Determination of binding potency of peptidic inhibitors of Grb2-SH2 by using the protein-captured biosensor method.
The growth factor receptor-binding protein 2-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, therefore, peptidic inhibitors of the Grb2-SH2 domain has been chosen as our target for the development of antiproliferative agents. The inhibitory effects of peptide analogs on the Grb2-SH2 domain have been determined by using surface plasmon resonance (SPR) technology developed with the BIACORE biosensor. Recently, we reported the analysis of interactions between peptides and the GST-Grb2-SH2 that was immobilized on the surface of sensor chip by using BIACORE biosensor (the protein-immobilized method). Herein, we analyze interactions of peptides with the GST-Grb2-SH2 that was captured by the anti-GST antibodies immobilized on the surface of sensor chip (the protein-captured method). Results obtained by both methods are in good correlation, indicating the immobilization of GST-Grb2-SH2 on the sensor chip did not significantly affect the binding of Grb2-SH2 with peptides. Both SPR-based assays are very sensitive bioanalytical methods and can be applied in screening inhibitors of target proteins or purifying GST-fusion proteins, however, considering the efficiency and the cost, the GST-Grb2-SH2-immobilized method is suggested for routinely determining the binding potency of inhibitors of Grb2-SH2.
Department of Chemistry, Tunghai University, Taichung, Taiwan, ROC. fdlung@thu.edu.tw
Feng-Di T
FD
Lung
Wan-Ching
WC
Li
Yung-Hsien
YH
Chang
Hui-Ming
HM
Chen
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D003365:Costs and Cost Analysis; D051380:GRB2 Adaptor Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000276:immunology; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000191:economics / Q000379:methods*
D000906:Antibodies; D051380:GRB2 Adaptor Protein; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase