L dopa

SK-MEL-2 cells, transiently transfected with expression plasmids of TYR, TYRP1, or their chimeric mutants, were lysed in tyrosinase lysis buffer (60 mM Tris-HCl (pH 6.8), 150 mM NaCl, 1.0% Triton-X), and then lysed in 6× SDS buffer (375 mM Tris-HCl (pH6.8), 30% glycerol, 6% SDS, 0.1% BPB) without reducing agents, before electrophoresis. Samples were separated on an 8% polyacrylamide SDS gel at 4 °C, and gels were equilibrated in 50 mM phosphate buffer (pH 6.0) for 3 h with gentle shaking. Colorimetric staining was carried out by incubating gels with a color formation buffer (10 mM) phosphate buffer (pH 6.8) containing 1.5 mM L-DOPA (Nacalai Tesque) and 4 mM 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH, Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) for 1 h at 37 °C.

The strains used in this study are summarized in Table 1. Yeast cells were grown in potato dextrose agar (PDA) [24 g l⁻¹ potato dextrose broth (Difco), 15 g l⁻¹ agar] or YPDA (1 % yeast extract, 2 % polypeptone, 2 % glucose, 2 % agar) for a couple of days at 25 °C. For the induction of laccase production, asparagine medium [1 g l⁻¹l-asparagine, 0.5 g l⁻¹ MgSO₄・7H₂O, 3 g l⁻¹ KH₂PO₄, 1 g l⁻¹ thiamine, 1 g l⁻¹ biotin, 1 mM L−DOPA (Nacalai), pH5.6] was used [17 (link)]. Escherichia coli DH5α was the host strain for the recovery of a plasmid and was grown in LB medium [10 g l⁻¹ polytone, 5 g l⁻¹ bacto yeast extract (Difco), 10 g l⁻¹ NaCl, pH 6.8] containing 25 µg ml⁻¹ kanamycin for 12 h at 37 °C at 150 r.p.m.