The BirA enzyme from E. coli strain K12 is one of the biotin protein ligases (BPLs), which catalyze the attachment of biotin to target proteins. In addition to enzymatic biotinylation, the target molecules can also be biotinylated using the appropriate chemical reagents. In chemical biotinylation, biotinylation of the target molecule can occur at multiple amino acid residues, whereas by using the enzyme BirA, biotinylation occurs at a single lysine amino acid residue within a 15 amino acid long sequence. This target sequence (GLNDIFEAQKIEWHE), named AviTag, can be fused to N- or C-terminus or within an exposed loop of a target protein using molecular cloning. Using BirA and AviTag, we can specifically biotinylate target proteins that can be used in further methods. Due to the very strong binding of biotin to (strept)avidin, biotinylated target proteins can be purified, multimerized, localized inside the cell, or immobilized. With our experimental work, we prepared an active recombinant BirA protein, which can biotinylate target proteins with an inserted AviTag. First, using molecular cloning, we prepared a His6-MBP-3C-BirA containing vector, which we used for the induced expression of recombinant protein in E. coli BL21[DE3] cells. There was a sufficient amount of recombinant protein in the soluble fraction, therefore we used only the soluble fraction for further work. Using Ni-affinity chromatography and size-exclusion chromatography, we purified the recombinant protein from the remaining components of the soluble fraction. Finally, we demonstrated that the prepared and purified recombinant protein is also active and catalyzes the biotinylation of the substrate fused with AviTag. Mouse cathepsin S (mCTSS) with AviTag was used as substrate. After incubation of the substrate and the enzyme with appropriate reagents, we used SDS-PAGE electrophoresis followed by western blot analysis to confirm successful biotinylation. We proved the activity of the prepared recombinant enzyme BirA, which can be further used for the biotinylation of substrates fused with AviTag.
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