If the protein is well-expressed, the easiest approach is to treat the cells the same way you would with a protein expressed in soluble form in the cytoplasm - break the cells open by sonication, French press or microfluidizer. You lose the advantage of a purer starting material, but simplify the procedure at the start and maximize the yield.

Good day,

I doubt it, but, have you checked for the presence of the protein on the media? Once the osmotic shock was performed, did your clear solution turn into a cloudy or milk-Like solution, if so, there is a big chance that your hypotonic buffer is not compatible with your protein of interest, yielding in denaturing and precipitation. Bit knowing what protocol are you following, is all I can think about. Next, you'll find an osmotic shock protocol you might try, also check for 26-28 references. In case you are not able to get them, send me a private message to share them with you.

Best regards.

Many thanks, Adam Shapiro and Adan Castillo. Here is the brief outline of the protocol. The cell pellet was resuspended in 100mM Tris, 20% Sucrose and 1mM EDTA, stirred for 30 min and centrifuged. Again resuspended in 5mM MgCl2 for 30 min and centrifuged. The supernatant from both spins were combined and dialyzed. The protein of interest could not be detected in supernatant but in cell pellet on SDS-PAGE.

Ok, correct me if I'm wrong. Once you grew bacteria and overexpressed your protein, you did one centrifugation run so biomass would precipitate as a pellet. Then, you resuspendended the pellet using what, sonicator? And once again, you did centrifugation run and resuspended one more time. Both supernatants were mixed and dialyzed (using membrane or amicon cartidges?). Then analyze both reactions through SDS-PAGE and found your target protein is in the precipitate. If this is the case, then your protein is not being correctly folded at the periplasm and it is most likely to be found at inclusion bodies, wich could be for different reasons.

Excessive overexpression speed; you can counter lowering the overexpression temperature. Also, You could try to change the IPTG concentration, try 1 mM, 2 mM, .5M and so on, depending on the results you are getting. If this doesn't solve the problem, try changing your E. coli strain, i.e. origami strain directs protein synthesis and folding to the periplasm. Moreover, if this still does not solve your issue, You could try changing overexpression media. And finally, if nothing else fixes the missfolding problem, you will have to go over protein re-folding technique. If you'd unfortunately get to this sad point, send me a private message and I'll get Back to you with a Good refolding protocol.

Good luck