Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae
Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae
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作者：
Sheff, MA;Thorn, KS
绿色荧光蛋白（GFP）已成为越来越受欢迎的用于确定蛋白质定位和丰度的蛋白质标签。随着具有改变的荧光光谱的GFP变体以及来自其他生物体的GFP同系物的可用性，现在可以使用蛋白质标签进行多色荧光，以及使用荧光共振能量转移（FRET）测量蛋白质相互作用。我们已经创建了一组酵母标记载体，其含有密码子优化的GFP变体、CFP（青色）、YFP（黄色）和Sapphire（UV可激发的GFP）。这些密码子优化的标签的可检测性是未优化标签的两倍。我们还创建了含有单体DsRed构建体tdimer2的标签载体，其比目前使用的标签可检测性高15倍。这些标签显着提高了酵母活细胞荧光成像的检测限，并提供了足够的四色成像可区分的荧光团。版权所有（C）2004约翰威利父子有限公司。
Green fluorescent protein (GFP) has become an increasingly popular protein tag for determining protein localization and abundance. With the availability of GFP variants with altered fluorescence spectra, as well as GFP homologues from other organisms, multi-colour fluorescence with protein tags is now possible, as is measuring protein interactions using fluorescence resonance energy transfer (FRET). We have created a set of yeast tagging vectors containing codon-optimized variants of GFP, CFP (cyan), YFP (yellow), and Sapphire (a UV-excitable GFP). These codon-optimized tags are twice as detectable as unoptimized tags. We have also created a tagging vector containing the monomeric DsRed construct tdimer2, which is up to 15-fold more detectable than tags currently in use. These tags significantly improve the detection limits for live-cell fluorescence imaging in yeast, and provide sufficient distinguishable fluorophores for four-colour imaging. Copyright (C) 2004 John Wiley Sons, Ltd.