Dear all,
I would like to know whether addition of IPTG before (37 degC) and after lowering induction temperature (16 degC) affect protein expression levels?
*add IPTG before = add IPTG when 37 degC and then cool down to 16 degC
*add IPTG after = add IPTG when temperature is 16 degC.
And another question, does prechilling your cultures at 4 degC before IPTG induction help solubilization or protein expression?
Regards,
Luke
Hi there,
Always cool down before adding IPTG as what you are aiming at is decrease the rate of protein synthesis. If IPTG is added when culture is at 37°C, protein synthesis will start at high rate which can be detrimental to protein folding and solubility.
Hi there,
Always cool down before adding IPTG as what you are aiming at is decrease the rate of protein synthesis. If IPTG is added when culture is at 37°C, protein synthesis will start at high rate which can be detrimental to protein folding and solubility.
Hi Luke,
Dominique is right, it is better first to cool down your culture before induction with IPTG, especially when you express toxic for E. coli proteins. There are various "protocols" for it - some people chill on ice for 2 min, some for 20 min. There are also people who first decrease the incubator temperature to 16 °C, and when your culture is cooled down, only then add IPTG.
However, in my hands, it works well both if I add IPTG before cooling or after that. There are a lot of factors and issues to decide about when and how much of the inducer to add. For example, one can use the autoinduction medium (no need for IPTG) and other can use Tuner BL21 or its derivatives (LacY permease mutant, for well-tuning of IPTG concentration);
one usually induce E. coli culture with 0.1 mM IPTG and another do it with 1 mM IPTG.
If one uses a fusion to molecular chaperones such as MBP or Trigger factor (Tig), or uses very soluble proteins such as GST, thioredoxin, CBM or NusA as a fusion partner - the influence on your protein's solubility will be very minor even if you add IPTG at 37°C.
Additional factors are the different levels of repression - e.g. pLysS (Novagen Merck), Lemo21 (NEB), BL21-AI (Invitrogen, Thermo, arabinose induction) for T7 promoter -based plasmids, or NEB express with Lac Iq for tac-promoter based expression, the presence of Lac Iq and lac operator at your expression plasmids et cetera. When using a very repressive system , you can not afraid for solubility or even toxicity problems when adding IPTG to the "warm" culture.
If you do not use MBP or TIG fusions, I suggest not lowering the temperature below 18°C, since E. coli chaperones work at 16°C at less extent. Alternatively, you can use Arctic BL21 for expression at 15 or 16°C.
The temperature law says that lowering for every 7°C should add 2 hours to the overall expression time, while "the golden standard" is expression at 37°C for 4 hours.
Best and success,
Yevgeny
If your goal is to express proteins at 16 degree Centigrade then grow your culture at 37 degree centigrade until your OD 600 reaches 0.4 and then bring down the temperature of the incubator. Add IPTG once the temperature has come down. Only then will the protein synthesis occur at a reduced rate thereby facilitating proper folding of thw protein and hopefully driving it into the soluble fraction.
Thank you everyone for your insights and thoughtful answers. I really appreciate your help. I will surely use your suggestions to improve my experiments.
Luke
Totally agree with Dominique Liger
I had several trails with the temperature of addition of inducer.
It should be less than the temperature of induction otherwise you will get a different results.
In some protocols, they suggest to set the OD of addition in ice bath, then to add inducer at this temperature, specially if you are comparing with control.
Good luck :)
This is a very protein-dependent question. I find in many cases that induction at reduced temperatures does not necessarily require cooling the culture prior to the addition of IPTG. Use a minimal amount of IPTG (0.1mM) with a log-phase culture in rich media (E.coli with OD600 of ~1-1.5 in rich 2xYT media). Growing your cells to a greater density will increase yield and limiting the amount of IPTG will prevent insoluble aggregates/ inclusion bodies. This is especially true for "toxic" proteins.
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