Dear all,
Sorry for the off-topic question...
What can be done to avoid a protein going inside inclusion body.The gene is
cloned in pET30a with C-ter his tag and expressed in BL21-DE3 from 37 to
18C for 3-4 hr with .5mM of IPTG,it is going to inclusion body.All
suggestions are welcome.
Thanx in
Lower temperature, use chaperones (e.g. TAKARA set), refolding?
Quoting shivesh kumar [EMAIL PROTECTED]:
Dear all,
Sorry for the off-topic question...
What can be done to avoid a protein going inside inclusion body.The gene is
cloned in pET30a with C-ter his tag and expressed in BL21-DE3
Also worth trying a lower IPTG level ~ 0.1mM and or different media,
e.g. TB, SOC or m9 instead of LB, it's all a bit random but sometimes
these things can make a difference. Adding 3% EtOH on induction
worked for me once, it's supposed to shock the cells and stimulate
chaperone
Hi Weijun and all,
Anyone tried the Stratagene's ArcticExpress^(TM) Competent Cells for
expression of proteins at low temperature (13 degree). The company
claims that the cells encode chaperonins Cpn60 and Cpn10 and they can
enhance the protein folding and solubility.
Hongmin
Dear All,
I
Dear All,
I got enclusion body in many cases when I tried to express human
proteins in E coli. I would like some suggestions on how to go about
it. I would also like to try co-expression of GroEL/GroES or
DnaJ/DnaK, and would like to know where to get the plasmids.
Any help or comments would be
Hi Weijun,
you can get a kit from Takara (chaperone kit #3340) which has five
different chaperone plasmids (combinations of various chaperone). It
worked for me assofar that I could express huge amounts of the chaperones,
but my target protein was either not expressed at all any more (seems as
if
] Inclusion body
Dear All,
I got enclusion body in many cases when I tried to express human
proteins in E coli. I would like some suggestions on how to go about
it. I would also like to try co-expression of GroEL/GroES or
DnaJ/DnaK, and would like to know where to get the plasmids.
Any help
Dear Weijun
Have a look at our REFOLD database - there are some examples of
chaperone assisted refolding as well as many protocols for refolding
of disulphide-rich proteins
http://refold.med.monash.edu.au
good luck
ashley
On 24/03/2007, at 12:23 PM, [EMAIL PROTECTED] wrote:
Hi Weijun,
