van den Bergh C J, Bekkers A C, De Geus P, Verheij H M, de Haas G H
Department of Biochemistry, State University of Utrecht, The Netherlands.
Eur J Biochem. 1987 Dec 30;170(1-2):241-6. doi: 10.1111/j.1432-1033.1987.tb13691.x.
The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.
编码猪胰原磷脂酶A2（proPLA）的cDNA已被克隆并在酿酒酵母中表达。只有将proPLA与酵母α-交配因子的前原序列融合后，才能实现proPLA的表达和分泌。分泌后，融合蛋白被KEX2蛋白酶切割，产生一种140个氨基酸的磷脂酶A2酶原样形式。该蛋白通过离子交换色谱法以高产率纯化。用胰蛋白酶进行有限的蛋白水解可切割“酶原”以产生活性磷脂酶A2，其与正宗的猪胰酶无法区分。这些结果表明，每124个氨基酸残基中含有高达7个二硫键的蛋白质可以被酵母分泌装置正确加工。
