Purification and Characterization of N-glycosylation Mutant Mouse and Human P-glycoproteins Expressed in Pichia pastoris Cells

P-glycoprotein confers multidrug resistance in mammalian cells and basic structure–function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al. (1999) J. Biol. Chem. 274, 34711–34718). The degree of glycosylation of these preparations was unknown, and was of relevance for crystallization studies. Therefore mutant proteins in which the N-glycosylation sites were eliminated (Asn → Gln in mouse MDR3 Pgp, Asn → Gln or Ala in human MDR1 Pgp) were expressed in P. pastoris and purified to homogeneity. Yields of mutant Pgp were the same as for parent wild-type proteins. Nucleotide-binding and catalytic (ATPase) characteristics were completely normal in the mutant proteins. Mass spectrometry indicated that mutant and wild-type proteins did not differ significantly in mass, demonstrating that the wild-type proteins contain no N-glycosylation.