Figure 2.
Optimization of the biotin synthase reaction medium from the in vitro heterologous system. Biotin synthase activity was measured by the conversion of [3H]DTB to [3H]biotin. Biotin formed was determined by TLC analysis and phosphorimaging quantitation (“Materials and Methods”). Complete reaction medium consisted of 1.5 mg of protein extract from BL21 E. coli cells overproducing Bio2 from Arabidopsis, 10 mm dithiothreitol (DTT), 0.5 mm Fe(NH4)2(SO4)2, 1 mm NADPH, 10 mm KCl, 0.2 mm Ado-Met, 5 mm Fru-1,6-bisP, 0.5 mm l-Cys, 0.1 mm thiamin pyrophosphate (TPP), 20 mm l-Asn, and 150 μm [3H]DTB in 50 mm Tris-HCl, pH 8 [lane BL21(DE3)/Bio2]. The effect of omissions, as indicated, was then investigated. An assay with complete reaction medium except for substitution of the protein extract from Bio2-overproducing strain by a protein extract from untransformed bacteria, was run as negative control [lane BL21(DE3)]. The data are from a representative experiment repeated three times. Fe2+ = Fe(NH4)2(SO4)2.