臺灣大學: 化學研究所陳平; 張智芬陳振誠Chen, Chen-ChengChen-ChengChen2013-04-112018-07-102013-04-112018-07-102011http://ntur.lib.ntu.edu.tw//handle/246246/257344RhoA為小GTP水解酶裡Rho蛋白質家族成員之一，它調控多種的細胞行為，包括細胞移動、分化、細胞週期的前進，和基因的轉錄；就像大部分的GTP水解酶，RhoA循環在活化態 (和GTP結合) 和非活化態 (和GDP結合) 之間，並且只有活化態的RhoA可以作用於下游受器蛋白，由於RhoA的活性受到多種調控機制進行動態控制，因此偵測活體細胞裡RhoA在時間及空間上活性的動態分布以及偵測細胞萃取液中RhoA活性的高低是非常重要的。 在本研究中，我在一段胜肽上接上了靈敏於環境改變的螢光分子，成功建造出只會結合到活化態RhoA的全新RhoA生物感測器，在in vitro實驗中，一旦生物感測器結合到GTPγS-RhoA，螢光強度會增加7.5倍，並且得到解離常數(Kd)為200 nM；該生物感測器也正應用於偵測細胞萃取液中RhoA活性的高低，除此之外，由於該生物感測器含有用來穿透細胞膜的反轉錄活化子片段(TAT domain)，因此也可應用在偵測活體細胞裡RhoA活性在時間及空間上的動態分布。RhoA, a member of the Rho family of small GTPase, regulates multiple cellular behaviors including cell migration, differentiation, cell cycle progression, and gene transcription. Like most of GTPases, RhoA cycles between the active form (GTP-bound) and the inactive form (GDP-bound). Only the active form of RhoA can interact with downstream effectors. Due to the dynamic control of RhoA activity by a variety of regulating mechanisms, it is important to determine the spatiotemporal dynamics of RhoA activity in living cell and the total steady-state level of RhoA activity in cell lysates. In this study, a new RhoA biosensor was successfully constructed by incorporating an environment-sensitive fluorophore onto the polypeptide that can only interact with the active form RhoA. Upon binding to GTPγS-RhoA in vitro, the biosensor showed a 7.5-fold increase in fluorescence intensity and with Kd value of 200 nM. The biosensor was applied to the detection of RhoA activity in cell lysates. Furthermore, the biosensor has been conjugated to the trans-activator of transcription (TAT) domain for cell delivery, so it might be used to determine the spatiotemporal activity of endogenous RhoA in living cells.1354582 bytesapplication/pdfen-US生物感測器RhoABiosensorthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/257344/1/ntu-100-R98223217-1.pdf