Mutagenesis and Characterization Studies to Develop Novel Bioluminescent Systems

摘要

The overall objective of this project was to discover, design and demonstrate the feasibility of bioluminescent materials for use in marking, tagging, and anti-tamper applications. Five major accomplishments were completed. (1) The feasibility of performing dual analyte reporter gene assays in mammalian cells grown at 37 C with thermostable red and green light-emitting mutants of firefly luciferase was demonstrated. (2) The gene for Ppy RE9, a novel thermostable red-light emitting firefly luciferase variant, was human codon optimized. The gene provided approximately 100-fold greater signal intensity than a commercial genetic reporter in a human cell line. (3) Mutagenesis studies of luciferase identified residues responsible for blue-shifted emission, pH and thermal stability. (4) A red-emitting luciferase variant Ppy RE10 was selectively labeled with near IR fluorescent dyes. Through the BRET process, the enzymes produced light with maxima at 705 nm or 778 nm. Fusion proteins containing these labeled enzymes were made and immobilized onto magnetic microspheres. (5) A fluorescent chlorophyll a derivative was isolated from the worm C. variopedatus. Also, mass spectral studies of a small molecule fraction of O. phosphorea mucous advanced the structure identification of a putative bioluminescence stimulating factor.