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.