Lamping Erwin, Monk Brian C, Niimi Kyoko, Holmes Ann R, Tsao Sarah, Tanabe Koichi, Niimi Masakazu, Uehara Yoshimasa, Cannon Richard D
Department of Oral Sciences, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
Eukaryot Cell. 2007 Jul;6(7):1150-65. doi: 10.1128/EC.00091-07. Epub 2007 May 18.
The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, ADDelta. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K(m) value and the V(max) value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.
真核膜蛋白的研究一直受到能够实现持续高水平功能性蛋白表达的系统匮乏的阻碍。我们报告了使用一种改良的膜蛋白超表达系统来表征三类真菌膜蛋白（ABC转运蛋白Pdr5p、CaCdr1p、CaCdr2p、CgCdr1p、CgPdh1p、CkAbc1p和CneMdr1p，主要促进剂超家族转运蛋白CaMdr1p，以及细胞色素P450酶CaErg11p），这些蛋白导致了五种致病真菌的耐药表型，并用于表达人P糖蛋白（HsAbcb1p）。该超表达系统由一组质粒组成，这些质粒可将表达盒的单拷贝稳定整合到改良宿主酿酒酵母菌株ADDelta的染色体PDR5位点。实现了异源蛋白在高达质膜蛋白29%水平的过表达。膜蛋白表达时带有或不带有绿色荧光蛋白（GFP）、单体红色荧光蛋白、His、FLAG/His、Cys或His/Cys标签。测试的大多数GFP标签蛋白在细胞内能够正确转运，His标签蛋白能够进行亲和纯化。ABC转运蛋白的动力学分析表明，ATP酶活性的表观K(m)值和V(max)值不受His标签添加的显著影响。七种真菌药物泵的外排特性通过它们的底物特异性以及八种异生物质对它们的独特抑制模式来表征，这些异生物质可使过表达ABC药物泵的酿酒酵母菌株对氟康唑产生化学增敏作用。改良的超表达系统在真核膜蛋白研究中具有广泛应用，也可用于制药行业的药物筛选。
