Arun K H S, Kaul C L, Ramarao P
Cardiovascular and Receptorology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Phase-X, Sector 67, S.A.S. Nagar (Mohali)-160 062, Punjab, India.
J Pharmacol Toxicol Methods. 2005 Jan-Feb;51(1):1-23. doi: 10.1016/j.vascn.2004.07.006.
In the last five years, green fluorescent protein (GFP) has emerged from being a mere curiosity to become a reliable tool for molecular pharmacological research. GFP produces an intense and stable green fluorescence noncatalytically by absorbing blue light maximally at 395 nm and emitting green light with a peak at 509 nm. It consists of 238 amino acids and its molecular mass is 27-30 kDa. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence to be utilised in nonnative organisms, wherein it can be used as a reporter. This use of GFP permits real-time analysis of receptor dynamics. The emitted fluorescence can be used as a nontoxic marker and detected using fluorescence-activated cell sorting (FACS), thus avoiding any staining procedure, expensive mRNA analysis or hazardous radiolabeled binding assays. The potential value of GFP has also been recognized in orphan receptor research, where various GFP-tagged therapeutic proteins have been constructed in an attempt to identify the endogenous ligand(s). These chimeric proteins have been used to determine the site and time course of receptor expression and to relate receptor dynamics with therapeutic outcome. The preparation of new GFP constructs for identifying germ layer cells (endodermal, ectodermal, and mesodermal), as well as neuronal, haematopoietic, endothelial, and cartilage cells, has provided a useful battery of tissue/receptor-specific screening assays for new chemical entities. Genetically engineered cells with GFP expression have provided a valuable tool for automated analysis, and can be adapted for high-throughput systems. GFP is being increasingly utilised for the study of receptor dynamics, where, having already proved beneficial, it will likely continue to contribute towards the search for new classes of drugs, as well as to "de-orphaning" orphan receptors.
在过去五年中，绿色荧光蛋白（GFP）已从一种单纯的新奇事物发展成为分子药理学研究的可靠工具。GFP通过最大程度地吸收395nm的蓝光并发射峰值为509nm的绿光，非催化地产生强烈而稳定的绿色荧光。它由238个氨基酸组成，分子量为27 - 30 kDa。GFP荧光的产生无需辅因子，这一特性使得GFP荧光可在非天然生物体中利用，在其中它可作为报告基因。GFP的这种用途允许对受体动力学进行实时分析。发射的荧光可用作无毒标记，并使用荧光激活细胞分选（FACS）进行检测，从而避免任何染色程序、昂贵的mRNA分析或危险的放射性标记结合测定。GFP在孤儿受体研究中的潜在价值也已得到认可，在该研究中，已构建了各种GFP标记的治疗性蛋白质，试图鉴定内源性配体。这些嵌合蛋白已用于确定受体表达的位点和时间进程，并将受体动力学与治疗结果相关联。用于鉴定胚层细胞（内胚层、外胚层和中胚层）以及神经元、造血、内皮和软骨细胞的新型GFP构建体的制备，为新化学实体提供了一系列有用的组织/受体特异性筛选测定。具有GFP表达的基因工程细胞为自动化分析提供了有价值的工具，并且可适用于高通量系统。GFP越来越多地用于受体动力学研究，在该领域，它已证明是有益的，可能会继续为寻找新型药物以及“解除”孤儿受体的“孤儿身份”做出贡献。
