Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway.
Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway.
复制标题
酿酒酵母表达、加工和分泌蛋白水解敏感的昆虫利尿激素需要使用缺乏编码分泌途径中发现的 Yap3 和 Mkc7 内切蛋白酶的基因的酵母菌株。
DOI：
10.1042/bj3301333
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Courchesne,WE
中科院分区：
文献类型：
--
作者：
Copley,KS;Alm,SM;Schooley,DA;Courchesne,WE
A system is described for the heterologous expression of peptides inSaccharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acidManduca sextadiuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then treated with peptidylglycine α-amidating enzyme to generate the α-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys↓-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on thein vivoactivity and specificity of Yap3 and Mkc7 expressed at physiological levels.
