Fig 1.
The overall scheme of the experiment.
Human TLR family members (1–10) are schematically shown at the starting point of the flowchart. Cytoplasmic TIR domains under consideration are shown by stars. At the first step, genetic constructs are created for TLRTIR to be expressed together with MBP protein-carrier (“MBP”) or H6 tag (hexahistidine tag, “H6”). Then, the design of experiment is implemented and cultivation parameters for the production of TLRTIR hybrids in the soluble form are selected and preparatively applied. In the next step, the protein purification protocols are developed to produce the milligram quantities of soluble TLRTIR. Finally, protein folding is confirmed by circular dichroism (CD) and NMR spectroscopies.
Fig 2.
Contour and surface response plots for H6-TLR1TIR production.
The effect of temperature and time after induction with different IPTG concentrations on the H6-TLR1TIR expression level. “H6-TLR1TIR“indicates protein yields in milligrams per liter of M9 minimal salts medium quantified based on the protein band intensities on the SDS-PAGE. Models were built based on 150 (A) and 23 (B) [27] experimental points.
Fig 3.
Best yields of recombinant soluble human TLR1-10TIR with either N-terminal His-tag (A, C) or MBP (B, D). A, B: Total (tot.) and soluble (sol.) protein fractions of cellular lysates are shown on the SDS-PAGE for H6-TLRTIR (A) and MBP-TLRTIR (B). Equivalents of 20 mkl of M9 are loaded to each lane. Target protein bands are marked by the red asterisk. C, D: Intensities of the protein bands, corresponding to the total (blue) and soluble (beige) H6-TLRTIR (C) or MBP-TLRTIR (D) are shown. Error bars indicate the standard deviations, statistical significance is provided according to the independent t-test (*—p<0.05, **—p<0.01, ***—p<0.001, ****—p<0.0001, ns denotes that the band intensity is not statistically significant compared to the background).
Table 1.
Summary on the TLRTIR production and purification protocols.
Fig 4.
Summary of TLRTIR purification.
Main purification steps for H6-TLR1TIR (A) H6-TLR2TIR (B), MBP-TLR3TIR (C), MBP-TLR7TIR (D). Purification protocol for each protein included the following steps: Cellular lysis (“Lysate” line for clarified lysate); immobilized metal affinity chromatography (“IMAC”, picture of a flask denotes a fraction of soluble protein that has no affinity to immobilized Ni2+ ions); hybrid protein digestion by thrombin (“Digestion” for classical H6/MBP-TLRTIR cleavage in the tube and a picture of scissors for on-column digestion successfully applied in case of H6-TLR1,2TIR); ion-exchange chromatography (“SP/Q” for TLR1,2TIR) or size exclusion chromatography (“SEC” for TLR3,7TIR). Purple check marks are for the lines illustrating the result of hybrid protein purification, and green check mark—for the result of target protein purification. Bands corresponding to hybrids and target proteins as well as molecular weight markers are signed.
Fig 5.
Structural analysis of TLRTIR.
(A) CD-spectra of the TLR1/2/3/7TIR samples in 30 mM MOPS, pH 6.3, 50 mM NaCl, 0.5 mM TCEP (TLR1TIR), in 60 mM MOPS pH 7.4, 10 mM NaCl, 1 mM TCEP (TLR2TIR), in 30 mM MOPS, pH 8.0, 125 мМ NaCl, 0.5 mM TCEP (TLR3TIR) and in 25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM TCEP, 0.001% NaN3 (TLR7TIR). Calculated secondary structure composition is shown for each protein. (B) 1H,15N-HSQC NMR spectra of TLR1/2/3TIR recorded at 303K in 30 mM MOPS pH 7.4, 64.4 mM KCl, 5.3 mM NaCl, 0.5 mM MgCl2, 0.5 mM TCEP, 0.001% NaN3 i.e. in a buffer designed to properly mimic cellular cytoplasm. D2O was added to the sample to a H2O/D2O ratio of 95 : 5. (С) Spatial structure of TLR1TIR obtained using NMR (PDB: 7NT7) [25], of TLR2TIR resolved by X-ray (PDB: 1FYW) [23] and structure of TLR3TIR predicted by AlphaFold2 [34]. α-helical regions are indicated in red, and the β-sheets are in blue.