Dear Howard,
I am presently making fusions between the C-terminal secretion signal of an E.coli
secreted haemolysin and GFPuv from Clontech (vector pBAD-GFPuv). I get extremely
good expression of a stable GFP-secretion signal fusion BUT it has very slow fluorophore
activation kinetics (around 10 hours) and is not secreted by the haemolysin secretion
apparatus. This is very unusual for Hly secretion signal fusions (we have secreted around
10 heterologous fusion proteins to the medium with our secretion system). I am assuming
that the problem is the fusion joint and so am now adding random length amino acid
linkers to try to recover a fluorescent and secreted protein. I believe that this approach has
been used before to regain fluorescent fusions from non-fluorescent ones.
I can't be of much more help than that, except to refer you to the July 1996 issue of Gene
(vol 173 N° 1) all on GFP. I found this a good background source.
Good luck,
Mark.
__________________________
Dr. Mark A. Blight,
Institut de Génétique et Microbiologie,
Bâtiment 409,
Université de Paris XI,
91405 Orsay cedex,
France.
Tel: +33 1 69 15 66 99
Fax: +33 1 69 15 78 08
e-mail: blight at igmors.u-psud.fr
__________________________
