Dj. Manfra et al., EXPRESSION AND PURIFICATION OF 2 RECOMBINANT STEROL-CARRIER PROTEINS - SCPX AND SCP2, Protein expression and purification, 6(2), 1995, pp. 196-205
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.