Drew David, Kim Hyun
Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London, UK.
Methods Mol Biol. 2012;866:41-6. doi: 10.1007/978-1-61779-770-5_4.
Expression plasmids for Saccharomyces cerevisiae offer a wide choice of vector copy number, promoters of varying strength and selection markers. These expression plasmids are usually shuttle vectors that can be propagated both in yeast and bacteria, making them useful in gene cloning. For heterologous production of membrane proteins, we used the green fluorescent protein (GFP) fusion technology which was previously developed in the Escherichia coli system. We designed an expression plasmid carrying an inducible GAL1 promoter, a gene encoding a membrane protein of interest and the GFP-octa-histidine sequence. Here we describe construction of multi-copy yeast expression plasmids by homologous recombination in S. cerevisiae.
用于酿酒酵母的表达质粒提供了多种载体拷贝数、不同强度的启动子和选择标记。这些表达质粒通常是穿梭载体，可在酵母和细菌中都能复制，使其在基因克隆中很有用。为了异源生产膜蛋白，我们使用了先前在大肠杆菌系统中开发的绿色荧光蛋白（GFP）融合技术。我们设计了一种表达质粒，它携带一个可诱导的GAL1启动子、一个编码感兴趣膜蛋白的基因以及GFP-八聚组氨酸序列。在此我们描述通过酿酒酵母中的同源重组构建多拷贝酵母表达质粒的方法。
