New chimera proteins for fluorescence correlation spectroscopy.
A new class of chimera proteins has been developed. They are ideally suited for detection by fluorescence correlation spectroscopy (FCS), a new technology to analyze molecular interactions. The molecular structure of these chimera proteins consists of four domains: a N-terminal (His)6-tag for affinity chromatography followed by an eight amino acid epitope for immunodetection, a polypeptide affinity domain (ADF) for target specific interaction and a C-terminal Green Fluorescent Protein (GFPuv) for reporting of interaction with the target by FCS. We designed, prepared and characterized a prototype of ADF-GFP proteins capable of specific interaction with DNA fragments bearing nuclear factor (NF)-kappaB sites. ADF NF-kappaB p50 and a non-DNA-binding deletion mutant (p35) combined with GFPuv were inserted in a procaryotic vector and expressed in E. coli. Following affinity purification the fluoroproteins p50-GFPuv and p35-GFPuv were employed in specific protein-protein and protein-DNA interaction studies. FCS analysis as well as EMSA showed that p50-GFPuv revealed a fully functional ADF. We present a model for the preparation of GFP fusion proteins capable of specific interaction with proteins, lipids or nucleic acids. The rational design allows any polypeptide fragment to be incorporated into the chimeric protein. So a new series of bio-molecules with different binding specificities and assays can be developed.
National Institute of Dental and Craniofacial Research, Pain and Neurosensory Mechanisms Branch, National Institutes of Health, Bethesda, USA.
Z
Z
Olah
U
U
Trier
B
B
Sauer
M
M
Schäfer-Korting
B
B
Kleuser
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA