Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in toll-like receptor and interferon signaling

Obayed Ullah, M., Valkov, Eugene, Ve, Thomas, Williams, Simon, Mas, Caroline, Mansell, Ashley and Kobe, Bostjan (2014) Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in toll-like receptor and interferon signaling. Protein Expression and Purification, 106 31-40. doi:10.1016/j.pep.2014.09.019


Author Obayed Ullah, M.
Valkov, Eugene
Ve, Thomas
Williams, Simon
Mas, Caroline
Mansell, Ashley
Kobe, Bostjan
Title Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in toll-like receptor and interferon signaling
Journal name Protein Expression and Purification   Check publisher's open access policy
ISSN 1046-5928
1096-0279
Publication date 2014-10-13
Year available 2014
Sub-type Article (original research)
DOI 10.1016/j.pep.2014.09.019
Open Access Status
Volume 106
Start page 31
End page 40
Total pages 10
Place of publication Maryland Heights, MO, United States
Publisher Academic Press
Collection year 2015
Language eng
Formatted abstract
TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the “BB loop” regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction.
Keyword Toll-like receptor adaptor protein
Innate immunity
Protein expression
Protein purification
Systematic construct design
GST pull-down assays
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes epub ahead of print - Available online 13 October 2014

 
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Created: Fri, 17 Oct 2014, 10:57:41 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences