Green fluorescent protein as a signal for protein-protein interactions.
Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.
Park
S H
SH
Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.
Raines
R T
RT
D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
D008164:Luminescent Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012259:Ribonuclease, Pancreatic