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:
- 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.
- Grow from single colonies in Starter Media (Minimal Media + 5% LB) at 30°C to OD600 = 0.6.
- Use the starter culture to inoculate large expression Minimal Media.
- Induce the cells in the same way as "parent cells."
- 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:
- Inoculate 3 x 20 ml of Starter Media. Grow overnight at 30 - 37°C.
- Using starter cultures inoculate 6 x 1.5 L Minimal Media each with 15 ml. Grow at 30 -37°C to OD600 = 0.6.
- 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).
- Induce for 2 - 6 hours.
- 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.