Full-length human TLR3, TLR7, TLR8 and TLR9 were cloned into a pENTR/TOPO vector with C-terminal FLAG- and His-tags. From this Gateway entry vector two expression vectors were generated for each TLR: one for baculovirus expression and the second one for stable mammalian expression. The HEK293 cells with stable expression did not produce enough protein, therefore we focused on the baculovirus expression system. The expression conditions were optimized and a general membrane-protein purification protocol was developed on small scale. Most full-length constructs showed extensive degradation that can be traced back to the expression. Full-length TLR9 could be purified on a very small scale, however any scale-up of the protocol resulted in too much degradation. For structural studies, full-length TLR3, 7, 8 and 9 might be further stabilized by forming a complex 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. Human UNC93B1 was cloned into a pENTR/TOPO vector with C-terminal FLAG- and His-tags. A purification protocol was developed for UNC93B1 and most of the protein has a tendency to form large molecular weight aggregates. Small quantities of pure, monomeric UNC93B1 were obtained for screening of crystallization conditions. Co-expression of hTLR3 with hUNC93B1 in insect cells slightly increased the yield and the stability of both proteins, however, large-scale batches showed too much degradation.
| Botos, Istvan; Segal, David M; Davies, David R (2011) The structural biology of Toll-like receptors. Structure 19:447-59 |
