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Growth Media and Induction protocol for production of biotinylated proteins (Avidity.com)

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NOTE: The following media is used for growth of cultures for the overproduction of biotinylated fusion proteins using the AVBIOL cell strain, which is transformed with a plasmid encoding the BirA gene (for production of Biotin Ligase) available from Avidity.com (original protocol is available here).  In order to take advantage of this method of biotinylating proteins you must have an oligonucleotide sequence encoding the biotinylation recognition sequence upstream or downstream of your expressed gene.

Media:

TYH Media, 1 Liter

  • 20 g tryptone
  • 10 g yeast extract
  • 11 g HEPES
  • 5 g NaCl
  • 1 g MgSO4
  • pH 7.2-7.4 with KOH

Protocol:

  1. Grow a 10 ml overnight culture from a single colony or glycerol stock in TYH media supplemented with 10 μg/ml chloramphenicol and the appropriate antibiotic (eg. ampicillin) needed to maintain the expression vector with shaking at 37˚C.
  2. Place 5 ml of the overnight into 1 L of TYH media in a baffled Fernbach flask with 100 μg/ml ampicillin. Note: Chloramphenicol is not included.
  3. Add 20 ml of a 20% sterile glucose solution (0.5% final conc.) and shake vigorously at 37˚C.
  4. When the OD600 of the mixture reaches 0.7, remove 1.5 ml as a pre-induction sample.
  5. Add 10 ml of 5 mM biotin solution (50 μm final). The biotin solution is made by adding 12 mg of d-biotin to 10 ml of warm (microwaved) 10 mM bicine buffer (pH 8.3) and filter-sterilizing the solution with a syringe and a 0.2 micron filter.
  6. Add 15 ml of 100 mM IPTG (1.5 mM IPTG final) to induce for 3 hr.
  7. Pellet cells in 4 x 250 ml centrifuge bottles at 5858 x g for 10 min.
  8. Pour off media from cell pellets, re-suspend each pellet in 10 ml B-PER (Pierce Chemical Company, Pittsburgh, PA) (40 ml total volume).
  9. Shake on a rotary shaker 10 min, RT.
  10. Combine suspensions into one bottle and centrifuge at 16,270 x g for 15 min.
  11. Save supernatant. Re-suspend pellet in 25 ml B-PER. Shake on a rotary shaker 10 min, RT.
  12.  Centrifuge at 16,270 x g for 15 min.
  13. Add supernatant to that previously saved. Discard pellet.

Pre-induction and induced samples of bacterial proteins can be analyzed by SDS-PAGE, western blotting with labeled StrepAvidin, or enzymatic means.


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