Laboissière M C, Chivers P T, Raines R T
Department of Biochemistry, University of Wisconsin--Madison 53706-1569, USA.
Protein Expr Purif. 1995 Oct;6(5):700-6. doi: 10.1006/prep.1995.1092.
Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and reduction of protein disulfide bonds. Saccharomyces cerevisiae cells lacking PDI are inviable. PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear. A cDNA that codes for rat PDI fused to the alpha-factor pre-pro segment was expressed in a protease-deficient strain of S. cerevisiae under the control of an ADH2-GAPDH hybrid promoter. The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus. The typical yield of isolated protein was 2 mg per liter of culture. The catalytic activity of the PDI from S. cerevisiae was indistinguishable from that of PDI isolated from bovine liver. This expression system is unique in allowing the same plasmid to be used both to complement pdi1 delta S. cerevisiae and to produce PDI for detailed in vitro analyses. Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.
蛋白质二硫键异构酶（PDI）是内质网中一种丰富的蛋白质，它催化蛋白质巯基的氧化以及蛋白质二硫键的异构化和还原。缺乏PDI的酿酒酵母细胞无法存活。PDI是许多不同蛋白质加工复合体的组成部分，而细胞存活所需的PDI实际活性尚不清楚。在ADH2 - GAPDH杂交启动子的控制下，将编码与α - 因子前体 - 前导序列融合的大鼠PDI的cDNA在蛋白酶缺陷型酿酒酵母菌株中表达。尽管所得蛋白质的C末端具有KDEL或HDEL序列，但细胞仍对其进行加工并将其作为单体分泌到培养基中。分离蛋白质的典型产量为每升培养物2毫克。酿酒酵母中PDI的催化活性与从牛肝中分离的PDI的催化活性没有区别。该表达系统的独特之处在于，同一质粒既可用于互补pdi1δ酿酒酵母，又可用于生产PDI以进行详细的体外分析。PDI体内行为与体外特性之间的相关性可能揭示具有生物学重要性的结构 - 功能关系。
