BirA Biotin Protein Ligase, Recombinant
Recombinant BirA Biotin Ligase (amino acids 3 – 321) with the N-terminal His-tag, MW = 36.2 kDa, expressed in an E. coli cell expression system. BirA can be removed after biotinylation by running the reaction mixture through a Ni-NTA column if the substrate does not contain a His-tag.
For in vitro biotinylation of proteins and peptides. E. coli Biotin Ligase BirA (EC 188.8.131.52) activates biotin to form biotinyl 5'-adenylate and transfers the biotin to biotin-accepting proteins and peptides. The natural substrate of BirA is the Biotin Carboxyl Carrier Protein (BCCP), requiring fusion of at least 75 residues to the target protein. However, several shorter peptide sequences have been described for BirA-mediated biotinylation such as 15 amino acids in length AviTag™ peptide (GLNDIFEAQKIEWHE). BirA site-specifically biotinylates a lysine side chain within this peptide. AviTag works at either the N or C terminus of the target protein.
Catalog # BBL-301
Applications: In vitro biotinylation of proteins and peptides. The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Enzymatic biotinylation with BirA is more specific than chemical biotinylation which often generates heterogeneous products that may have impaired function.
Storage buffer: 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 3 mM DTT, and 50% Glycerol.
Concentration: 1.0 mg/mL by A280
Purity: >90% (SDS-PAGE, Coomassie staining)
Activity: 5,000 Units/µg
Definition of Activity: 1 Unit is the amount of enzyme that will biotinylate 1 pmol of AviTag peptide substrate in 30 minutes at 30°C in the reaction buffer containing 10 mM Tris-HCl, pH 8.0, 10 mM ATP, 10 mM MgOAc, 50 µM d-Biotin, and 38 µM peptide substrate.
Storage is recommended at -20°C for longer periods of time.
This product is intended for research use only.
Shipping: Product requires shipping on ice packs. Please contact firstname.lastname@example.org for shipment estimates.
In addition, Amid Biosciences offers BirA Biotin Ligase version as a maltose-binding protein fusion enzyme (BirA Biotin Ligase, MBP-tag).
Application References:1. Mark Howarth & Alice Y Ting. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. (2008) Nature Protocols 3: 534
2. Askin et al. Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase. (2016) Anal. Methods 8: 418
3. Cai Huang and Ken Jacobson. Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation. (2010) BioTechniques 49: 881
4. de Boer et al. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. (2003) Proc. Natl. Acad. Sci. USA 100: 7480
5. Elif Nur Firat-Karalar, Tim Stearns. Probing mammalian centrosome structure using BioID proximity-dependent biotinylation. (2015) Methods in Cell Biology, 129: 153