Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency
Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency
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DOI：
10.1006/jmbi.1999.3130
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发表时间：
1999-10-08
影响因子：
5.6
通讯作者：
Wittrup, KD
中科院分区：
文献类型：
--
作者：
Shusta, EV;Kieke, MC;Wittrup, KD
酵母细胞表面展示的效率可以作为增强突变蛋白的热稳定性和可溶性分泌效率的代理筛选变量。对几种能够在酵母表面展示的单链 T 细胞受体 (scTCR) 单位点突变体及其双突变体和三突变体组合进行了分析，以了解酿酒酵母的可溶性分泌。虽然未检测到野生型 scTCR 的分泌，但在酵母上清液中产生了单突变体、双突变体和三突变体，并且双突变体和三突变体导致表达增加。可溶性分泌水平与酵母表面上与 Aga2p 融合的活性 scTCR 的数量密切相关。 scTCR 突变体的热稳定性与 scTCR 的分泌和表面水平直接相关，有证据表明内质网质量控制装置的细胞内蛋白水解决定了显示效率。因此，酵母展示是一种定向进化支架，可用于鉴定具有显着增强的稳定性和分泌特性的突变真核蛋白。 (C) 1999 年学术出版社。
Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties. (C) 1999 Academic Press.