Yang T T, Sinai P, Green G, Kitts P A, Chen Y T, Lybarger L, Chervenak R, Patterson G H, Piston D W, Kain S R
Cell Biology Group, Clontech Laboratories, Inc., Palo Alto, California 94303, USA.
J Biol Chem. 1998 Apr 3;273(14):8212-6. doi: 10.1074/jbc.273.14.8212.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.
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，可用于监测多种系统中的基因表达和蛋白质定位。由于具有改变光谱特性的突变体的出现，包括几种蓝色发射变体，使用GFP报告基因的应用得到了极大的扩展，所有这些变体在蛋白质的发色团区域都含有从酪氨酸-66到组氨酸的单点突变。然而，先前描述的“BFP”报告基因的效用有限，主要是因为在高等真核生物中使用此类变体时荧光相对较弱且表达水平较低。为了改善这些特性，我们将含有四个点突变（苯丙氨酸-64突变为亮氨酸、丝氨酸-65突变为苏氨酸、酪氨酸-66突变为组氨酸、酪氨酸-145突变为苯丙氨酸）的GFP蓝色发射突变体与一个合成基因序列相结合，该合成基因序列包含在高表达人类蛋白质中优先发现的密码子。选择这些突变是为了优化在37摄氏度培养的哺乳动物细胞中正确折叠的荧光蛋白的表达，并使信号强度最大化。改进的荧光和更高的表达水平相结合，产生了一种增强型蓝色荧光蛋白，它具有更高的灵敏度，适用于与绿色发射荧光团进行双色检测。
