Precipitating bactriophage

Michael Benedik benedik at uh.edu
Tue Jun 4 10:18:43 EST 1996


In article <4ovcsd$75a at bubba.NMSU.Edu>
dkim at nmsu.edu (DANIEL Y KIM) writes:

> Hello:
> 
> I have been trying to construct a filamentous phage display of a protein 
> fused to M13 gene III.  In my plasmid/phagemid, the gene III fusion is 
> interrupted by an Amber stop codon.  I imagine there is a fair amount of 
> protein being produced which is not fused to gene III and which will be 
> secreted into the growth medium, instead of being incorporated into a 
> phage particle.
> 
> Is there a way to precipitate the phage without the free protein?  It 
> seems that PEG will precipitate both phage and free protein (60 kDa) out 
> of the medium (positive Western signal in the absence of helper phage).  
> If not, is there a convenient way to filter out the larger phage from the 
> free protein?
> 
> Thanks
> 
> Daniel Kim
> dkim at nmsu.edu

In theory the free protein would be in the periplasm and not the growth
medium, however it is a legitimate worry since there generally is some
percentage of cells which lyse.

At the appropriate concentration I wouldn't think that PEG should ppt
60kDa proteins. Alternatively you could run some quick sizing column
(ppt your supernatant and resuspend in small volume, then run over a
spin column for example.) You should be able to find a spin column with
matrix size that will include the protein you are worried about yet
will exclude the phage which is massively larger. lastly you can purify
your phage particles on CsCl gradients or on an agarose gel.



Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at uh.edu



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