BirA biotinylation protocol

Biotinylation of avi-tagged proteins

This protocol uses a kit, but works fine with BirA we purify ourselves too.

Materials

• Biomix A: 0.5M bicine buffer solution, pH 8.3
• Biomix B: 100mM ATP, 100mM Mg(OAc)2, 500uM d-biotin
• 1 mg/mL BirA biotin-protein ligase

Protocol

1. Buffer exchange proteins into 10 mM Tris-HCl, pH 8.0. (Salt inhibits BirA.)

2. Dilute to 1 mg/ml protein solution.

3. Determine correct amount of BirA and buffer components for reactions.

   For MBP:
   
   1 nmol of 42 kDa protein = 42 µg.
   
   We want 10 nmol, so 420 µg.
   
   1 mg/ml protein solution has 500 µg protein in 0.5 ml.
   
   500 µg protein substrate/(420 µg substrate per 10 nmol) = 11.9 nmol substrate to be biotinylated.
   
   11.9 nmol substrate/(10 nmol per 2.5 µg BirA) = 2.97 µg BirA
   
   40 µM solution gives the most efficient reaction.
   
   1000 µg/1 ml avi-MBP WT = 66.6 µM
   
   (66.6 µM/40 µM)*(x/0.5 ml) => x = 0.83 ml.
   

4. Mix final reaction mixture. Bring up to 0.83 ml with Biomix A, B (10X), BirA, and buffer.

   • 500 µl substrate
   • 83 µl Biomix A
   • 83 µl Biomix B
   • 161 µl Tris buffer, pH 8.0
   • 3 µl BirA enzyme

5. Allowed reaction to proceed for 45 min at 30 °C.

6. Buffer exchange into fresh 10 mM Tris buffer to remove excess biotin.

7. Concentrate protein to final concentration of 1.7 mg/ml, ~40 µM.