title: Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique.
abstract: Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1-D2-D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Minsun
authors.initials: M
authors.lastname: Hong
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.|Department of Systems Immunology and Institute of Antibody Research, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Sung-Il
authors.initials: SI
authors.lastname: Yoon
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.|The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D005408:Flagellin / Q000276:immunology; D006801:Humans; D000090263:Leucine-Rich Repeat Proteins; D008024:Ligands; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification*; D051198:Toll-Like Receptor 5 / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000090263:Leucine-Rich Repeat Proteins; D008024:Ligands; D011506:Proteins; D011993:Recombinant Fusion Proteins; D051198:Toll-Like Receptor 5; D005408:Flagellin