Dave Dilyx

Hi Confuzzled26, this is an interesting phenomenon. Looks like the interior of the E.coli bacteria makes provides a solubilizing environment only when the bugs are fed well. Anyways, you've shown one very important aspect of your target protein, namely that it can be expressed in soluble form and that it remains soluble when you're purifying it. Therefore, there's nothing fundamentally wrong with the protein (such as exposed hydrophobic surfaces, missing binding partners...) that could render the protein insoluble. There may be a way to solubilize the protein that's expressed when using minimal media, resulting a non-soluble form. Where I'm getting at is this: the buffer composition that you're using to lyse the cells and purify may be sub-optimal. Changing the pH, salt type or concentration, having reducing agent around, amino acids, carbohydrates, detergent etc. There are a lot of physical chemical parameters to test. Doing this can be quite cumbersome. On the other hand, there are protein solubility kits on the market that you may want to try. For instance, there's the OptiSol protein solubility screening kit. You'll get a solubility profile out of this and can adjust the buffer you're using for lysing / processing the lysate. Would be great to hear if this helps. Regards, Dave