Yang T T, Cheng L, Kain S R
Cell Biology Group, CLONTECH Laboratories Inc., Palo Alto, CA 94303-4230, USA.
Nucleic Acids Res. 1996 Nov 15;24(22):4592-3. doi: 10.1093/nar/24.22.4592.
The green fluorescent protein (GFP) from Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of cells and organisms. Despite many early successes using this reporter, wild type GFP is suboptimal for most applications due to low fluorescence intensity when excited by blue light (488 nm), a significant lag in the development of fluorescence after protein synthesis, complex photoisomerization of the GFP chromophore and poor expression in many higher eukaryotes. To improve upon these qualities, we have combined a mutant of GFP with a significantly larger extinction coefficient for excitation at 488 nm with a re-engineered GFP gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yield an enhanced GFP which provides greater sensitivity in most systems.
来自维多利亚多管水母的绿色荧光蛋白（GFP）是一种多功能报告蛋白，可用于监测多种细胞和生物体中的基因表达及蛋白质定位。尽管早期使用这种报告蛋白取得了许多成功，但野生型GFP在大多数应用中并非最佳选择，原因在于其在蓝光（488nm）激发下荧光强度较低、蛋白质合成后荧光发展存在显著延迟、GFP发色团存在复杂的光异构化现象以及在许多高等真核生物中表达不佳。为了改善这些特性，我们将一种在488nm激发下具有显著更大消光系数的GFP突变体与一个经过重新设计的GFP基因序列相结合，该序列包含在高表达人类蛋白质中优先发现的密码子。改进后的荧光强度与更高表达水平相结合，产生了一种增强型GFP，它在大多数系统中具有更高的灵敏度。
