Hi there,

If inclusion bodies are formed it means the protein is not folded and has formed aggregates. If you want to recover protein from IB, detergent is not enough for solubilization. You need to use urea or GdmCl at first and then add detergent and finally to attempt protein refolding by removing urea/GdmCl.

I assume you tried expression in bacteria; then perhaps switching to a eukaryotic expression system may help. They have an appropriate membrane insertion machinery. Note that just solubilising the IB with denaturant will not help, because the protein is unlikely to fold correctly outside a biomembrane.

My suggestion is to try with different detergents and play with their concentrations. Recently, people started adding also lipids to the extraction buffer since they eventually can help to stabilize and solubilize your protein. Try to check how is the native mix of lipids in the cell membranes where your protein is nativelly expressed and add them also to your extraction buffer.

From personal experience in successfully purifying a 14-span membrane protein in native form, the zwitterionic sulfobetaine detergent (C14 length) performed best at preventing aggregation during solubilization, among many nondenaturing detergents (nonionic and zwitterionic) that I screened. (aka: SB 3-14 and Zwittergent 3-14).

See also: Nondetergent sulfobetaines remarkably stabilize proteins against aggregation. (Anal Biochem. 352 (2006) 299–301).

Hi Kumar,

I agree with Engelbert. And I can recommend the MP purification service provided by Creative Biostructure. I worked with them in the expression and purification of MP before. Creative Biostructure has a professional MP platform, and this website also has some resources for learning.

Hope it helps!