TIR domains of TLR family-from the cell culture to the protein sample for structural studies

• Published in: PloS one 2024-07, Vol.19 (7), p.e0304997
• Main Authors: Lushpa, Vladislav A, Goncharuk, Marina V, Talyzina, Irina A, Arseniev, Alexander S, Bocharov, Eduard V, Mineev, Konstantin S, Goncharuk, Sergey A
• Format: Article
• Language: English
• Subjects: Antifungal agents; Biology and Life Sciences; Cell culture; Design of experiments; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Functional morphology; Humans; Immune system; Innate immunity; Kinases; Magnetic resonance spectroscopy; Medicine and Health Sciences; NMR spectroscopy; Nuclear magnetic resonance spectroscopy; Physical Sciences; Protein Domains; Protein Folding; Protein purification; Proteins; Research and analysis methods; Spectroscopy; Structure-function relationships; TLR2 protein; TLR3 protein; Toll-like receptors; Toll-Like Receptors - chemistry; Toll-Like Receptors - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0304997

Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E. coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7TIR and MBP-tagged TLR3/5/7/8TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.