Protein solubility in E.coli with different media

[Confuzzled26]

Hello fellow scientists,

I have been having a slight problem with protein solubility, when i overexpress my protein of interest (14kda) using LB (luria bertani) broth, my protein is soluble and i am able to purify however when i do the same overexpression using minimal media (M9) the protein is insoluble, what kind of variations either to the media or growth conditions would anyone recommend in order to get this protein soluble, i plan to do NMR to resolve the full protein structure, any feedback would be most appreciated!

Edited November 24, 2011 by Confuzzled26

[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

 

Hello fellow scientists,

I have been having a slight problem with protein solubility, when i overexpress my protein of interest (14kda) using LB (luria bertani) broth, my protein is soluble and i am able to purify however when i do the same overexpression using minimal media (M9) the protein is insoluble, what kind of variations either to the media or growth conditions would anyone recommend in order to get this protein soluble, i plan to do NMR to resolve the full protein structure, any feedback would be most appreciated!

[CharonY]

Which system/protocol is used for overexpression and isolation?