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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-LB4
Session: LATE-BREAKING/Proteomics: New Methods
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Identification of the Interacting Protein of Heme-regulated Phosphodiesterase from Escherichia coli Using Protein Microarray

Yukie Sasakura1,2, Makoto Nogami1, Tokiko Yoshimura-Suzuki2, Shinichi Fukuzono1, Toru Shimizu2, Katsuhiro Kanda1

1 Hitachi High-Technologies Corporation, Hitachinaka, Japan
2 Institute of Multidisciplinary Research for Advanced Materials, Tohoku Univ., Sendai, Japan

Correspondence address: Yukie Sasakura, Hitachi High-Technologies Corporation, Bio-Medical Center, 882, Ichige, Hitachinaka, 312-8504 Japan.

Keywords: Affinity, Binding; Complex, Non-Covalent; Interactions, Protein/Peptide; Sample Preparation.

Novel aspect: To identify interacting proteins of the target by LCMS analysis, we developed a unique methodology by immobilizing the target on a solid surface by Ag-Ab interaction. The novel interacting protein and its physiological relationship was determined.

 

In our previous study, we have developed the novel protein immobilization method for the protein-protein interaction analyses on the solid surface. Anti-Tag antibody (Ab) was in advance, immobilized on the sold surface and tag-fused target proteins were captured by antigen-antibody interaction. Using this method, the already known interaction of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) was successfully detected on the solid surface. [1,2]
In this study, we adopted this novel immobilization method to identify the protein interacting with Ec DOS. Ec DOS was immobilized on the solid surface via anti-Tag Ab, and E.coli lysate was applied in it. Interacting proteins were captured on the surface, eluted from it, and analyzed by Nano-LC/LIT-TOF MS. We succeeded to identify GntR, which is the transcriptional regulator of Gnt operon, as the novel protein interacting with Ec DOS, and demonstrated their physiological relationships in vivo. These results suggested that this method would be useful to clarify the unknown protein-protein interactions.
[1] Sasakura, Y., et.al., Anal.Chem., 76, 6521-6527 (2004), [2] Sasakura, Y., et.al., Biochemistry, 44, 9598-9605 (2005)