Introduction

Biotinylating avi-tagged proteins while they’re being expressed is a convenient way to specifically biotinylate your protein of interest on just its avi-tag. To do this, you’ll:

1.    Clone or otherwise obtain expression vectors for your avi-tagged protein of interest and BirA.

2.    Co-transform your expression vectors into BL21 (DE3) competent cells.

3.    Grow up an overnight starter culture.

4.    Inoculate your main culture with the starter culture, add D-biotin to the media, and induce protein expression with IPTG.

5.    Harvest the cell pellet and store at -80 °C until you’re ready to purify your biotinylated protein.

In this protocol, we’ll provide step-by-step instructions on how to biotinylate your avi-tagged protein of interest while it is being expressed in Escherichia coli (E. coli). This general strategy is also applicable to insect cell and mammalian cell protein expression; though, the exact experimental details will vary according to your expression system (Erdmann et al, 2010; Ioannou et al, 2018).

 

Printable Protocol

For a print-friendly version of this protocol, click here.

 

Materials

BL21 (DE3) Chemically Competent E. coli Cells

or

BL21 (DE3) Electrocompetent E. coli Cells

Sterile microcentrifuge tubes

Ice bucket

Ice

E. coli Competent Cell Recovery Medium

Small volume culture tubes (~ 12 mL)

Ampicillin (Sodium), USP Grade, 100 mg/mL stock solution

Kanamycin Monosulfate, USP Grade, 50 mg/mL stock solution

or other antibiotics as required by your expression plasmid(s)

Bacterial agar plates with appropriate antibiotics

Sterile LB (or your expression media of choice)

D-Biotin, 1 M stock solution

IPTG, 1 M stock solution

250 mL flask

50 mL conical tube

4 L baffled flask

Temperature controlled shaking incubator

Bunsen burner

Glass beads or bacterial cell spreader

Centrifuge

1 L (or larger) centrifuge tubes

Bacterial Cell Lysis Buffer

or make your own cell lysis buffer using some of the following:

DTT

TCEP HCl

PBS

            Tris HCl

            HEPES

            Imidazole

 

Method

Clone or obtain expression vectors for your avi-tagged protein of interest and BirA (biotin ligase)

You need to have either (i) a single expression vector that expresses both BirA and an avi-tagged version of your protein of interest, or (ii) two compatible expression vectors that each express BirA and an avi-tagged version of your protein of interest.

Note 1: This protocol assumes you already have such expression vectors ready.

Note 2: This protocol is written assuming that you have two compatible expression plasmids that express BirA and an avi-tagged version of your protein of interest that have kanamycin and ampicillin resistance. Modify accordingly if you have one plasmid or different antibiotic selections.

 Co-transform your expression vectors into BL21 (DE3) competent cells

a.    Place sterile microcentrifuge tubes on ice. You’ll need one tube for each protein of interest that you’re expressing. So, if you’re expressing one protein of interest, you just need one tube. If using electrocompetent cells, place a sterile cuvette on ice.

b.    Remove competent cells from the -80 °C freezer and thaw on ice.

c.     Add 1 mL (~ 1 ng) of each plasmid (one plasmid for BirA and one plasmid for your protein of interest) to a microcentrifuge tube on ice.

d.    Add 25 mL of thawed cells to each DNA tube and mix gently by pipetting up and down a few times. Avoid air bubbles. You don’t have to pipette the entire volume. You just want to mix the cells and DNA together.

e.    Incubate DNA-cells mixture on ice for 30 minutes.

f.      If using chemically competent cells, heat-shock the tube at 42 °C for 30 seconds then put the tube back on ice.

g.    If using electrocompetent cells, transfer the DNA-cells mixture to the chilled cuvette without adding bubbles. Flick the cuvette downward to make sure there are no bubbles, and visually check to ensure the liquid spans between each metal-coated side to conduct the electricity. Electroporate the cuvette and put it back on ice.

h.    After heat or electrical shock, immediately add 1 mL of Recovery Medium to the tube or cuvette. Gently pipette up and down and then transfer the entire mixture to a culture tube.

i.      Incubate at 37 °C for 1 hour with shaking (~ 200 rpm) or rotation.

j.      Pour cell mixture into a prewarmed selective (50 mg/mL kanamycin + 100 mg/mL ampicillin) agar plate with the appropriate antibiotics. Use a sterilized spreader or autoclaved glass beads to spread the cells evenly on the plate.

k. Incubate the plate overnight at 37 °C.

 

Note 3: Use sterile technique when transforming your bacterial cells and transferring media between flasks. For example, wipe your bench, pipetter, and gloves with ethanol, and flame the caps or foil before putting on the flask. (Ethanol is flammable - be careful not to light it on fire when working with a Bunsen burner.) Although the antibiotics help prevent unwanted contamination, it is still good practice to exercise proper sterile technique.

