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Biochemical and structural characterization of innate immunity kinase complex proteins
Author(s) -
Call R. Jason,
Ghirlando Rodolfo,
Bell Jessica K.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.707.1
Subject(s) - dimer , activator (genetics) , microbiology and biotechnology , chemistry , kinase , scaffold protein , recombinant dna , in vitro , innate immune system , circular dichroism , receptor , biochemistry , signal transduction , biology , gene , organic chemistry
Protein‐protein interactions form the communication link from membrane bound receptors to their downstream cellular targets. Objective Our long‐term goal is to examine the protein‐protein interactions responsible for the activation and regulation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study we have targeted the kinase scaffold proteins, TRAF family member‐associated NF‐κB activator (TANK) and NAK‐associated protein 1 (NAP1). Methods Bacterial expression systems have been used to produce recombinant target protein and refolding methods developed for insoluble targets. Initial biochemical characterization of recombinant targets was completed using circular dichroism (CD), gel filtration (GF) and analytical ultracentrifugation (AUC). Results The protein domain comprising the N‐terminal region of NAP1 can be successfully refolded as assessed by CD, while TANK is soluble. Both constructs form homo‐oligomers as determined by GF. AUC experiments show that the NAP1 N‐term behaves as an elongated dimer in vitro . Gel filtration studies of TANK's kinase interaction site show that it elutes as a dimer species. Conclusions The scaffold proteins, TANK and NAP1, form stable homo‐oligomers via their N‐terminal regions. Funding from the Am. Cancer Society (RJC) and supported in part by the NIH intramural research program, NIDDK (RG).