Hi Laurie,
nuclear protein, cysteines, my thumbs prickle it might be a zinc-binding
protein.
Although 12 Cys per 257 aa is not yet that much. If you have always DTT
present oxidation should be no problem.
Reconstitution of zinc sites can be very tricky and is not
straightforward. Also E.coli
We are trying to express for structural studies a 257 AA
eukaryotic intracellular [...]
As you describe about your protein, I guest your protein may
required disulfide bonds to be folded correctly
As Laurie described her protein is intra-cellular, so it will not need
disulfide bonds,
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
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
Dear Laurie,
your cysteines might all be cytines, i.e. all paired and not really be the
reason for the aggregation. Since you have soluble protein as long as MBP is
tagged to it, why don't you test a large number of conditions for solubility?
I'd probably start with a 3-D screen testing 2-4
