Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells

Protein Expr Purif. 1991 Aug;2(4):256-65. doi: 10.1016/1046-5928(91)90081-s.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Animals
  • CHO Cells
  • Cell Membrane / chemistry
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cricetinae
  • Detergents
  • Drug Resistance
  • Electrophoresis, Polyacrylamide Gel
  • Membrane Glycoproteins / isolation & purification*
  • Solubility

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Detergents
  • Membrane Glycoproteins