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.