酪氨酸激酶受体多肽片段在SPR传感芯片表面的磷酸化及其与下游蛋白的相互作用  

Phosphorylation of Receptor-mimic Tyrosine Peptide on Surface Plasmon Resonance Sensor Chip Surface and Its Interaction with Downstream Proteins

作　　者：李雪玲[1] 黄明辉[2] 曹慧敏[1] 陈瑶[2] 赵建龙[1] 杨梦苏[2] 

机构地区：[1]中国科学院上海微系统与信息技术研究所,上海200050 [2]香港城市大学深圳药用生物芯片重点实验室,深圳518057

出　　处：《分析化学》2008年第10期1327-1332,共6页Chinese Journal of Analytical Chemistry

基　　金：上海重点基础研究资助项目(No.04JC14081);国家重点基础研究发展规划项目(973计划)(No.2005CB724305)

摘　　要：胰岛素样生长因子-1受体(IGF-1R)信号转导通路与各种正常生理活动及多种疾病发生密切相关。将IGF-1R来源的未磷酸化多肽序列LYASVNPEY固定于表面等离子体共振(SPR)生物传感器专用的CM5芯片表面,以含有相应蛋白激酶的细胞裂解液进行处理,利用SPR生物传感器和抗酪氨酸磷酸化抗体PY20进行酪氨酸磷酸化水平检测。结果表明:PY20能灵敏地检测多肽酪氨酸磷酸化水平变化,即能够区分不同孵育时间以及不同成分的细胞裂解液所引起的多肽酪氨酸磷酸化水平差异;研究了IGF-1R来源多肽的酪氨酸磷酸化对多肽-蛋白间相互作用的影响,证实了只存在磷酸化的多肽与其下游的胰岛素受体底物(IRS-1)相互作用,测定了此磷酸化的多肽与IRS-1的亲和常数(KA)为(2.02±0.09)×108Lmol-1、结合速率常数(ka)为(2.30±0.15)×106Lmol-1s-1以及解离速率常数(kd)为(1.14±0.13)×10-2s-1。这种新型的模拟受体的多肽酪氨酸磷酸化研究方法可用于受体磷酸化信号通路的研究以及基于这些信号通路的药物筛选研究。Insulin-like growth factor receptor-1 （IGF-1R） signal pathway is critical in many physiological activities and oecurance of some diseases. IGF-1R derived nonphosphorylated peptide LYASVNPEY was immobilized on the surface of CM5 sensor chip specialized for surface plasmon resonance （SPR） biosensor. The IGF-1R derived peptide surface was treated with cell lysates containing or not containing IGF-1R kinase. The results show that the antibody PY20 can sensitively detect the level of tyrosine phosphorylation using SPR biosensor, i. e. PY20 can discriminate the different level of peptide tyrosine phosphorylation treated with different incubation time and different lysates. The effect of the peptide tyrosine phosphorylation on peptideprotein interaction was further studied. The results demonstrated that only the phosphorylated peptide can interact with its downstream insulin receptor substrate-1 （IRS-1）. The binding affinity （KA） and association and dissociation kinetics constants （ka and kd） of the phosphorylated peptide with IRS-1 were measured by SPRbiosensor, with the results of KA = （ 2. 02 ± 0. 08 ） ×10^8 mol^-1, ka=（2.30±0.15） ×10^6Lmol^-1s^-1 and kd = （1.14 ± 0. 13） × 10^- 2s^-1, respectively. This new phosphorylation method of tyrosine peptide that simulates cell reeeptor has the characteristics of real-time, speediness, no labeling and convenience for detection, which is instrumental for the investigations on receptor phosphorylation signal pathways and the drug screening against these signal pathways.

关 键 词：表面等离子体共振 生物传感器 胰岛素样生长因子-1受体激酶 酪氨酸磷酸化 亲和力 

分 类 号：Q55[生物学—生物化学]