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Using T7 Phage Display to Select GFP-based BindersMingHua Dai, Emanuele Pesavento, Csaba Kiss, Nileena Velappan, Peter Pavlik, and Andrew Bradbury of B-9 and Jim Werner and Jemshid Temirov of MPA-CINT recently published an article in Protein Engineering, Design & Selection. This publication describes the use of T7 bacteriophage (viruses that infect bacteria) for the selection of fluorescent binders to proteins of interest. The green fluorescent protein (GFP)-based fluorescent binders fold in the cytoplasmic compartment of bacteria. The T7 bacteriophage also assembles in the same compartment. This co-localization made this a successful display approach for modified fluorescent proteins. This publication titled “Using T7 phage display to select GFP-based binders” (Protein Engineering, Design & selection pp 1-12, 2008,Epub May 10th) also describes the use of two single molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, to determine the number of fluorescent molecules displayed per phage. This has never been carried out for phage in the past, and it was found that only one to three fluorescent proteins were displayed per phage, significantly less than expected (five to fifteen). The researchers were able to optimize selection protocols and identify fluorescent affinity reagents for 8 different proteins. The binding of these fluorescent affinity reagents was mediated by a single binding loop, originally derived from antibodies. As a result of the intrinsic fluorescence of these ligands they were able to use secondary reagent free methods such as flow cytometry to study binding characteristics. Although the affinities of the reagents selected in these experiments was quite low (greater than 400nM), their potential is clear. Once methods to improve affinity have been developed, fluorescent affinity reagents will be extremely useful in diagnostic, detection and proteomics. By developing a successful protocol for selecting such reagents, this group has paved a way for identifying new binding molecules for health and national security interest targets.
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