Figure 6
Liberation of biologically active [35S]-biotin, and its verification and quantification in E. coli. (A) Autoradiograph of a SDS-PAGE gel showing acid hydrolysis of the flow through from the metal chelate column (FT1) and the elution fraction containing 35S-labeled PfBCCP-79 (E1). (B) Autoradiograph of cell lysate from a ΔbioB E. coli strain cultured with the protein hydrolysates shown in panel A. The 17 kDa BCCP protein from E. coli is specifically labeled by the E1 hydrolysate, showing that [35S]-biotin is taken up and incorporated into cellular proteins. (C) Standard curve showing cell growth of ΔbioB E. coli cultures supplemented with increasing concentrations of biotin. This curve was used to quantify the concentration (indicated by the red line) of [35S]-D-biotin produced.