Recommended reading: Use of glucose to control basal expression in the pET system(InNovations 13:8)
2.Pick a single colonyinto 2-3 ml LB medium in a sterile snap-cap tubecontaining appropriate selective antibiotics and grow at 37oC o/n.
You can use this starter for additional induction experiments for about 10 days.
You may want to keep a glycerol stock that might be good for a few months from this growth. However, in most clones, you can lose the expression after a few weeks/months, and you will have to start from a fresh de-novo transformation of DNA into the expressing bacterial strain.
3.Add 1.2 ml starter into 120ml 2xYT medium containing the antibiotics and grow at 37oC till OD600=0.6
(This may take 1.5-3h, depending on the bacterial strain).
4.Remove 2 x 1.5 ml of bacterial cells into sterile tube –on ice (un-induced sample), spin down aspirate sup and freeze pellets in –20°C
5.Split culture into 6 x 125 ml erlenmeier flasks (20ml each).
6.Place 3 flasks at Room Temp. shaker and 3 flasks at 37ºC
7.Add IPTG to the 37oc flasks to final concentration of 0.1 0.4 and 0.8mM. (See notes below for Glucose suppression)
8.Let the 23 oc flasks shake for 15 minutes. Then add IPTG(0.1 0.4 and 0.8mM) to these flaskstoo.
7.Run bacterial concentration proportional volumes of protein samples on PAGE-SDS gel.
(The bacterial concentration proportional volumes of protein samples are indicated by the OD measurements of the bacterial suspension at each time point before the cenrifugation at step 9 in the first protocol).
If protein stays in inclusion bodies after all those calibrations, go to:
Heat shock growth procedure
This protocol is based on : Diamant S, Eliahu N, Rosenthal D, Goloubinoff P. Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem. 2001 Oct 26;276(43):39586-91.
When proteins are sensitive to heat shock, try: De Marco A, Vigh L, Diamant S, Goloubinoff P. Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones. Cell Stress Chaperones. 2005, 10(4):329-39
1.Grow cells at 37oC till OD600 is 0.2-0.3 .
2.Add 0.1% glycerol and 0.1mM Potassium Glutamate directly into the medium (We use: SIGMA G-1501 L-Glutamic acid monopotasium salt)
3.Heat shock for 20-30min at 42oC
4.Transfer to original temperature (23oC or 37oC) and let cells grow to OD 0.7-0.8 .
5.Induce with various concentrations of IPTG
6.Follow same induction procedure as before (pick samples after 2h/4h/on etc.)
If suppression of the expressed protein is not complete, and the proteinis toxic to the cells or forms inclusion bodies, one can grow the DE3 based bacterial strains in the presence of 1% glucose to suppress basal expression. In this case, another calibration of IPTG induction is required, with higher amounts of IPTG (0.5mM, 1mM and 2mM), since the effective concentration of the IPTG is now lower.
Reagents and solutions
IPTG (MW: 238.3): Dissolve 238 mg IPTG into 10 ml of distilled H2O to a
concentration of 100 mM. Filter through a 0.22 µm disposable filter.
PMSF: dissolve in isopropanol at a concentration of 200 mM.
2xYT (For 1 liter): 16 g Bacto-Tryptone+10 g Bacto-yeast extract+5 g NaCl
Lysis buffer: 50mM Tris pH 8.0+10% glycerol+0.1% Triton X-100 (TX100 is Optional, might effect purification and/or activity of your protein)+100mg/ml lysozyme (Check if Lysosyme won’t mask your protein in coomassie staining, it is about 15Kd)+1mM PMSF
* Add lysozyme and PMSF right before the experiment.