Kupfer R, Beiche F, Schweizer M
Institut für Biochemie, Universität Erlangen-Nürnberg, Staudtstrasse. 5, D-91058 Erlangen, Germany.
Curr Genet. 1996 Feb;29(3):219-26. doi: 10.1007/BF02221551.
The 272 647-dalton polypeptide of fatty acid synthase (FAS) from Rattus norvegicus has been expressed in a proteinase-deficient strain of Saccharomyces cerevisiae. The seven overlapping cDNA clones for rat FAS spanning the entire coding region were the starting material for this undertaking. In a series of cloning steps an expression plasmid was constructed in which the cDNA was placed under the control of the yeast ADH1 promoter. Northern blotting of total RNA isolated from yeast transformed with this expression plasmid demonstrated a high rate of transcription of the 7.4-kb cDNA. However, a successful translation required further manipulation of the sequence immediately upstream of the rat FAS translational start codon. This was obtained when the 86 bp of the rat FAS cDNA immediately 5' to the start codon were replaced by a nonamer corresponding to the immediate 5'-vicinity of the translational start codon of the yeast ADH1 gene. Nevertheless, the translation product could be detected only by Western blotting. The FAS proteins of S. cerevisiae and rat are not functionally interchangeable. Using the purification protocol of rat FAS the heterologously expressed FAS could be enriched by at least one order of magnitude.
来自褐家鼠的脂肪酸合酶（FAS）的272647道尔顿多肽已在蛋白酶缺陷型酿酒酵母菌株中表达。跨越整个编码区的七个大鼠FAS重叠cDNA克隆是这项工作的起始材料。在一系列克隆步骤中构建了一个表达质粒，其中cDNA置于酵母ADH1启动子的控制之下。对用该表达质粒转化的酵母分离的总RNA进行Northern印迹分析，结果表明7.4-kb cDNA的转录率很高。然而，成功翻译需要对大鼠FAS翻译起始密码子上游的序列进行进一步操作。当大鼠FAS cDNA起始密码子5'端的86 bp被与酵母ADH1基因翻译起始密码子紧邻的5'区域对应的九聚体取代时，实现了这一点。尽管如此，翻译产物只能通过Western印迹检测到。酿酒酵母和大鼠的FAS蛋白在功能上不可互换。使用大鼠FAS的纯化方案，异源表达的FAS可以富集至少一个数量级。
