I have cloned 4 genes in Champion™ pET SUMO Expression System (invitrogen), expression of all 4 is very high but only one is soluble rest 3 are coming in pellet after sonication. I have to do crystallization of these proteins, hence I need high expression of soluble proteins. I have tried low iptg concentration up to 0.05mM and different growth temperatures(16, 25 , 37 degrees) also. I also tried T7 Express lysY/Iq Competent E. coli cells (NEB) as expression host, but nothing worked at all.
Thanks
f your protein expresses as inclusion bodies, you may haye to try refolding (see “Protein Expression and Refolding – a practical guide to getting the most out of inclusion bodies”, Cabrita and Bottomley, 2004, Biotechnology Annual Review, 10:31-50." for a review
If you want to solubilize the protein that is in the pellet, try urea or guanidine hydrochloride. If you want to obtain soluble protein without refolding, it may help to try a different tag such as GST or adding the tag to the opposite end of the protein relative to where you have it now. Moreover, if your protein of interest has multiple disulfide bonds in its native state, you may try using Shuffle cells that have been engineered to promote disulfide bond formation in recombinant soluble cytoplasmic proteins.
You can follow what was already been suggested. If your protein do not contains disulfide bond then you may wish to do a simple experiment. Dissolve your pallet with different conc of Urea or Guanidium HCl (1 to 6 M) and see at what minimum GuCl (suppose X) you can get highest yield. Then accordingly dissolve your pallet using the same lysis buffer with X GuCl. then run low slope gradient to finally 0 GuCl. if you get soluble then ok, otherwise you may wish to keep a minute amount of tween 20 in your final wash buffer and then elute with same amount of tween 20 containing buffer, and then keep if it works. We got success for one insoluble protein following this method; however it could be specific solution.
8M Urea or 8M Guanidium HCl can solubilize, and you can skip sonication. The proteins have to be refolded afterwards. I'd recommend the following strategy: http://www.mcponline.org/content/4/10/1558.short
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