pGEX/GST gene fusion problems-> HELP!

Pete pkursula at cc.oulu.fi
Wed Aug 14 02:21:51 EST 1996


>   Howdy, I'm using a GST fusion system and am getting a lot of fusion
> protein cleavage, which is highly undesireable.  The GST is attached to
> the cytoplasmic tail of a eukaryotic cell receptor.  I've tried using the
> protease inhibitor PMSF at a 1mM concentration with no effect.  I'm not
> sure if cleavage is occuring in the GST portion or the fusion portion of
> the protein.  I am getting a fusion protein, but also get equal to greater
> amounts of a degradation product.  If you have any leads or ideas, please
> let me know.  I also am unable to think of an experiment to determine
> where the cleavage is occuring.  I have the option of switching to a
> different GST vector but am unsure whether or not it would help.  Oh, the
> proteins are being grown up in E. Coli BL21 cells (tried DH5 cells also,
> no difference) and use IPTG as an inducer.
> 
Same thing happened to me... with a similar protein! I tried different 
amounts of IPTG and different induction times. The result was : I can't 
induce for over 2 hours, otherwise I get extensive degradation (>50 % in 
6 hours). It may be that the protein is somewhat toxic to the bacteria 
and they chew it up as it accumulates, mine was degraded after 2 hours 
induction. This is how it works for me nowadays: 

1) make a fresh transformation into BL21 and grow on amp plate overnight 
OR make a small scale liquid culture of previously transformed 
bacteria overnight (depending if you want to induce 1 ml or 1 l)

2) in the morning, start a fresh liquid culture either from a colony on 
your plate (small-scale) or by diluting your overnight culture 1:100 
(large-scale)

3) grow 4-5 h

4) add IPTG to 0,2 mM (works best for me), induce for 1-2 h

5) purify

The yield is not the best you can get, about 5 ug / ml culture, but the 
degradation is gone. Of course this is just my own experience and may not 
work for you.

Petri Kursula
Biocenter Oulu & Department of Pathology
University of Oulu
Finland




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