Intrinsically disordered regions affect the expression of Toll-like receptor TIR domains in soluble form

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Resumo

Here we investigate the effect of the C-terminal region on the solubility of TLR4 and TLR10 TIR domains upon the heterologous expression in Escherichia coli. We demonstrated that the absence of this region significantly enhances the yields of globular domains in a soluble form. These findings complement the protocols, previously established to synthesize the TLR TIR domains for the structural studies by various techniques, including by NMR spectroscopy.

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Sobre autores

V. Lushpa

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: ms.goncharuk@gmail.com
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 142290

М. Goncharuk

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: ms.goncharuk@gmail.com
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 142290

A. Arseniev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: ms.goncharuk@gmail.com
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 142290

К. Mineev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt

Email: ms.goncharuk@gmail.com
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 142290; Max-von-Laue-Strasse 9, Frankfurt am Main, 60438 Germany

S. Goncharuk

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Autor responsável pela correspondência
Email: ms.goncharuk@gmail.com
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 142290

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2. Fig. 1. Analysis of amino acid sequences and structures of TLR family proteins. (a) – Analysis of amino acid sequences of intracellular domains. Amino acids constituting the TIR domain according to the UNIPROT annotation are shown in bold. Solid underlining indicates the C-terminal regions and their size in amino acid residues (aa). In the case of TLR10, dotted underlining indicates the sequence described in the work of Nyman et al. [26]; (b) – structures of intracellular domains of TLR1 (PDB: 7NT7) and TLR4/10 according to AlphaFold2 data [29]. TIR domains are shown in red, and the N- and C-terminal regions are shown in green and blue, respectively.

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3. Fig. 2. Assembly of expression constructs. The genes encoding the TIR domains of the TLR4 and TLR10 receptors (TLR4-TIR and TLR10-TIR, respectively) were amplified by PCR, treated with restriction endonucleases, and cloned into the pGEMEX-1 vector treated with the same restriction endonucleases. TLR4-ICD and TLR10-ICD are the genes of the intracellular domains of TLR4 and TLR10, respectively, which encode the TIR domains of these proteins and the amino acid residues adjacent to them. H6-GS-TR is a sequence encoding a tag of six histidines, a flexible linker and a thrombin recognition site (HHHHHHGSGSGLVPRGS), the target gene is TLR4-TIR or TLR10-TIR, T7pr is the T7 promoter, T7ter is the T7 terminator, Ampr is the ampicillin resistance gene.

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4. Fig. 3. Selection of conditions for culturing bacterial strains. (a) – TLR4-TIR expression landscapes constructed using the DOI method [28]. A graphical interpretation of the effect of inducer concentration, temperature, and cultivation time on the accumulation of soluble TLR4-TIR in the cell is provided. The axes show the yield of soluble TLR4-TIR (mg/L of the minimum salt medium M9) and the studied parameters: inducer concentration, temperature, or cultivation time. The landscapes are constructed for the median parameter values: 20°C, 48 h, and 0.05 mM IPTG; (b,g) – selection of optimal parameters for culturing bacterial strains with expression of TLR10-ICD/TIR (b) and TLR4-ICD/TIR (g). Protein electrophoresis in 14% SDS-PAGE of total cellular protein (O) and clarified cell lysate (P) of strains expressing TLR10-ICD/TIR and TLR4-ICD/TIR is shown. The mobility of bands corresponding to the target protein is indicated by arrows. Bands corresponding to soluble target protein are marked with asterisks. Results are presented for an inducer concentration of 0.05 mM IPTG; (c, d) – yield of soluble (beige columns) and total (blue columns) protein of TLR10-ICD/TIR (c) and TLR4-ICD/TIR (d) under optimal cultivation conditions: 28°C, 0.05 mM IPTG, 32 h for TLR10-ICD, 20°C, 0.05 mM IPTG, 48 h for TLR10-TIR, 28°C, 0.05 mM IPTG, 24 h for TLR4-ICD and 25°C, 0.16 mM IPTG, 57 h for TLR4-TIR. The given yields (mg/L M9 minimal salt medium) are calculated from the intensities of the target protein bands on gel electrophoresis; n/a – background gel intensity (in an empty cell). All experiments were performed at least in triplicate, the error is indicated as standard deviation. ICD (IntraCellular Domain) – intracellular domain of the protein (TLR10-ICD or TLR4-ICD), TIR – TIR (Toll-Interleukin-1 Receptor) domain of the protein (TLR10-TIR or TLR4-TIR).

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