Gerard,
Solubility is not a trick and there is no simple or single solution
(pardon the pun). In fact, one might think that this is a field of its
own. You may want to refer to some of the previous posts on protein
solubility/solubilization in the archives by myself and a number of
other individuals at the URL: http://www.bio.net/archives.html
Among the things which I have seen in use for this dilemma:
1) slow, step-wise dialysis of denaturant from your solution ie
6M-4M-2M-1M-0M-0M...
2) rapid dilution of your protein (uM final conc) into "Native" buffer
conditions. We like to add the protein solution at ~50ul/min to the
Native buffer. The trick is trying a large number of "Native"
conditions to find out which one your protein prefers.
3) find another set of buffer conditions into which to dialyze...see
note for #2
4) if your protein interacts with a ligand, try the refolding process in
the presence of the ligand
5) try to vary the induction and growth conditions to minimize the
instability. You'd be amazed at how big an effect there can be with a
simple change in your media recipe or temperature of induction. If you
produce your protein more slowly, the cell may be able to handle it
better and won't put it into inclusion bodies.
6) try other constructs, larger/smaller, based on homology to other
systems. This can be tedious and meaningless in the long run and
requires a really good feeling for the mol.biol. mumbo-jumbo. (Gee, is
it obvious what I think of rational design)
Let me know if you come up with any other ideas and I'll report again
should I hear of anything else.
Good luck,
Randall C Willis, Researcher
Biochemistry, Hosp for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8 CANADA
416-813-5933 (ph) -5022 (fax)
willis at gandalf.psf.sickkids.on.ca