 

Note 4: You can plate a fraction of your cells in the recovery medium if you’re getting too many colonies on the plate.

 

Grow up an overnight starter culture

a.    The next day, prepare your overnight starter culture. Add 50 mL of sterile LB, 50 mL of 50 mg/mL kanamycin, and 50 mL of 100 mg/mL ampicillin to a sterile 250 mL flask.

b.    Use a sterile 200 mL pipette tip to pick a single colony from your overnight plate and place it into the 250 mL flask. Place the cap or a piece of foil over the flask.

c.     Allow the flask to shake (~ 200 rpm) at 37 °C overnight.

 

Note 5: Set up your starter culture towards the end of the day and let it go overnight. There is no advantage to having it go longer, and doing so may lead to ampicillin degradation and loss of the amp-selective plasmid. Prepare your starter culture during the last hour that you’re in the lab and harvest it first thing the next morning, which would allow for ~ 16 hours of incubation depending on your work schedule.

 

 Inoculate your main culture, add D-biotin to the media, and induce protein expression

a.    Set up your main culture. Add 1 L of LB (or other media of your choice), 1 mL of 50 mg/mL kanamycin, and 1 mL of 100 mg/mL ampicillin to a 4 L baffled flask. Prewarm flask at 37 °C while you process your overnight starter culture.

b.    Transfer your overnight starter culture from the flask to a 50 mL conical tube.  Centrifuge at 4,000 xg for 10 minutes to pellet the cells. Discard media supernatant back into the 250 mL flask and bleach before discarding the used media.

c.     Resuspend cell pellet by gently pipetting up and down with a few milliliters of media from your main culture, then transfer resuspended cell mixture to the main culture in the 4 L baffled flask.

d.    Shake the flask at 240 rpm and 37 °C and periodically measure the optical density of your culture (OD600).

e.    To measure the OD600 of your culture, pipette out 1 mL of culture and measure its absorbance at 600 nm wavelength of light using LB media only as a buffer blank.

f.      When the culture reaches an OD600 of ~ 0.3, adjust the temperature to 18 °C (or whatever your induction temperature is), slow shaking to 160 rpm, and add 1 mL of 1 M D-biotin to the culture. It will likely take approximately 2-3 hours for your main culture to reach this density. The exact time will depend on the density of your starter culture, the growth rate of your cells, and the growth media. 

g.    When your main culture reaches an OD600 of ~ 0.6, add 1 mL of 1 M IPTG to induce protein expression. Continue growing your culture overnight at 18 °C to express your avi-tagged protein of interest and BirA to biotinylate the avi-tag.

Note 6: The expression conditions (volume, growth medium, temperature, IPTG concentration, shaker speed, etc.) can be optimized for your protein of interest. If you’ve already optimized expression conditions for your protein, then use similar conditions here. If you need more guidance on how to optimize expression conditions, check out this article.

Note 7: Biotin has poor solubility in water. See this article for tips on solubilizing concentrated biotin stock.

 

Harvest the cell pellet and store at -80 °C until you’re ready to purify your biotinylated protein

a.    After expression, transfer your culture to 1 L centrifuge tubes and centrifuge at 4,000 xg, or faster, to pellet the cells.

b.    After centrifugation, transfer the supernatant media back to the 4 L baffled flask and bleach it before discarding.

c.     Option 1: Scrape the cell pellet out of the centrifuge tube and into a storage container and store the cell pellet at -80 °C until you’re ready to lyse the cells and purify your protein of interest.

d.    Option 2: Resuspend your cells in cell lysis buffer using gentle mixing and pipetting, then transfer the resuspended cell mixture to storage containers and store at -80 °C until you’re ready to lyse the cells and purify your protein of interest.

Note 8: GoldBio makes a Bacterial Cell Lysis Buffer that is especially formulated to facilitate robust cell lysis. Depending on your protein of interest, you may want to include DTT or TCEP-HCl in your cell lysis buffer to prevent protein aggregation.  However, you can also make your own lysis buffer using PBS, Tris HCl, HEPES, or other pH buffers and salt. If you make your own lysis buffer, keep in mind what your first purification step will be.

References

Erdmann, D., Zimmermann, C., Fontana, P., Hau, J. C., De Pover, A., & Chène, P. (2010). Simultaneous protein expression and modification: an efficient approach for production of unphosphorylated and biotinylated receptor tyrosine kinases by triple infection in the baculovirus expression system. Journal of biomolecular techniques : JBT, 21(1), 9–17.

Ioannou, M., Papageorgiou, D. N., Ogryzko, V., & Strouboulis, J. (2018). Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase. BMC research notes, 11(1), 390. https://doi.org/10.1186/s13104-018-3500-9

Li, Y., & Sousa, R. (2012). Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin. Biotechnology letters, 34(9), 1629–1635. https://doi.org/10.1007/s10529-012-0942-3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tags

biotin Biotin-NH2 Simon Currie

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