Functional expression of the human breast cancer resistance protein in Pichia pastoris

Abstract

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40–80 nmol P i/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[α- 32P]ATP was dependent preferentially on the presence of Co 2+ than Mg 2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[α- 32P]ADP by BCRP was much more significant in the presence of Co 2+ than that with Mg 2+. The K m and V max values of BCRP for [ 3H]E 1S transport were 3.6 ± 0.3 μM and 55.2 ± 1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.