Calibration of IPTG-induced protein expression in E. coli cells


Recommended reading: Use of glucose to control basal expression in the pET system(InNovations 13:8)

All procedures (from step 1-6) should be conducted in a semi-sterile atmosphere.

IMPORTANT:
For the best control for your induction: transform the vector only (without your gene)
into the bacteria, and take one sample of each induction condition for comparison of induction on coomassie.
  1. Transform the plasmid DNA into the appropriate bacterial strain For example: for pET vectors:(BL21(DE3)pLysS or BL21(DE3)
Pay attention to the specific strain antibiotic resistance!

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.

  1. Grow on shakers at 225rpm and take 2-3 x 1.5 ml samples from each temperature after 2h, 4h, 8h and over-night induction. (Times of induction can change according to your schedule and patience.) Check OD600 before spinning down samples in order to load on gel same amount of  bacteria from each induction.
  2.  Spin the samples at 4oC 15,000 rpm for 2 min and discard supernatant. Keep pellets at -20ºC
For further analysis of your samples, lysis is required. This protocol includes sonication. Alternative procedures are
·B-per detergent (for initial screening of several samples) 
This screen may show that most of your protein is found in the pellet, whereas following lysis by sonication your protein may be found in the sup.)
·French-press
Standard Lysis Protocol
1.Lyse pellets in 0.75 ml cold lysis buffer containing PMSF orother appropriate protease inhibitors. 
2.If lysis buffer contains lysosyme, and your protein does not tend to degrade, Incubate at 30oC for 15 minutes (to allow lysis by the lysozyme).Otherwise, incubate at 4oc for 30 minutes.
3.Sonicate 3x20’’(check the new sonication conditions for Hebrew Univ.) till sample clears up and is no longer viscous.
4.Centrifuge 12,000 rpm for 20’ at 4oC.
5.Collect sup to new tubes, and resuspend pellets in 750ml of 1x SDS sample buffer. Boil and aliquot into a few tubes for further analysis. (You can always resuspend in less Sample buffer, and load the appropriate relative amounts.)
6.Into new tubes: add21 µl of each sample and 7 µl of 4x SDS sample buffer containing 100mM DTT.Boil 4min. (Usuallymembrane proteins should not be boiled.)

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.)

Notes:

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.