Functional expression of the human breast cancer resistance protein in Pichia pastoris.
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 Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM 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.
Mao
Qingcheng
Q
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA 98195, USA. qmao@u.washington.edu
Conseil
Gwenaëlle
G
Gupta
Anshul
A
Cole
Susan P C
SP
Unadkat
Jashvant D
JD
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D008861:Microsomes / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases