Figure 1

Synthesis of radiolabeled biotin in E. coli. The E. coli biotin synthase (BioB) catalyzes the insertion of sulfur into desthiobiotin to form biotin, which is then ligated to the biotin carboxy carrier protein (BCCP) by the biotin protein ligase (BirA). The sulfur is donated from a [2Fe-2S] cluster on BioB which is formed by iron sulfur cluster biogenesis (Isc) proteins. E. coli supplemented with desthiobiotin and ³⁵SO₄ will produce small quantities of radiolabeled biotin bound to BCCP. The P. falciparum BCCP, which does not contain the amino acid cysteine, was overexpressed in the methionine auxotroph E. coli strain B834(DE3) to prevent the incorporation of ³⁵S into the BCCP protein. BirA, BioB and the Isc proteins were overexpressed to increase the yield of radiolabeled biotin.