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 chaperones might not help in this case.
As you already mentioned: I think expression insect cells is a good
idea to test.
Best,
Guenter
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
--
***********************************
Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz
e-mail: guenter.fr...@uni-konstanz.de
e-mail: guenter.fr...@uniklinik-freiburg.de
http://www.uniklinik-freiburg.de/neuropathologie/live/forschung/ag-g-fritz.html
Tel.: +49 761 270 5078
Fax.: +49 761 270 5050
