Bajinting Adam, Ng Ho Leung
St. Louis University School of Medicine, St. Louis, MO, USA.
Department of Biochemistry and Biophysics, Kansas State University, Manhattan, KS, USA.
Bio Protoc. 2019 Jun 5;9(11):e3261. doi: 10.21769/BioProtoc.3261.
Receptor tyrosine kinases (RTKs) are an important class of transmembrane receptors that mediate some of the most crucial biochemical pathways essential to the growth, differentiation, and survival of a cell and thus, are highly involved in cancers. Due to the complexity of RTKs having biochemically different domains including a transmembrane domain, an intact crystal structure of any of these proteins remain elusive as it is difficult to produce milligram amounts of intact functional RTKs for crystallography studies. A heavily studied RTK is fibroblast growth factor receptor 2 (FGFR2), which plays a key role in fibroblast growth regulation, differentiation, and oncogenesis. Previous studies have focused on expressing FGFR2's extracellular, transmembrane, and intracellular domains individually. For this protocol, we have focused on the extracellular and transmembrane domains of the FGFR2 protein. The function of the expressed protein is validated by demonstrating its ability to bind heparin and fibroblast growth factor 1 (FGF1). The primary contribution of our protocol is expressing two RTK domains together, including the transmembrane domain, in milligram quantities. Being able to express RTKs to define its crystal structures would enable pharmacologists to design cancer drugs that selectively target active conformations.
受体酪氨酸激酶（RTK）是一类重要的跨膜受体，介导细胞生长、分化和存活所必需的一些最关键的生化途径，因此与癌症密切相关。由于RTK具有包括跨膜结构域在内的生物化学不同结构域的复杂性，这些蛋白质中任何一种的完整晶体结构仍然难以捉摸，因为很难生产出毫克量的完整功能性RTK用于晶体学研究。一种被大量研究的RTK是成纤维细胞生长因子受体2（FGFR2），它在成纤维细胞生长调节、分化和肿瘤发生中起关键作用。先前的研究集中在分别表达FGFR2的细胞外、跨膜和细胞内结构域。对于本方案，我们专注于FGFR2蛋白的细胞外和跨膜结构域。通过证明其结合肝素和成纤维细胞生长因子1（FGF1）的能力来验证所表达蛋白质的功能。我们方案的主要贡献是能够以毫克量共同表达包括跨膜结构域在内的两个RTK结构域。能够表达RTK以确定其晶体结构将使药理学家能够设计选择性靶向活性构象的抗癌药物。
