We are trying to purify an artificial protein. Very good expressed, but mostly in the spun down pellet. If anyone has experience to make it a bit more soluble, with a kind of mild buffer, additives, will be helpful. Thank you.
Hi there,
If you want to have get native soluble protein, you'd better consider improving the culture condition (in terms of recipient strain, concentration of inductor, length of induction, temperature during induction aso) rather than adding additives to the lysis buffer which might in fact just solubilize aggregates of your proteins (unless it's what you want).
Hi there,
If you want to have get native soluble protein, you'd better consider improving the culture condition (in terms of recipient strain, concentration of inductor, length of induction, temperature during induction aso) rather than adding additives to the lysis buffer which might in fact just solubilize aggregates of your proteins (unless it's what you want).
A few things that sometimes can help a protein to be expressed in soluble form include (1) lowering the temperature during induction to as low as 16oC, (2) coexpressing with a chaperone, such as GroEL-GroES, and (3) including a high affinity ligand in the medium that can get into the cells. In some cases, the protein appears to be insoluble because it aggregates at high concentrations under certain conditions, but the insoluble protein can be redissolved with a change in ionic strength (salt) or pH, detergent, and/or dilution.
If the protein is truly insoluble, it may have to be fully denatured with urea or guanidine, then refolded. To do this, the protein is dissolved in the denaturant, diluted to a low concentration and refolded by gradually reducing the denaturant concentration, such as by dialysis. The correct formation of any disulfide bonds can be achieved during this process by including a mixture of reduced and oxidized glutathione. Certain additives can assist with refolding.
hey, if you have a fusion proteins, you can test some fusion tag that improve the solubility. For example the pDest-MBP for the gateway cloning or the SUMO vector for the normal cloning whit the restriction enzyme.
If the co expression or the low temperature expression does not work, change the construct.
Best
Riccardo
Or this article from 2004:
Method for enhancing solubility of the
expressed recombinant proteins in
Escherichia coli. Ghosh et al.
First-check the Q&A forums there are hundreds of similar threads on ResearchGate that that have already been answered very well!
Then..
Can you be a bit more specific about your tag, fusion protein, expression system etc?
As you already have a construct that expresses then there are a few things that you can try (I am assuming you are using T7, LB and IPTG at 37°C so far with a globular protein and not an IDP).
Firstly if you want to continue to use IPTG for induction use a richer, buffered medium , terrific broth is simple to make but can improve soluble yields dramatically over LB.
Try reducing temperatures post-induction to 16-20°
Try reducing IPTG concentration-although in most e.coli expression strains induction is not really titratable.
Try auto-induction media instead of IPTG
If none of these work then think of changing your fusion protein or tag, I particularly like SUMO, thioredoxin and Z-tag (and much prefer them to GST and MBP) and/or changing the boundaries of your construct to avoid e.g. unstructured domains. There is no sure way of getting soluble protein that stays soluble after cleavage from the fusion but these should help.
Of course the effects of all of these are dependent on your protein and it may be that you ultimately have to accept re-folding from inclusion bodies.
Good Luck
P.S this answer is a copy of my answer to a similar older thread
If you find your protein in spin down pallet, two possibility you should consider. The protein formed a inclusion body or they was membrane protein actually. In my case, my protein was not soluble in quanidine or urea solution. However it was soluble in ionic detergent solution. Therefore I considered it was a membrane protein. Most answers mentioned above were suitable to inclusion body. For membrane protein, it was not easy to make it soluble in aqueous solution since it might have a hydrophobic out surface. If you forced it to become soluble in aqueous solution, it might changed its native folding. I have read some article mentioned about expression of membrane protein in lipsome, but I never tried before.
I agree with Adam and Chiu that it may also be worth trying a couple of detergents e.g. 0.2% tween 20 in our lysis buffer can make a huge difference in soluble yield for some proteins. This level of detergent is not sufficient to extract proteins from inclusion bodies or membranes (if it is a true integral membrane protein) but may help to extract some 'apparently' insoluble proteins. We noticed this when HTP expression screening at the small scale using detergent/lysozyme/freze-thaw lysis and our results did not correspond to full-scale preps with mechanical lysis unless we also added the detergent.
You may be lucky and this is a simple fix for your protein without having to change too much else-worth a try.
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