FIG 3.

Characterization of purified MelC2 and the fusion protein MelC2h-MelC1. (A) Secreted tyrosinase activity in the unpurified supernatant of a large-volume culture of P. fluorescens harboring pDSK-hMelC2/C1 or pDSK-MelC2h-C1. Volumetric activity was measured via an l-DOPA assay by measuring the change in the A₄₇₅. (B) After purification, the molar specific activities of purified tyrosinases from the culture supernatants of P. fluorescens cells harboring pDSK-hMelC2/C1 and cells harboring pDSK-MelC2h-C1 were compared. Tyrosinase was purified using an Ni-NTA agarose column. (C) The purified tyrosinase from P. fluorescens harboring pDSK-hMelC2/C1 or pDSK-MelC2h-C1 was loaded onto an SDS-PAGE gel. (D) The MelC2h-MelC1 fusion protein from pDSK-MelC2h-C1 was analyzed by SDS-PAGE after long-term 4°C storage or diluted-trypsin treatment. (E) MelC2h-MelC1, after extended storage or trypsin treatment, was analyzed by Western blotting with a His tag antibody. (F) Culture supernatants of MelC2h/C1 and MelC2h-C1 with or without brief trypsin treatment were analyzed via Western blotting. (A and B) Error bars represent the sample standard deviation of the three samples. Lanes (D to F): 1, MelC2h-C1 (47.8 kDa); 2, MelC2h-C1 after 6-month storage at 4°C; 3, MelC2h-C1 after brief trypsin treatment; 4, MelC2h/C1 (32.6 kDa); 5, MelC2h-C1 (47.8 kDa).