A set of multicolored Photinus pyralis luciferase mutants for in vivo bioluminescence applications

Abstract

Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R and S284G) and one orange mutant (S293P) contained a single substitution. Although the remaining orange variant contained two mutations, L287I mainly contributed to the color change. Emission spectra collected on whole cells at pH 7.0 revealed that while a single peak of lambdamax approximately 605 nm accounts for red light production by the Q283R and S284G variants, orange light results from the contribution of two peaks of lambdamax approximately 560 and 600 nm. All spectra underwent a red-shift when cells were assayed under acidic conditions, whereas a blue-shift was observed at pH 8.0, indicating that the internal pH of Escherichia coli is close to the external pH shortly after imposition of acid or alkaline stress. In addition, changes in assay pH led to bimodal emission spectra, lending support to the idea that bioluminescence color is determined by the relative contribution of yellow-green and red-orange peaks. The set of multicolored luciferase mutants described here may prove useful for a variety of applications including biosensing, pH monitoring, and tissue and animal imaging.

In Vivo Cell Tracking with Bioluminescence Imaging

“…Also, Kitamaya et al [30] reported on the thermal instability and pH-sensitive spectral property of firefly luciferase which can affect its use as a sensitive multicolor luminescence label or bioluminescence resonance energy transfer donor. In addition, the isolation of bright, red-shifted variants of Photinus pyralis luciferase in order to minimize light absorption and scattering by tissue for in vivo animal studies has also been reported [31][32][33].…”

Use of ATP Bioluminescence for Rapid Detection and Enumeration of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System

“…The typical high energy yellow-green light emission with a peak at 562 nm at pH 7.5 and red light emission with a peak at 620nm when the pH decreases to 5 . This surprising phenomenon where Luciferase is able to emit light of different colors is not clearly understood but the isolation of colored luciferase variants shows that single amino acid substitution in www.intechopen.com Use of ATP Bioluminescence for Rapid Detection and Enumeration of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 101 N-terminal domain affects bioluminescence color by modulating slightly the polarity of the active site environment (Hosseinkhani, 2011;Shapiro et al, 2005). This interesting feature opens the way to wide applications in biotechnology (Branchini et al, 2005).…”

Molecular enigma of multicolor bioluminescence of firefly luciferase

“…Moreover, red-emitting variants of luciferase are also desirable for multiple tagging systems in whole cells as well as for dual-reporter systems [32,33,40]. Sample medium or environmental conditions produce high variability in the response, which is the main negative aspect encountered in an assay using luciferase as a reporter.…”