Site specific Biotinylation by BirA
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Site specific biotinylation using the Avi-tag and BirA

Updated: 18.01.23

The Avi-tag is a 15aa peptide sequence (glndifeaqkiewhe) recognized by the E. coli biotin ligase BirA, which biotinylates the lysine in the sequence. The tag can be used N- or C-terminal or be introduced in unstructured loops. For background information please check:

Targeted biotinylation can be a huge advantage for downstream applications like kinetic experiments.Biotinylation and tag removal can be done at same time


Method from -

Biotinylation of Membrane Proteins for Binder Selections


- BirA

- TEV or 3C Protease (Optional)

- 200 mM ATP (dissolve in buffer and adjust to pH 7)

- 1 mM Biotin (dissolve in 50 mM Bicine pH 8.3)

- 1 M MgOAc


Final reaction condition:

- 10 - 50 uM of target protein

- 1:1.5 (Protein:Biotin). ie. if 10 uM protein, then 15 uM Biotin

- 5 mm ATP

- 10 mM MgOAc

- Buffer (Recommended 150 mM NaCl, TRIS/HEPES pH 7.5-8, or PBS) to volume

- 20:1 (Protein:BirA)

- 50-100:1 (Protein:Protease)


Timing - 30-40 min @ 30 C, 1 hour @ RT, Overnight @ 4 C. Recommended: sometime at 30C then at 4C

Clean up - Reverse Ni-NTA and/or SEC.





Hothorn Reaction


The biotinylation reaction

1. Pipet the reaction:

0.15mM Biotin

0.6ul of a 50mM stock (Biotin needs to be pH to dissolve)

2 mM ATP

4ul of a 100mM stock

5mM MgCl2

1ul of a 1 M stock

2uM BirA

4.7ul of a 85uM stock

And your protein with the Avi-tag. Recommended is a concentration > 40uM; I used 15uM and that worked as well. Optional: add TEV at the same time. Fill with buffer to 200ul (I used 25mM Tris pH8, 150mM NaCl, 2mM b-Mercaptoethanol)

2. Incubate at 30 deg, gentle rocking for 1h 
3. ad the same amount of BirA and Biotin 
4. Incubate at 30 deg, gentle rocking for 1h 
5. seperate your protein through a His or SEC

The reaction conditions are derived from above mentioned publication and are aimed at full biotinylation. For Creoptix, it wouldn't be important to have 100% biotinylation, and one can work on less harsh conditions.

If you are using Strepavidin chips you may not even need to repurify, but it is highly recommended.

Purification of BirA in E.coli

  • Treat the protein as a normal Ecoli protein except the following
  • Add BMe during lysis and in final dialysis buffer + 10% glycerol.

Summary of an old purification: