SeMet Labeled Protein Expression via Metabolic Inhibition

Adapted from VanDuyne G.D. et al. (1993) J. Mol. Biol. Vol. 229, 105-124

 

Before starting you should check that expression is OK in minimal media.  Also it is best if all steps can be carried out at 37C the cells grow too slowly at 30 to do this in an efficient manner.

 

AS ALWAYS:

Use care in working with SeMet as it is toxic.  Gloves and a mask should be worn.

Disposal of SeMet Media must be done through the hazardous waste disposal program on campus. That means autoclave the used media, pour it in a waste bottle, and take it to the proper disposal site.

 

You’ll need:

Ø      1 L of M9 minimal media.

Ø      A BL21/(DE3) strain with your protein of interest in it, use a stock that expresses well!!

 

 

Day1

1.      Pick a single colony of your bacterial stock and inoculate a 40 mL stock of LB supplemented with the appropriate antibiotic.

2.      Grow overnight at 37 C with shaking.

 

Day2

1.      Pellet overnight culture at 3000 x g for 10 minutes at 4 C.

  1. Pour off supernatant and re-susped in 40 mL of M9 media with the appropriate antibiotic.
  2. Add 500 uL of re-suspended cells to 500 mL of M9 media. 
  3. Grow cultures to OD600 of 0.5 at 37 C with shaking.  This will take longer than in LB; 4-6 hrs minimum.
  4. Take a 50 uL sample and add to 50 uL of 2x protein loading buffer. Save this for later.

6.      Add the following to the cultures:

§         Lysine, phenylananine, and threonine to 100 mg/L

§         Isoleucine, leucine, and valine to 50 mg/L

§         L-selenomethionine to 60 mg/L

7.      Grow for 15 minutes at 37 C with shaking.

8.      Add IPTG to a final concentration of 1 mM.

9.      Lower temp to ~25 C. It’s best just to turn the incubator heater off.  Allow the cultures to grow overnight.

10.  Take a 50 uL sample the next morning and add it to 50 uL of 2x protein loading buffer.  Run a gel with the pre-induction sample.

11.  Spin down cells as you would normally.  Make your first purification attempt as you would purify Wt protein and tweak from there.  Consider raising the DTT or B-me content of your buffers, about 5mM B-me in all buffers should suffice.  If oxidation is still and issue protein may have to be stored under N2 gas.  Save some purified protein for mass spec to check the extent of SeMet incorporation.