PURIFICATION AND CHARACTERIZATION OF WHEAT ALPHA-GLIADIN SYNTHESIZED IN THE YEAST, SACCHAROMYCES-CEREVISIAE
PURIFICATION AND CHARACTERIZATION OF WHEAT ALPHA-GLIADIN SYNTHESIZED IN THE YEAST, SACCHAROMYCES-CEREVISIAE
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DOI：
10.1016/0378-1119(92)90507-l
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发表时间：
1992-07-15
期刊：
影响因子：
3.5
通讯作者：
GREENE, FC
中科院分区：
文献类型：
--
作者：
BLECHL, AE;THRASHER, KS;GREENE, FC
在异源微生物宿主中生产和纯化植物种子贮藏蛋白的有效方法的发展将促进这些蛋白的结构-功能研究。本报告描述了这种方法适用于生产和分离小麦α-麦醇溶蛋白，醇溶谷蛋白型种子贮藏蛋白，从酿酒酵母。从Neill等人[Gene 55（1987）303-317]的载体、生长条件和提取方法开始，我们进行了几项改进以增加每体积酵母细胞培养物的α-麦醇溶蛋白的产量。CYC 1：：Gli-A2-Y转录融合载体pAY 31（Neill等人，1987），通过用酵母2 μ质粒的ARS 1复制区替换ARS 1复制区进行修饰。我们配制了一种新的培养基，一种补充了几种氮源的合成确定（SD）培养基的衍生物，它允许选择维持质粒和生长到高细胞密度。携带修饰的表达载体并在该培养基中以甘油和乳酸盐作为碳源生长的细胞的稳定期培养物比在SD葡萄糖中生长的对数期培养物含有显著更高水平的α-麦醇溶蛋白。在80%乙醇中的超声处理选择性地和有效地从小规模或大规模培养物的细胞团中提取α-麦醇溶蛋白，允许在几个高产步骤中纯化每升几百μ g的小麦蛋白。从酵母中分离的α-麦醇溶蛋白在HPLC中与从小麦粉中纯化的A-麦醇溶蛋白级分相同的位置洗脱。N-末端氨基酸（aa）测序表明，信号肽从酵母细胞中的麦醇溶蛋白前体中去除。大约三分之二的分子具有与小麦胚乳细胞中产生的A-醇溶蛋白相同的N末端。α-麦醇溶蛋白的其余三分之一显然在靠近前肽的N末端三个氨基酸的类似加工位点处被切割。
The development of efficient methods for production and purification of plant seed storage proteins in heterologous microbial hosts would facilitate structure-function studies of these proteins. This report describes such methods applied to the production and isolation of wheat alpha-gliadin, a prolamine-type seed storage protein, from Saccharomyces cerevisiae. Beginning with the vector, growth conditions, and extraction methods of Neill et al. [Gene 55 (1987) 303-317], we implemented several improvements to increase the yields of alpha-gliadin per volume of yeast cell culture. The CYC1::Gli-A2-Y transcriptional fusion vector, pAY31 (Neill et al., 1987), was modified by replacing the ARS1 region of replication with that of the 2-mu-plasmid of yeast. We formulated a new medium, a derivative of synthetic defined (SD) medium supplemented with several nitrogen sources, that allows both selection for maintenance of plasmids and growth to high cell densities. Stationary phase cultures of cells bearing the modified expression vector, and grown in this medium with glycerol and lactate as carbon sources, contain significantly higher levels of alpha-gliadin than log-phase cultures grown in SD glucose. Sonication in 80% ethanol selectively and efficiently extracts the alpha-gliadin from cell pellets of small- or large-scale cultures, allowing the purification of several hundred-mu-g of the wheat protein per liter in just a few high-yield steps. The alpha-gliadin isolated from yeast elutes at the same position in HPLC as the A-gliadin fraction purified from wheat flour. N-terminal amino acid (aa) sequencing reveals that the signal peptide is removed from the gliadin precursor in yeast cells. About two-thirds of the molecules have the same N terminus as A-gliadin made in wheat endosperm cells. The remaining third of the alpha-gliadin is apparently cleaved at a similar processing site three aa closer to the N terminus of the prepeptide.