title: Expression of functional multidrug-resistance protein 1 in Saccharomyces cerevisiae: effects of N- and C-terminal affinity tags.
abstract: Studies of the multidrug-resistance protein 1 (MRP1) have been hampered by the lack of a simple expression system allowing for rapid generation of mutants and yielding milligram amounts of protein. Here, we describe a Saccharomyces cerevisiae expression system that meets those conditions. MRP1 was expressed under the control of the constitutive PMA1 (yeast proton pump) promoter. The best conditions for expression were determined, including the use of the chemical chaperone glycerol, which increased MRP1 expression. N-terminal poly-histidine or FLAG affinity tags reduce MRP1 expression, whereas the same tags fused to the C-terminus had no effect. All the fusion proteins were functional. We conclude that because of its low cost and simplicity, the S. cerevisiae-based MRP1-expression system will be useful for studies where a large number of mutants or milligram amounts of purified MRP1 are needed.
authors.affiliation: Membrane Protein Laboratory, Sealy Center for Structural Biology, and Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0437, USA.
authors.forename: Sung Haeng
authors.initials: SH
authors.lastname: Lee
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Altenberg
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D011993:Recombinant Fusion Proteins