Figure 7. Avidin blot of biotinylated BCCP catalysed by BPL.

(a) Biotinylation by BPL was carried in a reaction mixture containing 50 mM Tris pH-8.0, 3 mM ATP, 5 µM biotin, 5.5 mM MgCl₂, 0.1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin and 5 nM BPL and 15 µM BCCP over night at 4°C. The reaction mixture was then resolved on a 12% SDS PAGE and transferred to PVDF membrane. The membrane was then incubated with streptavidin HRP for 1 h at room temperature and developed with AEC/ H₂O_2. Lane 1: Molecular weight marker. Lane 2: MtBCCP was incubated with biotin, ATP and MgCl₂ but no MtBPL. The band observed was due to biotinylation of BCCP by endogenous host BPL. Lane 3: MtBPL catalysed biotinylation of BCCP. The intensity of the band was several folds higher than that of biotinylation by endogenous host BPL. (b) holoMtBPL catalyzed reaction was performed in the absence of biotin and ATP. MtBCCP was incubated with sample (peak 1, 2) independently in the absence of biotin and ATP for 30 min at 37°C and resolved on a 12% SDS-PAGE and transferred to PVDF membrane. Holo-MtBPL (MtbPL-bio-5′AMP) transfers biotin to BCCP which was detected by streptavidin –HRP. Lane 1: Peak 1, Superdex S200. Lane 2: Peak 2, Superdex S200.