EXPRESSION AND PURIFICATION OF 2 RECOMBINANT STEROL-CARRIER PROTEINS - SCPX AND SCP2

We report the cloning, expression, and purification of the rat sterol carrier proteins SCPX and SCPS. The cDNA's encoding rat SCPX and SCP2 were isolated from a lambda gt11 rat liver cDNA library. To maximize e xpression and to facilitate the purification of the recombinant protei ns, the SCPX and SCP2 proteins were expressed as carboxy-terminal fusi on proteins to the glutathione S-transferase (GST). The GST-SCPX and G ST-SCP2 fusion proteins contained a thrombin recognition site between the GST and SCPX or SCP2 polypeptides, The expression of the fusion pr oteins was controlled by the inducible tac promoter. Under optimal con ditions, the similar to 85-kDa GST-SCPX and the similar to 41-kDa GST- SCP2 proteins represented approximately 1-2% of the total cell lysate. Both fusion proteins were easily purified under nondenaturing conditi ons from the soluble fraction of total cell lysate by glutathione-Seph arose 4B affinity chromatography. Thrombin cleavage resulted in the re lease of the SCPX and SCP2 proteins from the GST-SCPX and GST-SCP2 fus ions, respectively. Amino terminal protein sequencing confirmed the au thenticity of the recombinant proteins. Furthermore, functional assay revealed that recombinant SCP2 is highly active in facilitating the co nversion of 7-dehydrocholesterol to cholesterol. Recombinant SCPX is a lso active in this assay but only 50% as active as SCP2. We anticipate that the preparation and purification techniques described in this st udy will facilitate further biochemical characterization of these prot eins. (C) 1995 Academic Press,Inc.