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 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.
Publication types
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Comparative Study
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP Binding Cassette Transporter, Subfamily G, Member 2
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ATP-Binding Cassette Transporters / biosynthesis*
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ATP-Binding Cassette Transporters / chemistry*
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / isolation & purification
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Adenosine Triphosphatases / chemistry
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Adenosine Triphosphatases / metabolism
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Cloning, Molecular
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Gene Expression Regulation, Fungal
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Humans
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Microsomes / metabolism*
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Neoplasm Proteins / biosynthesis*
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Neoplasm Proteins / chemistry*
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Neoplasm Proteins / genetics
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Neoplasm Proteins / isolation & purification
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Pichia / genetics
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Pichia / metabolism*
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Protein Engineering / methods
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
Substances
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ABCG2 protein, human
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ATP Binding Cassette Transporter, Subfamily G, Member 2
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ATP-Binding Cassette Transporters
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Neoplasm Proteins
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Recombinant Proteins
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Adenosine Triphosphatases