Protein insoluble in 8M urea or 6MGuHCl - (Apr/29/2007 )
I use BL21 STAR(DE3) to express three cellulase enzymes which are constructed in pET26 (Novagen). Good expression @RT overnight. Since our lab has no sonicator, I use lysozyme plus 3 freeze-thaw cycles to lyse the cell. Lysis buffer has 25mM TrisCl, 50mM NaCl, 2% Sucrose, 1mM EDTA, 1mM DTT, PMSF, 1mg/ml lysozyme. After lysis, proteins remain in the pellet. So of course I think, inclusion bodies. Then I tried 8M urea and 6M GuHCl lysis for up to 3hrs. And the proteins are still in the pellet.
That doesn't make any sense. Does anybody have similar experience to share with me? I appreciate it.
Both urea and GuHl are pretty strong denaturant. Giving you denative form of the protein.
If the proteins are still in the pellet, it means either it is EXTREMELY insoluble even though using the strong denaturant or it is not lysed properly. Hmmm.. you have protease inhibitor too.
Protein expression is always weird. Good luck! 
I hope the proteins were not lysed properly.
I'll try using either sonicator or microfluidizer to lyse the cell.
Microfluidizer? what is that? 
Why not freeze thaw method?
Maybe you can change the composition of your lysis buffer. 
I supplemented 2M NaCl, 1M Urea, 0.2% Tween, 0.1% Triton once at a time into the basic lysis buffer. And things were just not approved. So I suspect my way of doing lysozyme+freeze-thaw was not right.
Microfluidizer is a device that generate high shear fluid to lyse the cells.
Why not freeze thaw method?
Maybe you can change the composition of your lysis buffer.
That doesn't make any sense. Does anybody have similar experience to share with me? I appreciate it.
Decrease the expression time to 2-4h. We often use shorter times for "diva"-proteins, those which don´t want to cooperate with us
The expression level decrease too, the amount will be lower, but hopefully with less protein you could be lucky to find the protein in the supernant and not in the inclusion boddies. Microfluidizer is a device that generate high shear fluid to lyse the cells.
Yea, I am suspecting your lysozyme activity too. Perhaps it is not lysed properly. Just use the sonicator o the microfluidizer.
Are those vectors inducible with IPTG? If so, you may want to try inducing at lower concentrations of IPTG.
I also suggest, like the others, that you try to sonicate your extracts!
Good luck!
That doesn't make any sense. Does anybody have similar experience to share with me? I appreciate it.
what is a colour of your pellets?
If your check your targeted protein content in pellets so try to use strongest denaturant like Guanidine thiocyanate up to 6 M.
But it sounds strange that you could'nt dissolve your protein in 8M urea and 6M Gua. what's about grade of reagents you use. Recently I had a problem like that with low grade Gua. when I use ultrapure Gua from fluka the problem resolved. Otherwise try to increase incubation time till 2-4 hour at 37C.