A question on phage expression systems

Hayden Henry 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!
			Hayden 



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