Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luc...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2006-09, Vol.19 (9), p.391-400
Main Authors: Loening, Andreas Markus, Fenn, Timothy David, Wu, Anna M., Gambhir, Sanjiv Sam
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
DNA Mutational Analysis
enzyme stability
Luciferases, Renilla - biosynthesis
Luciferases, Renilla - blood
Luciferases, Renilla - genetics
Luminescence
Models, Genetic
Models, Molecular
Molecular Probe Techniques
Molecular Sequence Data
mutagenesis
Recombinant Proteins - biosynthesis
Recombinant Proteins - blood
Recombinant Proteins - genetics
Renilla
Renilla - enzymology
Renilla - genetics
Renilla luciferase
reporter gene
Sequence Alignment
Sequence Analysis, DNA
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Summary:Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output.These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzl023