HIS-tag proteins

Stephan Witte stephan.witte at uni-konstanz.de
Thu Jul 27 02:08:42 EST 1995


In article <3v3k25$t8u at sifon.cc.mcgill.ca>, anne-claude
<gingras at medcor.mcgill.ca> wrote:
> 
> Hi,
> 
> I am having a tough time trying to purify his-tagged proteins using 
> QUIAGEN system.  First of all, the expression levels I am getting are 
> quite low (<200=B5g/L), but still, the protein is >90% in inclusion 
> bodies.  When we purify this protein under denaturing conditions, we see 
> contaminant bands at an higher molecular weight.  Finally, almost all 
> the recovered protein stick to dialysis tubing and cannot be recovered 
> at the final stage.
> 
> Does somebody have nice tricks to try in order to get a protein in the 
> soluble fraction ?  Did somebody ever noticed the presence of other 
> bands on a Coomassie gel ?  What can I do to increase the yield ?  Any 
> idea of how I can do to avoid the protein to stick to the tubing ?
> 
> I will be most grateful if somebody can answer to one of more of my 
> problems.  Thank you a lot !
> 
> Anne-Claude    (gingras at medcor.mcgill.ca)

  
Hi,
for avoiding contaminations try first the isolation of inclusionbodies,
this
makes you get rid of alot of contaminations. Additional you can add 10-20
mM 
mercaptoethanol to all the chromatography-buffers, avoiding contaminants 
that are bound with disulphide bonds.

To avoid sticking to the tubing:
Don«t use dialysis-tubing made of cellulose hydrophobic proteins will stick
to it and you will loose a lot. There are tubings of cellulose-acetate
which overcome 
this problem. I usally get yealds greater 90% even with very hydrophobic
proteins.
Second you may add some tensides, like CHAPS, octylglucoside (both mild and
non denaturing)
 I get usually greater 95% active protein on dialysis from 6M GuCl with
octylglucoside.

Hope this helps,

Stephan
                    
Stephan Witte
Inst. of Immunology
University of Constance
stephan.witte at uni-konstanz.de



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