Madani Golnoush, Lamping Erwin, Lee Hee Ji, Niimi Masakazu, Mitra Alok K, Cannon Richard D
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago.
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago; Department of Microbiology, Faculty of Medicine, Chulalongkorn University.
J Vis Exp. 2021 Jun 13(172). doi: 10.3791/62592.
The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.
ABC转运蛋白成功的生化和生物物理特性很大程度上取决于异源表达系统的选择。在过去二十年中，我们开发了一个酵母膜蛋白表达平台，该平台已用于研究许多重要的真菌膜蛋白。表达宿主酿酒酵母ADΔΔ缺失了七个主要的内源性ABC转运蛋白，并且它含有转录因子Pdr1-3，该转录因子具有功能获得性突变，能够组成型过表达作为单拷贝稳定整合在基因组PDR5位点的异源膜蛋白基因。通用质粒载体的创建和一步克隆策略的优化使得异源ABC转运蛋白能够快速准确地克隆、诱变和表达。在这里，我们描述了一种新型蛋白酶可切割的mGFPHis双标签（即与六个组氨酸亲和纯化标签融合的单体酵母增强型绿色荧光蛋白yEGFP3）的开发和使用，该标签旨在避免标签对目标蛋白可能的干扰，并提高His标签与镍亲和树脂的结合效率。mGFPHis与膜蛋白开放阅读框（ORF）的融合使得通过检查聚丙烯酰胺凝胶和检测保留mGFPHis标签的降解产物能够轻松定量蛋白质。我们展示了这一特性如何促进用于膜蛋白溶解的去污剂筛选。本文介绍了一种高效、快速且可靠地分离在酿酒酵母ADΔΔ中过表达的C端标记的白色念珠菌多药外排转运蛋白Cdr1的小规模质膜制剂的方案。这种小规模质膜分离方案可在一个工作日内生成高质量的质膜。质膜制剂可用于测定Cdr1和Cdr1突变变体的酶活性。
