>>> 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, unless it has some really weird compartmentalization.
To add to the nice tricks about E.coli strains forming disulfide
bonds, if your institute is well-equipped for cell culture, just
secret it from HEK293 cells.
Its easier and cheaper than what most people think - all you need is a
good person to show you around the basic tricks, a hood, and an
incubator,
which most places have anyway.
A.
On Nov 16, 2010, at 18:22, van dat nguyen wrote:
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 <[email protected]
> 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
P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
