| Discovery and Application of a Bright Blue Fluorescent Protein Isolated from Structurally Targeted Library Screening | ||
| Marco A. Mena1, Thomas P. Treynor2, Stephen L. Mayo2 and Patrick S. Daugherty1, (1)Department of Chemical Engineering, UC Santa Barbara, Engineering II Rm 3357, Santa Barbara, CA 93106-5080, (2)Division of Biology, Division of Chemistry and Chemical Engineering, California Institute of Technology, Caltech 114-96, 1200 E. California Blvd, Pasadena, CA 91125-9600 The utility of blue fluorescent protein (BFP) as a marker of gene expression has traditionally been limited by poor photophysical properties. We have designed and screened libraries of BFP variants targeting positions neighboring the histidine-derived chromophore. Sorting the clones using FACS yielded several variants that were considerably brighter than the parental BFP and contained previously unreported substitutions. One of these variants, “Azurite” contained a considerably enhanced quantum yield (0.55 vs. 0.34), enhanced pH stability, and a 40-fold improvement in photobleaching half-life. The variant is well-suited for whole-cell cytometry, microscopy, or other fluorescent assays. Its effectiveness as a FRET donor to several other fluorescent protein pairs has been evaluated, and may lead to the development of novel FRET-based methods with multiple simultaneous acceptors for tracking of complex interactions. Extended Abstract Status: Not Uploaded | ||