I want to optimize the protein expression by inducing at a low temperature. I am just wondering what is the best induction time at 16 degree. I am using BL21 and 0.5 M IPTG. Thank you guys.
In my hands, 48h-72h top get maximal accumulation of the expressed protein, but I think that this depends of the protein nature.
At 16 deg, you could incubate for anywhere from 18-72 h. It is also dependent on the inducer concentration and the nature of protein.
Every situation is different, so do a small scale expression test and take time points monitor by either Coomasie or silver stained SDS-PAGE and identify the best time point for yourself. You can also vary IPTG concentration and temperature, OD at induction so as to identify the best over-expression condition.
I agree about varying IPTG concentration- I usually decrease the IPTG concentration at lower temps, and when I do overnight or longer inductions. The goal is to express your protein slowly, giving the bacteria time to fold the protein correctly and keep it soluble.
Hi,
You might want to think about autoinduction media. In a great deal of cases the IPTG uptake is more of a step function. Which means all your doing is affecting the number of cells in your population that are "on" rather than tuning each one to produce more slowly. John Dunn tried to manipulate the transporter to make act more like a rheostat to no avail. The autoinduction media plays with the uptake of IPTG by through changing glucose concentration as the growth proceeds. The media has glucose (limiting, but an inhibitor of lactose), glycerol (second carbon source) and lactose.
I know that it is popular to lower the IPTG concentration and, over a certain regime you do see slower accumulation of the target protein, but this rarely due to all of the cells expressing protein more slowly but instead it does so by reducing the number of cells that are expressing the protein at any one time (there are always some situations where the standard transport of IPTG cannot explain what is seen but not many). This transporter issue and the accompanying population behavior of IPTG induction is a well studied phenomena at the individual cell level.
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