A 15–amino acid BAP (GLNDIFEAQKIEWHE) was fused to the N terminus of the Hoc protein. The E. coli enzyme biotin ligase (BirA) sequence specifically ligates biotin to BAP. Briefly, His6-tagged BirA and Hoc-BAP were induced in BL21 RIPL cells and purified via a HisTrap HP column and SEC. For Hoc-BAP biotinylation, 1 μM recombinant BirA ligase was added to 30 μM Hoc-BAP monomer [in PBS-MgCl2 buffer (pH 7.4)] in the presence of 0.3 mM biotin and 5 mM ATP. The reaction proceeded at room temperature (RT) for 3 hours. Biotinylation was confirmed by streptavidin-HRP Western blot analysis. Free biotin was removed using Zeba Desalt spin columns (Thermo Fisher Scientific, MA). The biotinylation reaction mixture was loaded onto the center of the compact resin bed and centrifuged at 1000g for 2 min to collect the desalted sample. Purified Hoc-BAP-biotin was incubated with avidin at a 1:3 ratio for 1 hour at 4°C. HBBA was purified by SEC. The peak fractions were concentrated and stored at −80°C.

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