion protein, with trypsin, yielded the MBD. This fragment was further purified by RP-HPLC to a final yield of approximately 10 mg/l.
Begum
R R
RR
Laboratory of Structural Biochemistry, Molecular and Cellular Biology, Queen Mary and Westfield College, University of London, UK.
Newbold
R J
RJ
Whitford
D
D
D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015786:Cytochromes b5 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006868:Hydrolysis; D009532:Nickel; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D009532:Nickel; D015786:Cytochromes b5; D014357:Trypsin