Purification of human cholesteryl ester transfer protein by affinity chromatography on immobilized triazine dyes.
Human cholesteryl ester transfer protein was purified from lipoprotein-depleted serum or plasma in a three-step procedure utilizing commercially available triazine dyes immobilized on agarose. The method used consisted of successive chromatography steps on Reactive Red 120 agarose (Procion Red H-E3B, Cibachron Brilliant Red 4G-E), CM Sepharose, and Reactive Yellow 86 agarose (Procion Yellow M-8G). Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resultant protein preparation displayed two bands of variable intensity. The two components had apparent molecular weights of approximately 64,000 and approximately 65,000, respectively. Both bands reacted strongly to a monoclonal antibody directed against an epitope consisting of the last eight amino acids at the carboxy terminus of human CETP. Yields of cholesteryl ester transfer activity are 10-40% of the activity present in lipoprotein-depleted serum. The activity is approximately 50,000- to 100,000-fold purified relative to the starting material.
Rehberg
E F
EF
Department of Molecular Biology Research, Upjohn Company, Kalamazoo, Michigan 49001.
Greenlee
K A
KA
Melchior
G W
GW
Marotti
K R
KR
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000097:blood; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004396:Coloring Agents; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006023:Glycoproteins; D006801:Humans; D015151:Immunoblotting; D008074:Lipoproteins / Q000097:blood / Q000302:isolation & purification; D008969:Molecular Sequence Data; D014227:Triazines
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D004396:Coloring Agents; D018076:DNA, Complementary; D006023:Glycoproteins; D008074:Lipoproteins; D014227:Triazines