Cabantous Stéphanie, Pédelacq Jean-Denis, Mark Brian L, Naranjo Cleo, Terwilliger Thomas C, Waldo Geoffrey S
Bioscience Division, Los Alamos National Laboratory, MS-M888, Los Alamos, NM 87545, USA.
J Struct Funct Genomics. 2005;6(2-3):113-9. doi: 10.1007/s10969-005-5247-5.
We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691-695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102-107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.
我们改进了绿色荧光蛋白（GFP）折叠报告技术[Waldo等人，（1999年）《自然生物技术》17卷，691 - 695页]，以改造结核分枝杆菌中难以处理的蛋白质。将目标蛋白插入GFP的支架结构中，消除了由目标RNA内部隐蔽核糖体结合位点处截短的蛋白质变体表达所导致的假阳性假象。同时，我们基于GFP的微结构域开发了一种新的定量荧光蛋白标记和检测系统。这种分裂GFP系统在体内和体外均有效，适用于蛋白质表达和溶解度的高通量检测[Cabantous等人，（2005年）《自然生物技术》23卷，102 - 107页]。GFP折叠报告技术和分裂GFP技术共同提供了一个用于操纵和改善蛋白质折叠及溶解度的综合系统。
