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 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.
Loening
Andreas Markus
AM
The CRUMP Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Fenn
Timothy David
TD
Wu
Anna M
AM
Gambhir
Sanjiv Sam
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D049410:Luciferases, Renilla / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D049449:Luminescence; D008957:Models, Genetic; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D048790:Renilla / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D049410:Luciferases, Renilla