Figure 6.

Development of a high throughput screening- compatible and luciferase based assay to monitor BB0238-BB0323 interaction. (A) Vector map of pCMV N-Tluc-bb0238 for the expression of BB0238 fused to TurboLuc (Tluc) Luciferase. The cytomegalovirus (CMV) promoter allows strong expression of TurboLuc-BB0238. (B) Production of BB0238 or derivatives in transfected mammalian cells. Immunoblot analyses of 293 T cells transfected either with empty vector (lane 1), or luciferase fused BB0238 (full length or truncated BB0238-∆104, lanes 2 and 3) probed with BB0238 specific antisera. (C) Purification of BB0323. BB0323-N-terminus (residues 22–225) was cleaved from GST using PreScission protease and the supernatant fraction carrying purified BB0323 and post cleavage beads were probed with anti-BB0323 and anti-GST antibody to rule out GST contamination in BB0323. (D) BB0238 binding to BB0323 measured by a luciferase assay. A 96-well white opaque plate was coated with BB0323 (0.1 µg/well), blocked with 5% BSA and incubated with increasing amount of lysates expressing BB0238 in fusion with luciferase (Tluc-BB0238). After washing, bound proteins were detected using luciferase substrate solution and the relative luminescence unit was measured using a luminometer. Binding was calculated after subtracting the blank (wells incubated with increasing amount of extracts transformed with empty vector) mean ± standard error of the mean. (E and F). Competition of BB0238-BB0323 binding. The binding of Tluc-BB0238 with immobilized BB0323 was determined in the presence of recombinant BB0238 purified from E. coli (*p < 0.05, ns - non significant) (E) or antibody specific to BB0323 (*p < 0.01). (F). The luciferase activity in the absence of any competitor was considered as 100%, and the results are expressed as the percentage of Tluc-BB0238 bound to BB0323. One- or two-tailed Student’s t tests were used to compare the mean values using GraphPad Prism.