Figure 4.

Characterization of VTN binding to SE36. (a) Biophysical interaction analysis of the binding of VTN to SE36 using surface plasmon resonance (SPR). The raw data (color lines) were fitted to 1:1 binding and bivalent analyte model (black lines) using the Biacore T200 Evaluation software. The fit parameters from these models are summarized in (b). These experiments were performed in triplicate, and representative data are shown. (b) Kinetic and equilibrium constants for the binding of VTN to SE36. (c) Schematic representation of two schemes in bivalent binding model. (i) VTN contains two binding sites. (ii) VTN contains a single binding site and forms a dimer. (d) (i) Purity of VTN-1 to -4 recombinants confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. Two micrograms of each recombinant was run in a gel. Arrows indicate target proteins. Since estimated molecular weights were 18, 21, 14, and 12 kDa in VTN-1 to -4, respectively, it was presumed that these recombinants were glycosylated in Pichia pastoris. (ii) Reactivity of SE36 against each truncated VTN recombinant. Each recombinant was adsorbed to microtiter plate (1 μg/mL) and SE36 was added at the indicated concentrations. (e) (i) Purity of VTN-2-1 to -2-3 recombinants confirmed by SDS-PAGE and CBB staining. Two micrograms of each of recombinant was run in a gel. Arrows indicate target proteins. Since estimated molecular weights were 8 kDa in VTN-2-1 to -2-3, it was, likewise, presumed that these recombinants were glycosylated in P. pastoris. All recombinant proteins of VTN (d and e) were detected by anti-His tag antibody in western blotting and was purified by His GraviTrap. (ii) Reactivity of SE36 against each truncated VTN recombinant. Each recombinant was adsorbed to microtiter plate (1 μg/mL) and SE36 was added at the indicated concentrations.