Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems

Abstract

Bovine adrenal chromaffin granule cytochrome (cyt) b 561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-β-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b 561 in insect and yeast cell systems. The bovine cyt b 561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (∼0.5 mg detergent-solubilized cyt b 561/L culture). For the yeast system, the cyt b 561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b 561 expression (∼0.7 mg detergent-solubilized cyt b 561/L culture). Recombinant His-tagged cyt b 561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni–NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.