Figure 9.

Biochemical characterization of the recombinant BBLa protein produced in the Pichia cell culture. A, BBLa purified from the culture medium of P. pastoris was glycosylated. The purified protein was treated with (+) the endoglycosidase EndoHf and analyzed by SDS-PAGE. Separated proteins were stained with Coomassie Brilliant Blue (CBB) or the carbohydrate-staining PAS reagent after being transferred to a polyvinylidene difluoride membrane. Approximately 1 μg of the native protein and approximately 5 μg of the deglycosylated protein were loaded in the lanes. B, Immunoblot analysis of the deglycosylated recombinant BBLa protein produced from the Pichia culture (lane 1) and BBL proteins present in MeJA-treated wild-type BY-2 cells (lane 2). The antiserum against BBL was used for detection. C, Absorbance spectra of the deglycosylated recombinant BBLa protein (0.23 mg mL⁻¹ in 10 mm sodium phosphate buffer, pH 7.0) and a standard solution of FAD. D, Fluorescence emission spectra of the deglycosylated recombinant BBLa protein before (control) and after the treatment with sodium dithionite. The protein samples (0.23 mg mL⁻¹ in 10 mm sodium citrate buffer, pH 4.0) were irradiated at 450 nm.