Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.
P-glycoprotein, a hydrophobic 170-kDa integral protein overexpressed in the plasma membrane of multidrug-resistant cells, is proposed to function as an ATP-dependent drug efflux pump. Plasma membrane preparations highly enriched in P-glycoprotein were isolated from multidrug-resistant cells by discontinuous sucrose gradient and Ca2+ precipitation methods. Several strategies were used for P-glycoprotein purification, with the goal being to achieve both good yields and purity, while keeping experimental manipulation to a minimum. P-glycoprotein was solubilized from the plasma membrane using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Immunoaffinity chromatography using C219 monoclonal antibody produced low yields of moderately pure protein. Sequential lectin affinity chromatography on RCA-120 followed by lentil lectin resulted in a P-glycoprotein preparation that showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A fraction of P-glycoprotein did not bind to RCA-120, most likely as a result of heterogeneous glycosylation. A combination of chromatography on RCA-120 followed by immunoaffinity chromatography on C219 resulted in low yields of very pure P-glycoprotein.
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
C A
CA
Doige
F J
FJ
Sharom
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006224:Cricetinae; D003902:Detergents; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D012995:Solubility
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003902:Detergents; D008562:Membrane Glycoproteins