inclusion bodies HELP!!

Tim Dunn 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
> 
> HELP!!!!
> 
> 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
synthesis/secretion rates.
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
etc.  We
get plenty of protein for this kind of experiment.
Good Luck!

Tim Dunn
Centre for Molecular and Cellular Biology
University of Queensland
Australia 4075
e-mail t.dunn at mailbox.uq.oz.au
phone  (07) 365 4565
fax    (07) 365 4388 



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