Schultz L D, Hofmann K J, Mylin L M, Montgomery D L, Ellis R W, Hopper J E
Department of Virus and Cell Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Gene. 1987;61(2):123-33. doi: 10.1016/0378-1119(87)90107-7.
High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.
酿酒酵母中源自GAL启动子的高水平、半乳糖诱导型表达是由GAL4编码蛋白与核苷酸序列之间的高度特异性相互作用介导的。重组GAL启动子/外源基因构建体用于酵母中外源蛋白的可调控高水平表达的潜在用途已得到充分认可。然而，由于GAL4编码蛋白的水平非常低，以及如果组成型扩增GAL4蛋白水平会导致诱导性丧失，在这种构建体的多个拷贝的情况下，该系统的用途受到严重限制。为了克服这些限制，我们构建了一种新型酵母菌株，该菌株以可调控的方式过量产生GAL4蛋白。这种“整合体”菌株包含一个杂交基因的整合拷贝，该杂交基因由与GAL4结构基因融合的半乳糖诱导型GAL10启动子组成。在没有半乳糖的情况下，整合体菌株和同基因的“非整合体”亲本菌株仅显示来自组成型活性染色体GAL4基因的基础转录水平。然而，在向培养基中添加半乳糖后，“整合体”菌株合成的GAL4 mRNA至少比“非整合体”菌株多20倍，GAL4蛋白也显著更多。将含有GAL10启动子和与爱泼斯坦-巴尔病毒gp350结构基因融合的α交配因子前导肽的高拷贝数表达载体引入两种类型的细胞中。在半乳糖诱导后，所得转化的“整合体”细胞产生的gp350 mRNA比转化的“非整合体”细胞多约五倍，gp350相关蛋白多十倍。这种“整合体”菌株应该被证明普遍适用于由半乳糖诱导型启动子驱动的结构基因在酵母中的最大程度的可调控表达。
