Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase
Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase
复制标题
DOI：
10.1002/bit.20762
复制
发表时间：
2006-03-05
影响因子：
3.8
通讯作者：
Meagher, MM
中科院分区：
文献类型：
--
作者：
Inan, M;Aryasomayajula, D;Meagher, MM
美洲钩虫分泌蛋白（Na-ASP1）是一种潜在的候选疫苗，可对抗钩虫感染，已在毕赤酵母中表达。 Na-ASP1 是一种含有 20 个半胱氨酸的 45 kDa 蛋白质，通过将蛋白质与酿酒酵母 α 交配因子的前原序列融合，将其引导到细胞外。单拷贝克隆产生的大部分蛋白质被分泌到细胞外。然而，毕赤酵母中 Na-ASP1 蛋白基因拷贝数的增加使分泌能力饱和，因此，含有多个 Na-ASP1 基因拷贝的克隆中分泌蛋白的量减少。内质网 (ER) 驻留同源伴侣蛋白、蛋白二硫键异构酶 (PDI) 的过度表达能够增加高拷贝克隆中 (Na-ASP1) 蛋白的分泌。在具有不同 PDI 基因拷贝数的克隆中检查了 PDI 水平对 Na-ASP1 蛋白分泌的影响。 Na-ASP1 分泌量的增加与 PDI 拷贝数密切相关。 PDI 水平的增加也增加了所有克隆中 Na-ASP1 蛋白的总体产量。尽管如此，在过表达Na-ASP1和PD1蛋白的巴斯德毕赤酵母克隆中，细胞内Na-ASP1蛋白仍然积累。 (c) 2005 年 Wiley 期刊公司。
A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PD1 proteins. (c) 2005 Wiley Periodicals, Inc.