GFP/RFP and fusion proteins

Christian Monnerjahn cmonner at abt150.mpibpc.gwdg.de
Fri Feb 16 07:08:18 EST 2001


Hello experts!

We are interested to construct fusion proteins with RFP and EGFP in
order to check the expression level an enzyme in human cells. The final
goal is to correlate expression and enzyme activity. Thus, I would like
to ask some questions:

1) We would like to do N-terminal fusions (RFP/GFP-enzyme), as we know
that N-terminal fusions do not interfere significantly with the activity

of our enzyme. What kind of spacer (length/amino acid composition)
between the two proteins would be optimal?

2) The enzyme we want to work with is active as a dimer. Do also the
RFP/GFP form oligomers? Does anyone has experience if they can interfere

with oligomeric fusion partners?

3) Before transfecting human cells, we would like to test the enzymatic
activity of the fusion proteins in E. coli. As RFP is unsoluble in
bacteria (Clontech manual page 7), does anyone have any experience with
RFP
fusion proteins and their solubility in bacteria? Especially when the
fusion partner is soluble by itself?

Thank You very much for Your help!

Chrisitan Monnerjahn
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