Why is it recommended an OD600 of 0.6-0.8 for protein expression? My E.coli cells have a maximum OD600 of 2.2 and when I induced them at a high OD600 (around 1) the protein was expressed in high quantities. However, when I induced at a low OD600 ( around 0.5) - little to no protein was produced. What is the rationale behind induction of protein expression at an OD600 between 0.6-0.8 ? Is everyone using this specific range, or have you found, like me, that a different OD600 works better for your proteins?
The protein I am using:
PKA - kinase that phosphorylates non-specifically is rather toxic to the cell. At a very low OD600 the protein is not produced at high levels, whereas, at a high OD600, the concentration is satisfactory.
Hi Anca, if I recall correctly, inducing at 0.6 -0.8 is done in order to induce expression during log-phase growth when cells aren't too sparse or too crowded and while they have plenty of nutrients.
Inducing at higher OD's may result in more PKA abundance for a number of reasons including optimal PKA folding, stability, etc at higher cell densities. This could hypothetically be caused by higher expression of chaperones, PKA inhibitors, different metabolism, reduced growth rates and such when cells are at higher densities. Maybe the deleterious effects of PKA (which might be causing cell death and/or protein degradation at low densities) can be managed better by cells at higher densities. --all of this assumes you are letting cultures grow to OD600 2.2 whether they were induced at OD600 0.6-0.8 or 1. If they aren't allowed to both grow to OD600 2.2, then the results (after a given number of hours in the incubator) might just be due to having more cells in those that were induced at 1.
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