Journal
JOURNAL OF BIOTECHNOLOGY
Volume 157, Issue 1, Pages 75-81Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2011.10.007
Keywords
NOD1; Protein expression; FPA; NLR; Nucleotide binding; Bid
Categories
Funding
- NIAID NIH HHS [R01 AI056324-01, R01 AI056324-04, R01 AI056324-08, R01 AI091967-02, R01 AI056324-05, R01 AI056324-09, R01 AI056324, R01 AI082629-03, AI-91967, U01 AI078048-04, AI-82929, U01 AI078048-01, U01 AI078048, R01 AI056324-07, F32 AI082929, R01 AI082629-02, R01 AI056324-03, AI-78048, R01 AI056324-06A1, R01 AI091967, R01 AI056324-02, U01 AI078048-03, R01 AI082629, R01 AI082629-01A1, R01 AI091967-01, U01 AI078048-05, R01 AI082629-04, AI-56324, U01 AI078048-02, R01 AI056324-10] Funding Source: Medline
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NOD1 (NLRC1) is a member of the NLR family of innate immunity proteins, which are important cellular sensors of various pathogens. Deregulated NOD1 signaling is involved in various autoimmune, inflammatory, and allergic diseases, making it a potential target for drug discovery. However, to date, the successful high-yield purification NOD1 protein has not been reported. Here we describe the large-scale expression of recombinant NOD1 protein in non-adherent mammalian cells. One-step immunoaffinity purification was carried out, yielding highly pure protein with excellent yields. Gel-sieve chromatography studies showed that the purified NOD1 protein eluted almost exclusively as a monomer. Addition of the NOD1 ligand (gamma-Tri-DAP) stimulated NOD1 protein oligomerization. Using purified NOD1 protein for nucleotide binding studies by the Fluorescence Polarization Assay (FPA) method, we determined that NOD1 binds preferentially to ATP over ADP and AMP or dATP. We also documented that purified NOD1 protein binds directly to purified pro-apoptotic protein Bid, thus extending recent data that have identified Bid as an enhancer of NOD1 signaling. This expression and purification strategy will enable a wide variety of biochemical studies of mechanisms of NOD1 regulation, as well as laying a foundation for future attempts at drug discovery. (C) 2011 Elsevier B.V. All rights reserved.
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