Ms. Tara L. Southworth Profile

Tara L. Southworth

at Connecticut College

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Proceedings Article | 14 June 2004 Paper

Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

Bruce Branchini, Tara Southworth, Neelum Khattak, Martha Murtiashaw, Sarah Fleet

Proceedings Volume 5329, (2004) https://doi.org/10.1117/12.530725

KEYWORDS: Bioluminescence, Proteins, In vivo imaging, Biological research, Magnesium, Acquisition tracking and pointing, Genetics, Manufacturing, Error analysis, Biomedical optics

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Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

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