Zapata-Hommer Otilia, Griesbeck Oliver
Max-Planck-Institut für Neurobiologie Am Klopferspitz 18a 82152 Martinsried, Germany.
BMC Biotechnol. 2003 May 22;3:5. doi: 10.1186/1472-6750-3-5.
The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.
By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells.
"T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.
绿色荧光蛋白（GFP）已在细胞生物学中广泛用作基因表达的标记、细胞结构的标签、融合标签或作为基因编码生物传感器的关键组成部分。野生型基因的诱变产生了许多改进的变体，如增强型绿色荧光蛋白（EGFP）或适用于荧光共振能量转移（FRET）的颜色变体。然而，当这些突变体中的一些靶向特定细胞器或与其他蛋白质融合时，其折叠仍然是一个问题。
通过定向理性诱变，我们产生了一种具有改进折叠特性的GFP蓝宝石突变体的新变体，当在细胞器内表达或作为融合标签时，该变体特别有益。其吸收光谱对pH稳定，发射的pKa为4.9，使其在细胞内对pH扰动具有很强的抗性。
“T-蓝宝石”及其环形排列可作为蛋白质或细胞结构的标签，并可与红色荧光受体蛋白（如DsRed）结合用作FRET供体，这使得在基于强度的FRET实验中能够完全分离供体和受体的激发和发射。
