I want to optimize protein purification. My protein is expressed in periplasmic space of E.coli. I particularly want to know what is the routine protocol for these kind of proteins. Is it 1mM IPTG, 3h, 37 degrees?
Hello,
Thank you Sebastian. I am going to optimize the conditions myself. I am going for IPTG range from 0.1 to 1mM. For time and temperature, I agree. there are some specific temperatures in which I can assess the expression.
Thank you for your respond.
That helped
Hey Nazanin
As I have already experienced it with BL21, it is showing really high expression in 25 degrees and 0.2mM IPTG but to be honest the best thing is to play around with temperature and IPTG concentration to find the best condition, just keep it in your mind for thermophile enzyme I found higher temperature better for expression but this is not a rule. anyway, I wish you success and joy in your study.
Hey Hamid,
so nice to hear from you here!! Thanks for the tip. The protocol for the protein I'm using is 1mM IPTG,3h,26 degrees. But it's a very challenging protein.The yield is relatively low and the solution to that problem is just produce more (ie. 5-10 liters of bacteria culture). So, I was wondering if there is something that could be done about it.
I'll do the optimization and let you know the results.
Thanks, the thing which makes me questioned is why 3 hours? Normally the protocols for the protein expression which I am working with are for 18 to 24 hours. Nazanin did they mention why you have to give such a short time to the bacteria for expression?
Not specifically. But I assume the time of induction depends on the expression stability of your protein. For my protein, which is PA83, part of Anthrax toxin, stability and solubility seems to be an issue. If I induce overnight, I would get aggregates of my protein instead of soluble protein.
Anyway, I tried different concentrations of IPTG and 1mM seems to be the best concentration.
You wish to get your protein in Periplasm or it goes automatically? Periplasmic space is lesser than cytoplasm so you may get more protein if express in cytoplasm. There are two purposes of producing recombinant protein in Periplasm of E.coli: Less contamination with other protein and Disulphide bond formation. You should produce your protein in Cytoplasm if you are no facing problem of cytoplasmic protein contamination and no need of disulphide bond formation.
Hello Gopal,
Actually my protein forms inclusion bodies if expressed in the cytoplasm. So, I'm using an engineered plasmid which directs expression into the periplasmic space. That's the only way I could get my protein.
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