Protein Microarray System for Detecting Protein−Protein Interactions Using an Anti-His-Tag Antibody and Fluorescence Scanning:  Effects of the Heme Redox State on Protein−Protein Interactions of Heme-Regulated Phosphodiesterase from Escherichia coli

• Published in: Analytical chemistry (Washington) 2004-11, Vol.76 (22), p.6521-6527
• Main Authors: SASAKURA, Yukie, KANDA, Katsuhiro, YOSHIMURA-SUZUKI, Tokiko, MATSUI, Takuya, FUKUZONO, Shinichi, HAN, Moon Hi, SHIMIZU, Toru
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Antibodies - metabolism; Biological and medical sciences; Chemistry; Escherichia coli; Escherichia coli - enzymology; Exact sciences and technology; Fluorescence; Fundamental and applied biological sciences. Psychology; General, instrumentation; Heme - metabolism; Histidine - immunology; Oxidation-Reduction; Phosphoric Diester Hydrolases - metabolism; Protein Array Analysis; Proteins - metabolism; Spectrometric and optical methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac048832t

A highly sensitive microarray system for detecting protein-protein interactions has been developed. This method was successfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS). To immobilize (His)6-Tag fused Ec DOS, anti-(His)6-Tag monoclonal antibody (anti-(His)6-Tag mAb) was initially immobilized on the solid surface, and (His)6-Tag fused Ec DOS was fixed by antigen-antibody interactions. For this experiment, ProteoChip, generally suitable for antibody immobilization, was used as solid substrate. In this report, we confirm the antibody immobilization ability of ProteoChip and specific binding to the F(c) region of the antibody. Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PAS domain were investigated on the solid surface. Ec DOS immobilized via anti-(His)6-Tag mAb maintained interactions with the PAS fragment, in contrast to directly immobilized Ec DOS in the absence of anti-(His)6-Tag mAb. Heme-redox-sensitive interactions between Ec DOS and the PAS fragment were additionally detected using anti-(His)6-Tag mAb as a mediator. Our results collectively suggest that the immobilization method using anti-Tag antibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures and functions on solid surfaces.