Purification and reconstitution of human P-glycoprotein.
Human Pgp from the vinblastine-resistant cell line, KB-V1, can be purified by sequential conventional chromatography on DEAE-sepharose CL-6B resin followed by a wheat germ agglutinin column. By including glycerol (osmolyte protectant) and lipid during the solubilization and chromatography procedures most of the biological activity of Pgp can be retained. The activity of Pgp in the detergent extract or in the concentrated column fractions is stable for at least 8-10 months when stored at -80 degrees. However, repeated cycles of freezing and thawing of fractions result in considerable loss of activity. We have purified Pgp from KB-C1 (a subclone of KB 3-1 that is resistant to 1 microgram/ml colchicine) by following the same protocol. When this method was used for purification of Pgp from MDR1-transfected NIH 3T3 transfectants (N3-V2400, grown in the presence of 2.4 micrograms/ml vinblastine), the protein was eluted with 0.1 M NaCl from the DEAE-Sepharose CL-6B column as usual. However, during WGA lectin chromatography, the protein was eluted with a lower concentration of sugar (0.1 M instead of 0.25 M NAG). This altered elution pattern appears to be due to a difference in the glycosylation of human Pgp in mouse NIH 3T3 cells. This is consistent with the observation that human Pgp expressed in NIH 3T3 cells migrates faster compared to the protein from KB-V1 cells on 8-10% acrylamide gel. Similarly, other workers have purified Chinese hamster Pgp either by a single-step chromatography on Reactive Red 120 agarose or by a combination of anion exchange and immunoaffinity chromatography (see the article by Senior et al. for the purification and properties of ATPase activity of Chinese hamster Pgp). The high level of drug-stimulated ATP hydrolysis by Pgp (Table I), like other ion-transporting ATPases, indicates that this is a high-capacity pump that can function as an effective multidrug transporter. This is further supported by the qualitative demonstration of ATP-dependent vinblastine transport in proteoliposomes reconstituted with pure Pgp (see Fig. 2). Thus, these experiments provide strong evidence that purified Pgp retains its activity and that it functions as an ATP-dependent drug transporter.
Ambudkar
S V
SV
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Lelong
I H
IH
Zhang
J
J
Cardarelli
C
C
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000302:isolation & purification* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D002999:Clone Cells; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003630:Daunorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007202:Indicators and Reagents; D007624:KB Cells; D007700:Kinetics; D008081:Liposomes; D051379:Mice; D011510:Proteolipids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014162:Transfection / Q000379:methods; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000633:toxicity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007202:Indicators and Reagents; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D014747:Vinblastine; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003078:Colchicine; D003630:Daunorubicin