Full-length human TLR3 was subcloned into a pENTR/TOPO vector with C-terminal FLAG- and His-tags. From this Gateway entry vector two expression vectors were generated: one for baculovirus expression and the second one for stable mammalian expression. The baculovirus expression system and HEK293 cells with stable expression will be used, that provide correct glycosylation and folding of the target proteins. Full-length TLR3 is expected to be present in the membrane fraction of the cell lysate. Optimization of the expression conditions in insect cells is still in progress, while a purification protocol is being developed on small batches of the protein. Our goal is to determine the high resolution crystal structure of the full-length TLR3 complexed with double-stranded RNA. Since TLR3 has a single membrane-spanning alpha-helix, the signaling complex with 2 membrane-spanning helices may be a more stable candidate for crystallization experiments and structure solution. For structural studies, full-length TLR3 might be further stabilized by complexing it with hUNC93B1. Human UNC93B1 is a membrane protein essential in signaling by TLR3, TLR7/8 and TLR9. It is a relatively poorly characterized protein localized in the ER. A missense mutation in the mouse Unc93b1 gene abrogates signaling via the nucleotide-sensing TLRs. Cells from UNC93B1-deficient patients have compromised signaling via TLR3, 7, 8 and 9. It was shown that UNC93B1 binds to TLRs via their transmembrane domains. It was also shown that the UNC93B1-TLR complex may reach lysosomes without passing through the Golgi, and the interaction between UNC93B1 and TLRs also controls TLR trafficking. Co-expression of hTLR3 with hUNC93B1 in insect cells slightly increases the yield and the stability of both proteins. A purification protocol is also being developed for UNC93B1.
|Botos, Istvan; Segal, David M; Davies, David R (2011) The structural biology of Toll-like receptors. Structure 19:447-59|