MechanismsLandmark
Landmark purification establishes human CETP as a unique 74-kDa hydrophobic plasma apolipoprotein (J Biol Chem 1987)
Original title: Purification and characterization of a human plasma cholesteryl ester transfer protein
CETP was purified 55,000-fold with 27% recovery of activity from the density-greater-than-1.21 g/ml fraction of human plasma. In the final step, partially purified CETP was bound to a synthetic lipid emulsion of phosphatidylcholine, triglyceride, and fatty acid, and separated from unbound proteins by gel filtration on Sepharose 4B, yielding a single protein of apparent molecular weight 74,000 on SDS gel analysis; fatty acid was needed in the emulsion to prevent a contaminant from co-binding, though lipid peroxides in the emulsion caused substantial covalent degradation of the protein unless antioxidants were included. Solvent extraction of the emulsion-bound protein yielded active, delipidated CETP, and antibody raised against the purified 74-kDa protein completely removed transfer activity from partially purified fractions. Amino acid analysis showed an unusually high nonpolar residue content (45%), with calculated hydrophobicity exceeding that of any other known plasma apolipoprotein, establishing CETP as a unique, self-associating, peroxide-sensitive hydrophobic plasma protein and providing the foundational purified-protein characterization on which subsequent CETP structural and functional work was built.
Original abstract
The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and promotes transfer of cholesteryl esters between the lipoproteins. CETP has been purified 55,000-fold, with a 27% recovery of activity, from the d greater than 1.21 g/ml fraction of human plasma. In the final purification step, partially purified CETP is incubated with a synthetic lipid emulsion consisting of phosphatidylcholine, triglyceride, and fatty acid, and the bound activity, which elutes in the void volume, is separated from nonbound proteins by gel filtration on Sepharose 4B. Sodium dodecyl sulfate-gel analysis of fractions containing bound activity shows the presence of a single protein with an apparent Mr of 74,000. Inclusion of fatty acid in this emulsion was required to prevent the binding of a contaminant protein. However, incubation of CEPT with fatty acid emulsions containing lipid peroxides resulted in substantial inactivation and covalent degradation of the 74-kDa protein. This could be prevented by the inclusion of antioxidants during preparation of the emulsion. Solvent extraction of emulsion-bound CEPT gave a delipidated, active preparation. Purified IgG from a rabbit immunized with the 74-kDa protein completely removed activity from partially purified fractions. Amino acid analysis of the purified protein showed it to contain an unusually high content (45%) of nonpolar residues; the calculated hydrophobicity was greater than that of any other plasma apolipoprotein. These results show human CETP to be a unique plasma apolipoprotein with an apparent Mr of 74,000 which is hydrophobic, self-associating, and susceptible to covalent degradation by lipid peroxides.
Summary written by cetpinhibition.org from the published abstract; figures as published. Page updated 18 August 2026. Methods.