Expression of a Selenomethionine Variant in E. coli

 


Preparation of Minimal Media:
 
 

Step Component per 1L per 1.5L
1. Add water to the following, autoclave, and let cool. (NH4)2SO4 1.0 g 1.5 g
KH2PO4 4.5 g 6.75 g
K2HPO4 10.5 g 15.75 g
Na citrate 0.5 g 0.75 g
amino acid mix (19 L-amino acids minus methionine) 0.8 g (~42 mg each of 19 amino acids) 1.2 g (~63 mg each of 19 amino acids)
nucleotide mix (adenine, guanosine, thymine, uracil) 0.5 g (125 mg of each base) 0.75 g (187.5 mg of each base)
2. Add 40% (w/v) glucose (sterile filtered) 12.5 ml 18.75 ml
3. Add 1M MgSO4 (sterile filtered) 1.0 ml 1.5 ml
3. Add 10 mg/ml thiamine (sterile filtered) 0.4 ml 0.6 ml
4. Add 2 mg/ml d-biotin (sterile filtered) 2.0 ml 3.0 ml
5. Add 10 mg/ml L-SeMet 3.0 ml 4.5 ml
6. Add antibiotic 100 mg/ml ampicillin or carbenicillin 1.0 ml 1.5 ml

Expression:
 

  1. Plate out transformed B834(DE3) cells (methionine auxotroph, Novagen) on LB plates.  Minimal media is not necessary here and cells don't grow well on it.
  2. Grow from single colonies in Starter Media (Minimal Media + 5% LB) at 30°C to OD600 = 0.6.
  3. Use the starter culture to inoculate large expression Minimal Media.
  4. Induce the cells in the same way as "parent cells."
  5. If inclusion bodies are produced, try growing cells in Minimal Media + 0.5 M sorbitol + 2.5 mM betaine.  Filter sterilize the sorbitol and betaine before adding to the media.


Typical expression scheme:
 

  1. Inoculate 3 x 20 ml of Starter Media.  Grow overnight at 30 - 37°C.
  2. Using starter cultures inoculate 6 x 1.5 L Minimal Media each with 15 ml.  Grow at 30 -37°C to OD600 = 0.6.
  3. Cool to 30°C for 30 min if necessary.  Induce with 200 uM IPTG (use freshly made stock of 200 mM IPTG and add 1 ul per ml of media).
  4. Induce for 2 - 6 hours.
  5. Centrifuge cells for 15 min at 4000 RPM.  Flash freeze pellets in liquid nitrogen and store at -80°C, if desired.


Purification:

Follow normal purification procedures however keep a reducing agent present after lysing the cells.  Use 1 - 5 mM DTT or 1 - 5 mM TCEP [tris(2-carboxyethyl)phosphine].  TCEP should be used when purifying with a nickel IMAC column since it does not reduce the metal.  Send wild-type and SeMet variant proteins for mass spec analysis to confirm selenium incorporation.
 

Reference for SeMet proteins using MAD


[Hart Lab]

[X-ray Lab]