inclusion bodies HELP!!
t.dunn at mailbox.uq.oz.au
Wed Sep 27 19:13:29 EST 1995
In article <25SEP199513053868 at vax2.concordia.ca>,
susanai at vax2.concordia.ca (AITKEN, SUSAN-MARIE) wrote:
> I am in desperate need of some advice on how to avoid getting inclusion
> bodies. I have about a month left to finish my masters project work and
> 11 overexpressed proteins to purify and most of them are making inclusion
> bodies on me
> I have heard that growing the cells and inducing them at 30 degrees
> will help and plan to try this tomorrow. Perhaps also I will induce
> for less time and/or with less IPTG.
> If anybody has any advice or any other ideas please feel free to let
> me know and I will be most appreciative.
> Thanks in advance for the help
> Susan Aitken
Sometimes there is nothing whatever that you can do to avoid incusion
bodies, a complex interaction between protein solubility and
BUT it is true that slowing down the rate of synthesis may help. Dropping
the temp to 30 degrees is a start. I have also talked to people who have
had sucess leaving out IPTG entirely and letting the *induction* go
overnight. (There is a small amount of lactose in LB broth). This didn't
work for me tho'.
Playing with IPTG/Temp is your best bet, or clone into a different vector
... probably not an option at this stage.
I guess another option, depending on how much protein you need, is to take
the small amount of soluble stuff and throw away the inclusion bodies. We
do this for a lot of our GEX fusion protein we use in gel shift assays
get plenty of protein for this kind of experiment.
Centre for Molecular and Cellular Biology
University of Queensland
e-mail t.dunn at mailbox.uq.oz.au
phone (07) 365 4565
fax (07) 365 4388
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