Nucleic Acids Research, Vol 26, Issue 21 4946-4952, Copyright © 1998 by Oxford University Press
RM Biondi, PJ Baehler, CD Reymond and M Veron
The green fluorescent protein (GFP) is currently being used for diverse
cellular biology approaches, mainly as a protein tag or to monitor gene
expression. Recently it has been shown that GFP can also be used to monitor
the activation of second messenger pathways by the use of fluorescence
resonance energy transfer (FRET) between two different GFP mutants fused to
a Ca2+sensor. We show here that GFP fusions can also be used to obtain
information on regions essential for protein function. As FRET requires the
two GFPs to be very close, N- or C- terminal fusion proteins will not
generally produce FRET between two interacting proteins. In order to
increase the probability of FRET, we decided to study the effect of random
insertion of two GFP mutants into a protein of interest. We describe here a
methodology for random insertion of GFP into the cAMP-dependent protein
kinase regulatory subunit using a bacterial expression vector. The
selection and analysis of 120 green fluorescent colonies revealed that the
insertions were distributed throughout the R coding region. 14 R/GFP fusion
proteins were partially purified and characterized for cAMP binding,
fluorescence and ability to inhibit PKA catalytic activity. This study
reveals that GFP insertion only moderately disturbed the overall folding of
the protein or the proper folding of another domain of the protein, as
tested by cAMP binding capacity. Furthermore, three R subunits out of 14,
which harbour a GFP inserted in the cAMP binding site B, inhibit PKA
catalytic subunit in a cAMP-dependent manner. Random insertion of GFP
within the R subunit sets the path to develop two-component FRET with the C
subunit.
ARTICLES
Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum
Unite de Regulation Enzymatique des Activites Cellulaires, CNRS-URA 1773, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris Cedex 15, France.
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