A question on phage expression systems
henry at whio.lincoln.ac.nz
Mon Oct 7 13:54:50 EST 1996
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!
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