Hi all.
I wrote some times here about a unfold protein topic. The case is I have a pure protein but It is together with DNAk and In the exclusión molecular column I have several peaks that I cant divide. So We think our protein is unfolded and these peaks stand for differents folding states of my protein ( this also explain the unión of the DNAk).
We want to know some options/suggestions for get better the folding. I searched some protocols for protein expresión with a Heat shock step ( an additional step of 42ºC during 15-20 min before the induction for increased the concentration of Chaperones) and the use of GroEL/GroES plasmid.
Thank you.
In general I would recommend lower temperature for expression (e.g. 28°C ON) (but depends on system used and many other parametrs). To be able to give you more detailed answer you need to provide us with more details (used vector, bacteria, current expression conditions,...)
Hi I expressed on E.coli BL21(DE3)
the plasmid is pet21d
The conditions which I expressed are E.coli growth at 37ºC until OD600 is 0,6-0,7 then I induced with 0,1mM IPTG and change the Temperature at 20ºC and let it Overnight.
Purification steps: Ni column-anion exchange and exclusion molecular.
The problem is that In Superose6( exclusion molecular column) I have two peaks which are together ( Ve: 9,4ml and 10,5ml) and I can't separate them. In SDS-PAGE gel we have two bands, one my protein and the other is DNAk in a relation protein:DNAk 10:1. So we think the protein doesn't complete its folding.
Thank you
Hi Mohamed. No, we dont think our protein is toxic because the e coli growth in a normal time.
Yes, Our protein has a HisTag in N-terminal.
the proteins is prokaryotic and its MW is 134kDa (monomer)
Hi David,
than you could consider express it as a fusion protein together with some solubility enhancer partner...may be you can have some ideas here (or in cited or in similar papers):
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928792/
Thank you for that article Kristyna. There are some ideas which I want apply in my assays.
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