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

Mohammad Obayed Ullah, Eugene Valkov, Thomas Ve, Simon Williams, Caroline Mas, Ashley Scott Mansell, Bostjan Kobe

Research output: Contribution to journalArticleResearchpeer-review

4 Citations (Scopus)


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
Original languageEnglish
Pages (from-to)31 - 40
Number of pages10
JournalProtein Expression and Purification
Publication statusPublished - 2015

Cite this