Molecular cloning, overexpression, and characterization of the ligand-binding D2 domain of fibroblast growth factor receptor

被引:10
作者
Hung, KW
Kumar, TKS
Chi, YH
Chiu, IM
Yu, C [1 ]
机构
[1] Natl Tsing Hua Univ, Dept Chem, Hsinchu 300, Taiwan
[2] Univ Arkansas, Dept Chem & Biochem, Fayetteville, AR 72701 USA
[3] Ohio State Univ, Dept Internal Med, Columbus, OH 43210 USA
[4] Ohio State Univ, Dept Mol & Cellular Biochem, Columbus, OH 43210 USA
关键词
growth factor; receptor; expression; NMR; beta-sheet;
D O I
10.1016/j.bbrc.2004.03.028
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Fibroblast growth factors (FGFs) regulate a wide range of important cellular processes. The biological activities of FGFs are mediated by cell surface receptors (FGFRs). In the present Study for the first time we report the cloning, expression, and characterization of the ligand (FGF)-binding D2 domain of human FGFR2. D2 domain is expressed in Escherichia coli in high yields (10mg/L) as inclusion bodies. The protein is recovered by dissolving the inclusion bodies in 8 M urea and Subsequently refolding oil nickel affinity column. The protein is purified (to similar to97% purity) to homogeneity using heparin-Sepharose affinity column. Far-UV circular dichroism data and chemical shift index plot based on H-1-alpha, C-13-alpha, C-13-beta, and (13)carbonyl group chemical shifts suggest that D2 domain is an all beta-sheet protein consisting of 9 P-strands. lsothermal titration calorimetry and equilibrium urea unfolding experiments show that recombinant D2 domain is in a biologically active conformation and binds strongly to its ligand (FGF) and to the heparin analog, sucrose octasulfate (SOS). Using a variety of triple resonance NMR experiments, complete assignment of H-1, N-15, and C-13 resonances in D2 domain has been accomplished. The findings of the present study not only pave way for an in-depth investigation of the molecular mechanism(s) underlying the activation of FGF signaling but also provide avenues for the rational design of potent inhibitors against FGF-rnediated pathogenesis. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:253 / 258
页数:6
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