Hello,
I'm doing optimization of an extracellular protein cloned in the pET26b vector along with PelB leader sequence. I want to know the best time at which cells can secrete most of the protein with highest activity. I did this step till dead phase but protein concentration along with activity was still increasing. Can anyone guide me?
Hello Sana,
E. coli doesn’t secrete proteins into the extra cellular medium, with rare exceptions. Adding a leader sequence will likely direct secretion to the periplasmic space, but the protein will remain cell- bound. However, if you wait long enough, the cells will lyse and release their content into the medium. There is no point in waiting that long, as degradation of the protein may occur. I would suggest to harvest the cells after 3 h max after induction (if induction proceeds at 37 oC) and either to lyse them and proceed with purification from the crude extract or recover the protein from the periplasmic space using osmotic shock, for example.
Hi, you can try different strains of E.coli or just try different times with different lyses conditions or heat shock or osmotic shock as Pierre sad or electric shock. What is available. Than try renaturation conditions. I do not know what is wrong with your expression , you have large amount of protein. What kind of protein is it?
Best to you,
T.R.
Hello Pierre Béguin and Tijana Radovic
I have cloned my gene in pET26b vector along with PelB leader sequence that can direct protein secretion into the periplasmic space. I want to optimize conditions like best time, temperature, IPTG and lactose for protein expression. As my protein will be secreted in periplasmic space so after harvesting cells at different time I'm using supernatant for PAGE and protein activity analysis. My main goal is to get more protein with high activity. But as you said if I'll do expression for long time protein can degrade and obviously its activity will decrease.
My main problem is that I'm getting more protein in the dead phase with the highest activity.
I can't understand the logic behind it.
If ,as is most likely the cas, your protein is within the periplasmic space, it is cell-bound and absent from the culture supernatant. Only after cells die and lyse can the enzyme leak into the extracellular medium.
Sana Tehreem ,
You should not use the culture supernatant for your analyses. The protein is still inside the cell, just in a different compartment (the periplasm). That is the only logic behind you getting higher activity in stationary phase cultures (more cells dying, lysing and releasing their content into the medium).
If you really want to know at what point you are going to get more active protein, you have to either a) lyse the cells, possibly by sonication, and assay the resulting supernatants, or b) make periplasmic protein preps by osmotic shock, and assay them.
Hope it helps!
Dear Sana, environmental and nutritional cues direct metabolite synthesis and secretion. If overproduction of a metabolite occurs through genetic, metabolic and environmental conditions in Escherichia coli, the bacterium will most certainly secrete the excess since there is understandably no intracellular space to store baggage in microorganisms generally. So, simply manipulate genetic, nutritional and environmental conditions to favour overproduction and you will see what follows.
Thank you so much for your kind suggestions. I'll follow them to get better results.
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