Figure 3.
Biotinylation of PfBCCP-79. Western blot analysis of B834(DE3) E. coli cell lysate from cultures expressing his6-tagged PfBCCP-79, in combination with either the E. coli biotin ligase, BirA, the E. coli biotin synthase, BioB, or the iron sulfur cluster (Isc) proteins from Azotobacter vinelandii. Biotinylation levels of the E. coli BCCP (17kDa) and P. falciparum BCCP (11kDa) were assessed by affinity blotting with streptavidin-HRP (SA-HRP, upper panel). Biotinylation can be enhanced 160-fold by coexpressing PfBCCP-79 with BirA and BioB. Loading samples ten times more concentrated shows that there was some biotinylation under all conditions (SA-HRP 10X load, middle panel). Re-probing the blot with PfBCCP antiserum shows that PfBCCP-79 was expressed at similar levels in all samples (α-BCCP, bottom panel).