Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells and characterization of nucleotide binding properties of wild-type, Walker A, and Walker B mutant proteins

P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermenter culture, and purified. The final pure product is of high specifi c ATPase activity and is soluble at low detergent concentration. 120 g of c ells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single f ermenter run, Properties of the pure protein were similar to those of previ ous preparations, except there was significant ATPase activity in absence o f added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and p urity. This procedure should open up new avenues of structural, biophysical , and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wildtype mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophe nyl) adeno sine tri- and diphosphate. Both analogs were found to bind with K-d in the low micromolar range, to a single class of site, with no evidenc e of cooperativity. ATP displacement of the analogs was seen. Similar bindi ng was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showi ng that these Walker A and B mutations had no significant effect on affinit y or stoichiometry of nucleotide binding. These residues, known to be criti cal for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp.