A question on phage expression systems

Paul N Hengen pnh at ncifcrf.gov
Mon Oct 7 12:02:22 EST 1996


Hayden Henry (henry at whio.lincoln.ac.nz) 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?   

Huh? This is a very confusing question. I think you are mixed up with
the basic biology of M13/f1 phage and the life cycle of this phage
with respect to F pili (the means by which infection proceeds).
I suggest reading a review of M13 and phage display systems.

@article{Rasched.Oberer1986,
author = "I. Rasched
     and E. Oberer",
title = "Ff coliphages: structural and functional relationships",
journal = "Microbiol. Rev.",
volume = "50",
pages = "401-427",
comment = "review on M13 phages",
year = "1986"}

@article{Hill.Stockley1996,
author = "H. R. Hill
     and P. G. Stockley",
title = "Phage presentation",
journal = "Mol. Microb.",
volume = "20",
number = "4",
pages = "685-692",
month = "may",
comment = "review article on phage display",
year = "1996"}

> 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.

There are fusions done with either the III gene or the VIII gene of M13.
They will produce phage particles with different properties. There are
also lambda vectors used for phage display.

> 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.

What is a phage pilus?!!

1. You need a helper phage to correctly package the recombinant M13 because
some wildtype gpIII or gpVIII are required for correct arrangement of the
displayed proteins.

2. The F pilus is necessary on the indicator bacteria for the subsequent round
of infection by the phage particles. They infect by anchoring onto the tip of
the F pilus and infection proceeds through it.

--
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