Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking

• Published in: Journal of molecular recognition Vol. 28; no. 7; pp. 438 - 446
• Main Authors: Li, Qian, Wang, Jing, Yang, Lingjian, Gao, Xiaokang, Chen, Hongwei, Zhao, Xinfeng, Bian, Liujiao, Zheng, Xiaohui
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.07.2015; Wiley Subscription Services, Inc
• Subjects: affinity chromatography; Antibodies; Antibodies - metabolism; antigen-antibody interaction; Binding; Binding sites; Bridges (structures); Chromatography, Affinity - methods; diazotization; Docking; Fluorescence; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - metabolism; Ligands; molecular docking; Molecular Docking Simulation - methods; oriented immobilization; Protein Binding; protein-protein interaction; Proteins; affinity chromatography; oriented immobilization; protein-protein interaction; antigen-antibody interaction; diazotization; molecular docking
• ISSN: 0952-3499; 1099-1352
• DOI: 10.1002/jmr.2460

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro‐porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount‐dependent analysis in exploring protein–protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10‐10 M and (1.39 ± 0.12) × 109 M‐1. Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount‐dependent analysis is capable of exploring the protein–protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. Copyright © 2015 John Wiley & Sons, Ltd. Oriented immobilized green fluorescence protein (GFP) was used to investigate the interaction between GFP and GFP antibody. Hydrogen bonds and salt bridges were found to be the main driving force for GFP–GFP antibody complex by high‐performance affinity chromatography and molecular docking. The injection amount‐dependent analysis method was capable of exploring the protein–protein interactions with the advantages of ligand and time saving.