摘要
目的 确定人成纤维细胞生长因子受体 1(FGFR1)的配体精细结合位点。方法 合成肽文库筛选 ,定向点突变 ,原核细胞重组蛋白质表达及受体 配体结合实验。结果 采用1 2 5 I标记的酸性成纤维细胞生长因子 (aFGF) ,自合成肽文库中筛选到一个五肽序列 (WGPGM) ,其序列及立体结构和FGFR1细胞外段的一个基序 (WTSPEKM)相似。为了证明FGFR1的WTSPEKM基序是结合FGF的重要结构 ,采用定向突变技术将WTSPEKM基序中的P(CCA)突变成A(GCA) ,并在原核细胞中表达了野生型和突变型FGFR1细胞外段重组蛋白质 ,受体 配体结合实验显示突变的FGFR1细胞外段结合aF GF的能力明显降低。结论 FGFR1的WTSPEKM基序是配体结合的一个重要部位。
Objective To determine fine binding site of ligand to fibroblast growth factor receptor 1 (FGFR1). Methods Screening of synthetic peptide library, site directed mutagenesis, Flag 2 fusion protein expression in E. coli and cell free binding assay. Results A 125 I acidic fibroblast growth factor (aFGF) bound peptide with the sequence of WGPGM was fished out from a synthetic peptide library. The sequence was homologous to a motif (WTSPEKM) in extracellular domain of FGFR1. To confirm the role of the motif for the binding of aFGF to FGFR1, site directed mutagenesis technique was used. Both wild type and mutated (P CCA to A GCA ) extracellular domain of FGFR1 were expressed in E. coli as Flag 2 fusion proteins. The cell free binding assay showed that wild type extracellular domain of FGFR1 bound to 125 I aFGF efficiently whereas and mutated extracellular domain of FGFR1 did not. Conclusion The motif (WTSPEKM) in FGFR1 was one of key structures for the binding of aFGF to FGFR1 and therefore a target site for developing an antagonist of FGFR1.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第2期117-120,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目!(批准号 :3 95 70 19)
