In vitro activity of recombinant GH3-6.

(A) The expression of recombinant GH3-6 protein, which was used for the inhibition assays shown in Figs. 2 and 4, was tested by separating 10 µl of His GraviTrap column elution fractions (E1–E3) on a 4–12% polyacrylamide gel followed by Coomassie Brilliant Blue staining (upper panel) or immunodetection using a monoclonal antibody raised against poly-histidine (lower panel). The molecular mass standards (Precision Plus Protein all blue, BioRad) are indicated. The band with the size of approximately 70 kDa corresponds to the His-tagged GH3-6 protein. (B) TLC analysis of GH3-6 enzyme reactions with IAA and 20 amino acids (single letter code). The spot near the origin for the reactions with Trp represents the unbound amino acid. Plates were stained with Ehmann's reagent to detect indole compounds.