Bodmer M W, Angal S, Yarranton G T, Harris T J, Lyons A, King D J, Pieroni G, Riviere C, Verger R, Lowe P A
Biochim Biophys Acta. 1987 Aug 25;909(3):237-44. doi: 10.1016/0167-4781(87)90083-2.
The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.
本文描述了编码人胃脂肪酶的cDNA的分子克隆及其在酵母中的表达。对人胃吸出物中存在的一种脂肪酶进行了纯化，并测定了其N端氨基酸序列。发现该序列与大鼠舌脂肪酶的N端序列同源。用人胃组织分离的mRNA构建了一个cDNA文库，并用克隆的大鼠舌脂肪酶DNA进行探测。一个名为pGL17的克隆，约由1450个碱基对组成，包含人胃脂肪酶的完整编码序列。从分离的蛋白质获得的氨基酸序列和从克隆基因获得的DNA序列表明，人胃脂肪酶由一个379个氨基酸的多肽组成，其无糖基化的Mr为43162。人胃脂肪酶和大鼠舌脂肪酶的氨基酸序列高度同源，但除了猪胰脂肪酶必需的Ser-152周围的一个6氨基酸序列外，与人胰脂肪酶无关。构建了一个含有磷酸甘油酸激酶启动子和终止子序列以及人胃脂肪酶基因的酵母表达质粒。用该载体转化的酵母合成了具有脂解活性的酶。
