Expression and purification of two recombinant sterol-carrier proteins: SCPX and SCP2

We report the cloning, expression, and purification of the rat sterol carrier proteins SCPX and SCP2. The cDNA's encoding rat SCPX and SCP2 were isolated from a lambda gt11 rat liver cDNA library. To maximize expression and to facilitate the purification of the recombinant proteins, the SCPX and SCP2 proteins were expressed as carboxy-terminal fusion proteins to the glutathione S-transferase (GST). The GST-SCPX and GST-SCP2 fusion proteins contained a thrombin recognition site between the GST and SCPX or SCP2 polypeptides. The expression of the fusion proteins was controlled by the inducible tac promoter. Under optimal conditions, the approximately 85-kDa GST-SCPX and the approximately 41-kDa GST-SCP2 proteins represented approximately 1-2% of the total cell lysate. Both fusion proteins were easily purified under nondenaturing conditions from the soluble fraction of total cell lysate by glutathione-Sepharose 4B affinity chromatography. Thrombin cleavage resulted in the release of the SCPX and SCP2 proteins from the GST-SCPX and GST-SCP2 fusions, respectively. Amino terminal protein sequencing confirmed the authenticity of the recombinant proteins. Furthermore, functional assay revealed that recombinant SCP2 is highly active in facilitating the conversion of 7-dehydrocholesterol to cholesterol. Recombinant SCPX is also active in this assay but only 50% as active as SCP2. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of these proteins.