Additives to bacterial cultures to improve soluble expression of proteins

We are considering trying to add additives to bacterial cultures to see if they improve solubility of some proteins we express: L-arginine, glycyl-glycine, sorbitol and betaine. I would be glad to receive recommendations and experiences, also if they haven't worked.
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?).


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  • Patrick Van Gelder · Ghent University
    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...
  • Mark Van Raaij · Spanish National Research Council
    There are many papers that claim they do, see for example:
  • Patrick Van Gelder · Ghent University
    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
  • Mark Van Raaij · Spanish National Research Council
    We've tried betaine/sorbitol (BS-medium...:-)) for a couple of projects before, without any improvements in those cases.
  • Jai Kaushik · National Dairy Research Institute
    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.
  • Eduardo Ceccarelli · National Scientific and Technical Research Council
    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.
  • David Horita · Wake Forest School of Medicine
    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.
  • Bogar Araujo · Fundação Oswaldo Cruz
    In our case, we are trying to obtain soluble recombinant protein according to a technique used here:
    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!
  • Reema Dhoke · Institute of Microbial Technology
    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.

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