I working on the expression of a protein in BL21 cell using IPTG as inducer. Usually after adding the inducer, overnight culture is suggested followed by centrifugation to pallet down the cells. I want to know that if culture time (in presence of inducer) gets longer, is there any chance to get poor yield of the expressed protein?
Yes, this can happen but of course it does not happen in each case. In any case, I would optimize the induction time and then stick to the optimized protocol to obtain reproducible yields. Maybe there is also a window in which the yield is comparable, e.g. 3 - 5 h after induction.
Yes, this can happen but of course it does not happen in each case. In any case, I would optimize the induction time and then stick to the optimized protocol to obtain reproducible yields. Maybe there is also a window in which the yield is comparable, e.g. 3 - 5 h after induction.
The cells will not be happy if you induce for long times at 37oC and this may reduce significantly the protein yield. Usually for long time induction is best to reduce incubation temperature up to 16 -25oC. However, one should note that each protein behaves differently and sometimes what is good for a particular protein may be completely different for another one. It is always good to test different times and temperature for induction and check which conditions will give you better yield with less inclusion body formation.
It depends on your gene product whether it is going to be expressed in soluble or insoluble form. For soluble proteins it is going to be very critical and better to standardize the duration of induction and stick to it. Insoluble protein expression is very robust and induction can be performed for longer duration.
Yes it could affect the yeild as mentioned by others above. I would suggest that you if you are not already doing this, reduce the temperature to 18 degree once you add IPTG to your media. And to optimize the time for incubation after induction, I suggest that you take samples (500ul aliquotes) out at 0 hour and say every 2 hours and run them on the gel to check if you see an increase in your target protein (this is a quick dirty test) to make sure you are actually inducing the target protein. I have noticed most of my -GST tagged proteins are good after 4 hours of induction, whereas -HIS tagged proteins usually take/can withstand longer induction times.
Hi
As others said the expt needs to be optimized with difft concn of inducer, temp etc with alternatives, other tecq is NATIVE PAGE Electrophoresis to show native status of protein
Usually proteins degraded due to proteases so if any protocol says to add proteases
you can proceed with basic steps to see protein expression in electrophoresis and western blot, MALDI TOF
Thanks to all for your suggestions. I am retrying the expression in small aliquotes with different induction times. Let's see.
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