Fig. 4
Determination of the Ki value of the P. pastoris-expressed BLIP against the TEM-1 β-lactamase. 1.5 nM TEM1 β-lactamase was pre-incubated with varying concentrations of BLIP (0–15 nM) in 50 mM sodium phosphate buffer containing 1 mg/mL bovine serum albumin for 2 h at 25 °C. Remaining concentration of free β-lactamase at varying concentrations of BLIP was then estimated by the spectrometric β-lactamase assay using nitrocefin as a substrate. The plot of concentrations of free β-lactamase versus varying amount of BLIP represents the nonlinear regression fit of the data to Eq. (1) for the Ki calculation using the program OriginPro 6.0. Each point represents a single measurement. The experiment was repeated in duplicate. The determined Ki was 0.55 ± 0.07 nM