My general approach to making a protein more soluble has been to tag it as a fusion protein (maltose-binding protein is usually good) and express overnight at 15C. I'm an NMR spectroscopist so I pretty much always use minimal M9 media, but I haven't seen too many cases where media substantially impacts soluble/insoluble. What to try depends on your protein and current expression conditions. If you are inducing at 37C, I'd try lower temperatures (32, 22, 15) to see what that does to solubility and expression level. Most of mine have been 37C growth/15C induction, but we had one that had to be 32C growth/22C induction to get reasonable yield. There are a couple cell lines that can help with refolding and redox/disulfide formation. I have yet to come across a single protocol that is optimal for everything, though.

I dont get it I am afraid, do you want to get secreted proteins more soluble?, if your protein is intracellularly expressed than addition of the mentioned compounds outside the cell want help much...

There are many papers that claim they do, see for example:

http://www.ncbi.nlm.nih.gov/pubmed/17126029

OK, but the effect described is indirect by increasing the osmotic stress, thus you start extracting water out of your cell which is then countered by osmolytes inside the cell. I think in that case, it just doesnt matter what you add outside, th effect is just due to high enough concentrations. To be honest I have my doubts about this technique, the molecular crowding inside a bacterial cell is already incredible high. Anyway good luck with the experiments

We've tried betaine/sorbitol (BS-medium...:-)) for a couple of projects before, without any improvements in those cases.

You can give a try by adding Tween-20 or Tx-100 at 0.01-0.05% in culture at the time of induction. Lowering temperature to 16-20 deg celsius may further improve yield of soluble expression.

We have observed that for some proteins good nutrients supply as in the case of “terrific broth” medium results in a heterologous protein yield increase. However you may consider other factors that could improve proteins solubility as inductor concentration, temperature during the induction of the protein expression, codon frequencies on your protein and more important, the host strain that you are using for the expression. We have excellent results using very low induction, long periods of induction time at low temperatures (14-16 oC) with good oxygenation in terrific broth.

My general approach to making a protein more soluble has been to tag it as a fusion protein (maltose-binding protein is usually good) and express overnight at 15C. I'm an NMR spectroscopist so I pretty much always use minimal M9 media, but I haven't seen too many cases where media substantially impacts soluble/insoluble. What to try depends on your protein and current expression conditions. If you are inducing at 37C, I'd try lower temperatures (32, 22, 15) to see what that does to solubility and expression level. Most of mine have been 37C growth/15C induction, but we had one that had to be 32C growth/22C induction to get reasonable yield. There are a couple cell lines that can help with refolding and redox/disulfide formation. I have yet to come across a single protocol that is optimal for everything, though.

In our case, we are trying to obtain soluble recombinant protein according to a technique used here:

http://www.ncbi.nlm.nih.gov/pubmed/?term=Enhancement+of+the+Solubility+of+Proteins+Overexpressed+in+Escherichia+coli+by+Heat+Shock

We have tried the heat shock at 42 °C and induction at both 37 and 18 °C, however, we could not obtain the soluble form of the protein. I think that when it comes to improve solubility of proteins, there could be several hundred protocols and conditions to test. The tedious part is to define which one is the right one. Hope the paper helps, good luck!

I had tried using betaine to increase solubility of my protein which was expressing as inclusion bodies. I had used normal betaine but it did not work for my system so I simply went for refolding.

We have tried doing a 43C heat shock during expression to induce stress responses such as heat shock and thus internal chaperones, etc. It didn't work for the proteins we tried expressing. We've had partial success for some but not all proteins by over-expressing chaperones (GroES/GroEL seemed to work best in the case of these particular proteins, which are of phage/viral origin). We've also had some limited success by playing with different lysis/extraction buffers.

I have invested some time in this topic, and in my hands, only the fusion-tag/solubility enhancing fusion domain gives reasonable effects on solubility. For short proteins, I would suggest you use dnaK as fusion domain and induce in minimal medium, since this nicely brings up the corresponding chaperone, dnaJ in high amounts.

Larger proteins might likely require GroES/EL.

"Side-question: should we get cell culture-certified compounds, or would "normal", much more economical ones suffice? (i.e. are they likely to contain side-products that would affect bacterial growth?)."

Especially E. coli eats close to anyting you throw at it if it is not toxic. Most chemicals you can acquire commercially are by far more than pure enough for Colis. "Cell-culture" applies for eukaryotic cells. You should go for chemicals labelled "for microbiology", if you want. But p.a. grade chemicals are totally OK for E. coli.

Hi,

I'm also facing the problem of protein insolubility. Even at the 15 degree Celsius, it is forming inclusion bodies. The protein size is 97kDa. How can I get the protein into soluble form? Will these additives help me to get protein in soluble form?  

Inducing protein expression at 18°C with lower IPTG concentration and 2% or 3% ethanol as additive works good in many cases.