Wang Qian, Wang Zhixiang
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada, T6G 2H7.
Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada, T6G 2H7.
Methods Mol Biol. 2017;1652:135-143. doi: 10.1007/978-1-4939-7219-7_10.
As an essential tool in molecular biology and biochemistry, in vitro mutagenesis is important for mechanistic and functional studies of protein at the cellular level. Through site-directed substitution, insertion, or deletion of potential function residues, we may evaluate the specific biological role of proteins and understand the underlying mechanism. EGF ligand binding will activate EGFR and initiate signaling transduction for cell proliferation, differentiation, and survival; meanwhile, it will also induce the EGFR endocytosis for degradation to maintain cellular homeostasis. During this complicated process, some key residue(s) or sequences of EGFR molecule play essential regulatory roles. We describe in this chapter the inverse PCR-based site-directed mutagenesis protocol used in our studies on EGFR signaling and endocytosis.
作为分子生物学和生物化学中的一种重要工具，体外诱变对于在细胞水平上进行蛋白质的机制和功能研究至关重要。通过对潜在功能残基进行定点取代、插入或缺失，我们可以评估蛋白质的特定生物学作用并了解其潜在机制。表皮生长因子（EGF）配体结合将激活表皮生长因子受体（EGFR）并启动细胞增殖、分化和存活的信号转导；与此同时，它还将诱导EGFR内吞作用以进行降解，从而维持细胞内稳态。在这个复杂的过程中，EGFR分子的一些关键残基或序列发挥着重要的调节作用。在本章中，我们将描述基于反向PCR的定点诱变方案，该方案用于我们对EGFR信号传导和内吞作用的研究。
