Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen
Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen
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DOI：
10.1038/nprot.2006.373
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Rose, Jocelyn K. C.
中科院分区：
文献类型：
--
作者：
Lee, Sang-Jik;Kim, Byung-Dong;Rose, Jocelyn K. C.
分泌蛋白和细胞表面蛋白在真核生物的许多基本生物学过程中发挥重要作用，但通常比定位于细胞内区室的蛋白更难以分离和鉴定。然而，已经开发了几种高通量的“基因陷阱”技术来表征这些“分泌体”，包括酵母分泌陷阱（YST）屏幕。该方法包括将来自感兴趣的组织或细胞类型的cDNA文库与酵母（酿酒酵母）转化酶报告基因融合，将所得融合文库转化成转化酶缺陷型酵母菌株，并将转化体接种在含有蔗糖作为唯一碳源的培养基上。带有编码分泌蛋白或细胞表面蛋白的转基因的酵母细胞可以合成分泌型转化酶融合蛋白，该融合蛋白可以拯救突变体，然后可以对质粒DNA进行测序以鉴定编码它的基因。我们描述了这种筛选的最近改进版本，其允许在1-2个月内鉴定编码分泌蛋白的基因。
Secreted and cell surface proteins play essential roles in numerous essential biological processes in eukaryotic organisms, but are often more difficult to isolate and identify than proteins that are localized in intracellular compartments. However, several high-throughput 'gene-trap' techniques have been developed to characterize these 'secretomes', including the yeast secretion trap (YST) screen. This method involves fusing cDNA libraries from the tissue or cell type of interest to a yeast (Saccharomyces cerevisiae) invertase reporter gene, transforming the resulting fusion library into an invertase-deficient yeast strain and plating the transformants on a medium containing sucrose as the sole carbon source. A yeast cell with a transgene encoding a secreted or cell surface protein can synthesize a secreted invertase fusion protein that can rescue the mutant, and the plasmid DNA can then be sequenced to identify the gene that encodes it. We describe a recently improved version of this screen, which allows the identification of genes encoding secreted proteins in 1-2 months.