Purification of all proteins in an experssion library

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Wed Oct 14 10:31:04 EST 1998


Mr. M.J. Lush wrote:
> 
>         We are looking for proteins that stimulate a T-cell line.
> To do this we plan to generate a cDNA expression library and test
> the T-cells with pools of expressed proteins.
> 
>         Unfortunately bacterial proteins will also stimulate the
> T-cells, therefore we want to separate the expressed proteins from
> the bacterial ones.   What we are looking for is a tagging system
> that uses a single set of conditions to purify all proteins (if it
> failed to purify certain proteins they would be lost from the library)
> Can anyone suggest suitable systems for this (cost is a consideration
> because we'll have to purify at least 20 pools per round of cloning).
> 
> One possibility we are considering is making a phage display library
> and purifying the M13 phage by PEG precipitation,  how good is this
> at removing bacterial contamination and are there better ways of
> purifying M13 to 'homogeneity'?
> 
> TIA^6
> 
> --
> 
> Michael
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> NPC rights activist           |      Nameless Abominations are people too.

Hi Michael,

if you had a c-terminal poly his-tag with your expressed proteins they
could be affinity purified on a nickel column. Another thing would be
in vitro expression cloning where protein is made from a coupled 
transcription/translation assay (Promega's TNT) and pools are tested for
their biological acitvity. In this case you have no problems with
bacterial
background without any further purification. There was a nice overview 
on  this approach in the latest issue of Promega's Notes (see also on 
website).

Hope this helps
Ricky

P.S. Of course there are millions of other brands selling in vitro
translation
kits which definitively are as good as TNT :-)

******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de



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