I've found slow and cold protein synthesis to be the most effective, by using T

Hi,

I think that it is well worth exploring culture conditions first as I am not aware of any definitive studies that show these types of additives have a general use in solubilising proteins. They may help during lysis to prevent aggregation or they may stabilize a particular protein or protein family by acting as ligands etc. but as to them being generally useful in solubilising proteins I am not so sure.

 If you want to look at other parameters you can check the RG Q&A forums there are hundreds of similar threads on the theme of solubilising proteins that have already been answered very well!

Can you be a bit more specific about your tag, fusion protein, expression system etc?

Is 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 try adding a detergent to your lysis buffer we routinely use 0.2-1% Tween 20

Then..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° for longer periods (16-18hrs)

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 an edited copy of my answer to a similar older thread

First, many thanks for the answers.

Next, I had already used many of the tips that you give here, yet none of them had helped me obtain soluble protein. That is why I am searching for novel ways of preventing proteins from forming inclusion bodies. Apparently, this is easier than solubilising inclusion bodies and recovering active protein by in vitro refolding.

Last, some osmolytes (such as sorbitol) seem to be accumulated in E. coli by uptake from the growth medium. So, they can act as chemical chaperones in vivo, improving the solubility of recombinant proteins (see the link below for the relevant article).

aem.asm.org/content/77/13/4603.full

Hi,

To get soluble protein, I did co-transformation of my target plasmid with plasmid of GroES and GroEl which help protein folding during the cell growth. I also grew  my E.coli Culture around  16 degree at very low IPTG concentration( 0.1-0.2mM) for one of my membrane protein . I got pretty good results. Hope these tips help you.

Good luck

Weiping 

Kerem, the paper you referenced got an interesting result with the His6–HypF-N protein, it looked like a very good proportion went to the soluble cellular fraction with some omsolyte additions (of course "soluble" does not necessarily mean "native and fully active" ) Their result with GFP was a little less impressive, with at most a ~3 fold increase in fluorescence with additions.  The fraction of correctly folded GFP versus insoluble (non fluorescent) inclusion bodies could be very small. Its definitely woth a try although some additions, like trehalose, could be quite expensive and your E. coli may just eat it and still not correctly fold up your protein.  I found a reference which said multiple "problem" proteins in E. coli could be made soluble by a simple change in the lysis procedure, but it never worked for any proteins I tried. If there was an easy (and patenetable!) universal solution to this problem everyone would be using it I think.  All published positive results are worth a try, especially if easy, good luck!

To prevent inclusion bodies, the most effective method is to slow down expression - low temperature, arabinose inducible promoters etc.

People have had success with using strains in which one or more chaperone proteins is overexpressed with the target protein - although this seems to work best with bacterial proteins.

It is also possible to have a tag with a signal sequence that exports the protein out of the cell, or into the periplasm thereby preventing intracellular inclusion formation. This generally results in lower yields.