A question on phage expression systems

Deb Britt Deborah_Britt at brown.edu
Tue Oct 8 14:17:22 EST 1996


Hi Hayden-
There seems to be some confusion here-During infection, the phage coat
protein III binds to the F-pilus, which is present on the bacterial cell. 
If you insert a small peptide sequence into the coat protein III, the
phage can still assemble properly, and can still infect E. coli.  The
ablility of the phage to carry out its normal fuctions probably depends on
the size of the insert, i.e. if you put a large protein there, the coat
protein III might no longer bind to the F-pilus, so in this case you would
have to make phage that had both normal coat protein and your fusion
protein.  

When making phage-displayed ScFv, you usually clone the ScFv DNA into the
coat protein gene in a phagemid vector and transform this into the
bacteria.  You then use helper phage to produce  phage particles that
carry the ScFv/phagemid vector as their genome, and display the protein
III/ScFv.  You don't want to have phage that display the ScFv but carry a
wild-type genome, because then you have no way of recovering your clone.  

I hope this clarifies things-Good luck with your paper.

In article <henry.12.000DEA04 at whio.lincoln.ac.nz>,
henry at whio.lincoln.ac.nz (Hayden Henry) wrote:

> Hi all! I'm writing an essay on phage expression of scFV (single chain 
> antibody variable region fragments), and I have a question.
> 
> The primary question is: Can the phage be correctly packaged in a 
> bacteria, without ANY phage pillus?   
> 
> The background to this question is as follows: 
> In the early papers e.g. McCafferty et al., Nature 348, 552-554, they 
> inserted a synthetic sequence they wanted to express, into an fd-CAT1 
> vector and interrupted the coat protein III gene (though if you look at the 
> diagram it has incorrectly been labelled a coat protein VIII gene!). The 
> idea was that they could transfect this into an E.coli and express a phage 
> with the scFV attached on the phage surface.
> 
> Now my confusion arises from the fact that the coat protein III gene 
> encodes the anchor for the phage pillus. My understanding is that the phage 
> pillus is required for phage assembly. However, all of the three to four 
> copies of the cpIII protein will have an scFV attached to them and thus 
> would be unable to have the pillus attached. So how did the phage form 
> without a pillus?????
> 
> Later papers e.g. Barbas et. al. 1991 Proc.Natl. Acad. Sci. 88, 7978-7982 
> make mention that they HAD to use a helper phage in addition to the 
> modified phage vector, in order to express the packaged phage. It should be 
> noted that these papers were trying to achieve a monovalent display of the 
> scFV, i.e. one scFV and three to four pillus. 
> 
> If anyone can help me, my email is Henry at ono.lincoln.ac.nz 
> Thanks for your time!
>                         Hayden

-- 
Deborah Britt, Ph.D.
Department of Medical Oncology
Rhode Island Hospital




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