Simple purification of highly active biotinylated P-glycoprotein: enantiomer-specific modulation of drug-stimulated ATPase activity.
A simplified method for the expression and purification of P-glycoprotein (Pgp) is presented. This method is based on the in-frame fusion of both a polyhistidine tail and a 100-amino acid residue biotin acceptor domain of oxaloacetate decarboxylase from Klebsiella pneumoniae at the carboxyl terminus end of Pgp (Pgp-H6BD). The expression/purification protocol for Pgp-H6BD involves high-level expression of the fusion protein in the yeast Pichia pastoris, biotinylation in vitro with biotin ligase, solubilization of crude membrane fractions in detergent, and affinity purification by a combination of nickel and avidin chromatography. Biotinylated Pgp binds to immobilized monomeric avidin and can be eluted with free biotin in a high state of purity. This protocol is rapid and efficient and yields purified Pgp which shows robust ATPase activity, as determined by vanadate-induced trapping of photoactive nucleotides and by direct measurement of ATP hydrolysis by Pgp-H6BD. This method should be useful for structural studies of the protein by spectroscopic or crystallographic approaches. This purified Pgp-H6BD preparation has been used to study the enantiomer-specific effects of inhibitors of Pgp-mediated drug transport on the drug-stimulated ATPase activity of the protein. A series of 1, 4-disubstituted piperazine derivatives with a central chiral carbon and modified at the head and tail groups are shown to stimulate Pgp ATPase activity in a dose-dependent fashion. Some of these compounds are also capable of inhibiting either vinblastine or verapamil stimulation of ATPase activity of Pgp in an enantiomer-specific fashion. The enantiomeric specific inhibitory activity of these compounds suggests complex interactions at a single substrate binding site(s) on Pgp.
Julien
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Department of Biochemistry, McGill University, Montréal, Québec, Canada.
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D018435:ATP Binding Cassette Transporter, Subfamily B / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D018432:Drug Resistance, Multiple; D020536:Enzyme Activators / Q000494:pharmacology*; D006639:Histidine; D006801:Humans; D007711:Klebsiella pneumoniae / Q000235:genetics; D051379:Mice; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics; D010879:Piperazines / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013844:Thiazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; C403205:CP 147673; C403203:CP 162398; C403204:CP 172732; D020536:Enzyme Activators; D010455:Peptides; D010879:Piperazines; D011804:Quinolines; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; C033223:polyhistidine; D006639:Histidine; D014747:Vinblastine; C095122:multidrug resistance protein 3; D014700:Verapamil; D000251:Adenosine Triphosphatases