Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science.
Acta Naturae. 2011 Jul;3(3):77-84.
The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their(15)N- and [(15)N,(13)C]-isotope-labelled derivatives, were described. Each peptide was produced inEscherichia coliBL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.
成纤维细胞生长因子受体 3（FGFR3）是一种属于受体酪氨酸激酶家族的蛋白质。FGFR3 在人类骨骼发育中起着重要作用。该蛋白中的突变，包括跨膜（TM）区域中的 Gly380Arg 或 Ala391Glu 取代，可导致骨骼发育中的不同疾病。确定正常蛋白和具有单个 Gly380Arg 和 Ala391Glu 突变的蛋白中的 FGFR3 TM 结构域的空间结构对于理解控制二聚化和受体酪氨酸激酶信号转导的机制至关重要。描述了在细菌中表达真核基因的有效系统以及生产毫克量的正常 TM 片段 FGFR3 以及具有单个致病变异 Gly380Arg 和 Ala391Glu 的 TM 片段及其（15）N-和[(15)N，(13)C]-同位素标记衍生物的纯化方案。每个肽均作为硫氧还蛋白 A 的 C 末端延伸在大肠杆菌 BL21(DE3)pLysS 细胞中产生。该纯化方案涉及固定金属亲和层析、阳离子和阴离子交换层析，以及与人类肠激酶轻链的融合蛋白切割。使用 NMR 光谱证实了目标肽整合到 DPC/SDS 和 DPC/DPG 胶束中的效率。使用所描述的生产正常和单个 Gly380Arg 和 Ala391Glu 突变的 FGFR3 TM 结构域的方法，可以使用高分辨率异核 NMR 光谱研究其空间结构。
