Why does my protein stay in the pellet of the lysate?
I expressed a KPC-2 Protein in NP6 cells the vector is PqE-2. After lysis (8M urea in a tris buffer pH 8), the protein is in the pellet. I have tried many different things like different IPTG concentration 0.1Mm,0.2,0.3,0.4 and 0.5mM. sonication, microfluidizer, triton x-100, lyzozyme, and SDS. But I am unable to obtain satisfactory result. What else can I do to bring my protein into solution?
If you are referring to the beta-lactamase enzyme KPC-2, it is a soluble periplasmic protein of a Gram negative bacterium. It can be expressed as a soluble protein in E. coli using a periplasmic secretion signal sequence.
You probably funcatinally inactive protein expressed that is the reason to have proteins in pellets.
What temperature are expressing your proteins at?
Is is 37C?
Dear Laurence Stuart Hall
Thanks Alot for your courageous help in my experiment. yes i did iptg induction in different lower temp like 16, 18, 20 25 and 30 for overnight incubation but did not find result satisfactory. if possible please send me your protocol which you have used and got result. my email id : [email protected]
I would appreciate if you could the same.
thanks
ABID
Dear Abid, you have already gotten good advice but let me share my story... We also had difficulties getting our protein to the soluble fraction, and variations of the purification techniques did not solve the problem. What finally worked for us was that during the fermentation, we used a combination of low temperature (+16), low IPTG concentration (0.1 mM), and addition of the cofactor necessary for the correct conformation of the protein.
Good luck for the experiments!
Dear Tarja Kokkola ,
Thanks alots for your nice answer of my question, really your idea may be solve my puzzle. previously I did twice my experiment 16c and 0.1 mM IPTG on over night incubation but i was not add any cofactor,might be cofactor solve my problem.
Thanks
with regards
ABID ALI
You might try a pelB leader sequence.
https://en.wikipedia.org/wiki/PelB_leader_sequence
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