Expression of fusion proteins w/ GIII

Peter Wang plw at
Tue Jan 9 19:01:57 EST 1996

"MD. Finucane" <M.Finucane at> wrote:
>I am expressing a protein fused to Gene 3 of phage;
>There is an amber stop codon between the protein and the GIII,
>which theoretically should allow for either full length expression
>of the fusion protein (protein-GeneIII) or of just the protein, with no
>Gene III, depending on the bacterial strain used.
>So far so good, except that I can never seem to get the full-length
>expression.  Always the protein comes out alone, with no Gene III 
>attached.  Asking around, I get the impression that this has
>been seen before, but nobody knows why.


How are you determining whether the protein is fused to gIII or not?  By 
Western blotting?  As I'm sure you know, gIII is membrane-bound so in 
supernatants or osmotic shock there would only be the unfused form (note 
that even in bacteria with an amber suppressor, there is often a 
significant amount of unfused material due to incomplete suppression).  
Also, some gIII fusions exhibit a high degree of proteolytic cleavage 
that can vary from protein to protein or even different constructs.

Presumably you want to display your protein on phage.  Can you do a 
phage-ELISA to check if there is functional protein on the phage 
produced?  Do you know if your protein folds properly in the periplasmic 
space of E. coli?

- Peter

Peter Wang, M.D., Ph.D.
MRC Centre for Protein Engineering,
Hills Road, Cambridge, CB2 2QH, England

Tel (01223) 402104  (international calls +44-1223-402104)
Fax (01223) 402140  (     "          "   +44-1223-402140)

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