M. Julien et al., Simple purification of highly active biotinylated P-glycoprotein: Enantiomer-specific modulation of drug-stimulated ATPase activity, BIOCHEM, 39(1), 2000, pp. 75-85
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 po
lyhistidine tail and a 100-amino acid residue biotin acceptor domain of oxa
loacetate decarboxylase from Klebsiella pneumoniae at the carboxyl terminus
end of Pgp (Pgp-H6BD). The expression/purification protocol for Pgp-H6BD i
nvolves high level expression of the fusion protein in the yeast Pichia pas
toris, biotinylation in vitro with biotin ligase, solubilization of crude m
embrane 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 puri
ty. This protocol is rapid and efficient and yields purified Pgp which show
s robust ATPase activity, as determined by vanadate-induced trapping of pho
toactive nucleotides and by direct measurement of ATP hydrolysis by Pgp-H6B
D, This method should be useful for structural studies of the protein by sp
ectroscopic or crystallographic approaches. This purified Pgp-H6BD preparat
ion 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 centra
l chiral carbon and modified at the head and tail groups are shown to stimu
late Pgp ATPase activity in a dose-dependent fashion. Some of these compoun
ds are also capable of inhibiting either vinblastine or verapamil stimulati
on of ATPase activity of Pgp in an enantiomer-specific fashion. The enantio
meric specific inhibitory activity of these compounds suggests complex inte
ractions at a single substrate binding site(s) on Pgp.