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∼16 500). 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 ∼10 mg/l.