2.10 Analysis of protein-protein interaction
Proteins are selected prokaryotic expression in vitro and the method as described previously (Jing et al., 2019) (Supplementary Table 2 and Supplementary Figure 1). GOBPs proteins were titrated with Tris-HCl buffer solution by using isothermal titration calorimetry (ITC) (MicroCal ITC200, GE Healthcare, UK). All solutions were thoroughly degassed prior to the titrations to avoid the formation of bubbles in calorimeter cell. The sample cell and reference cell were filled by GOBPs and Tris-HCl buffer of pH=7.4, respectively. Measurements were carried out at 25.0 ± 0.1 °C with a continuous string (600 rpm), a maximum number of injections of 2 μL volume were 20. Control experiments were performed by titrating the Tris-HCl buffer with buffer and subtracted it from the respective GOBPs- Tris-HCl titration before data fitting. The analysis of ITC data was performed using Microcal Origin 7.0 software following the instrument’s manufacturer. The data were best fitted for one set of binding sites, and values enthalpy (ΔH), dissociation constant (KD), entropy (ΔS), and stoichiometry (n) were obtained. The affinity (∆G) was calculated from the Gibbs equation (Haman et al., 2019).
All the solutions involved in the reaction are desalted and then can be used for surface plasmon resonance (SPR) testing. The mixture of 400 mM EDC with100 mM NHS was injected over series S sensor chip CM5 (BR-1005-30, GE Healthcare, USA) at a flow rate of 10 μL/min. GOBP1 (10 μg/mL) and GOBP2 (10 μg/mL), including CSP1, CSP5, CSP10, and OBP17 were injected over CM5 chip at a flow rate of 10 μL/min, respectively. After each run, the dissociation and the regeneration were performed as described above. Following that, the proteins that could interact were serially diluted based on the initial binding, and the evaluation was carried out as described above.