Hi Laurie, What E. coli strain did you use? As you describe about your protein, I guest your protein may required disulfide bonds to be folded correctly. E. coli cytoplasm is a reduced environment, which is not suitable to make disulfide bonded proteins. to solve this problem it is recommended to use E. coli strain which a less reduced cytoplasm, ex SHuffle® T7 strain (from NEB) or Rosseta Garmi strain, or express rich Cysteine proteins in the periplasm of E. coli. using those strain with co-expression of Protien Disulfide Isomerase will be good to try. However, Recently our group have made a very good system in E. coli to expressed disulfide bonded protein in E. coli cytoplasm, please have a look at this paper:
http://www.ncbi.nlm.nih.gov/pubmed/20836848 A better system will be published soon. Best Wishes, Dat On Tue, Nov 16, 2010 at 6:13 PM, Laurie Betts <laurie.betts0...@gmail.com>wrote: > All - > > We are trying to express for structural studies a 257 AA eukaryotic > intracellular (also possibly nuclear) protein (predicted to be single domain > all-helical) that has 12 Cysteines. No known metal-binding function not > that it couldn't happen. So far (E. coli) it expressed solubly as MBP > fusion (with an N-terminal region deleted predicted disordered) until > cleavage of MBP, then it's not soluble, including detergents added. THe MBP > fusion is usually soluble aggregate so we assume that our part is not folded > right. We have so far assumed it needs a lot of reducing agent (5 mM DTT or > TCEP). Thinking of trying chaperones and insect cells next. > > Any experience out there that might help? Mostly I wonder about all the > cysteines. Don't really know if that is the problem. > > Laurie Betts >
