Expression of fusion proteins w/ GIII

Peter Wang plw at mrc-lmb.cam.ac.uk
Tue Jan 9 19:01:57 EST 1996


"MD. Finucane" <M.Finucane at bristol.ac.uk> 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.

Michael:

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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