PEDS Advance Access originally published online on October 21, 2005
Protein Engineering Design and Selection 2005 18(12):581-587; doi:10.1093/protein/gzi066
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A set of multicolored Photinus pyralis luciferase mutants for in vivo bioluminescence applications
Departments of 1Chemical Engineering and 2Bioengineering, University of Washington, Box 351750, Seattle, WA 98195-1750, USA
3 To whom correspondence should be addressed. E-mail: baneyx{at}u.washington.edu
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 ß5–
10–ß6 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 
max 
605 nm accounts for red light production by the Q283R and S284G variants, orange light results from the contribution of two peaks of max 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.
Keywords: biosensor/firefly/imaging/luc/luminescence/lux
Received June 1, 2005; revised August 22, 2005; accepted September 12, 2005.
Edited by Ashutosh Chilkoti