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.
Manfra
D J
DJ
Schering Plough Research Institute, Department of Immunology, Kenilworth, New Jersey 07033, USA.
Baum
C L
CL
Reschley
E
E
Lundell
D
D
Zavodny
P
P
Dalie
B
B
D000101:Acetyl-CoA C-Acetyltransferase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism
D002352:Carrier Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C072087:sterol carrier proteins; D000101:Acetyl-CoA C-Acetyltransferase; D005982:Glutathione Transferase; D013917:Thrombin