Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.
Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
Promega Corporation, Madison, Wisconsin 53711, United States.
Mary P
MP
Hall
James
J
Unch
Brock F
BF
Binkowski
Michael P
MP
Valley
Braeden L
BL
Butler
Monika G
MG
Wood
Paul
P
Otto
Kristopher
K
Zimmerman
Gediminas
G
Vidugiris
Thomas
T
Machleidt
Matthew B
MB
Robers
Hélène A
HA
Benink
Christopher T
CT
Eggers
Michael R
MR
Slater
Poncho L
PL
Meisenheimer
Dieter H
DH
Klaubert
Frank
F
Fan
Lance P
LP
Encell
Keith V
KV
Wood
D000818:Animals; D002460:Cell Line; D003445:Crustacea / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D049408:Luminescent Agents / Q000032:analysis / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering; D011719:Pyrazines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D048790:Renilla / Q000201:enzymology; D013696:Temperature
D049408:Luminescent Agents; D011719:Pyrazines; D011993:Recombinant Fusion Proteins; D008156:Luciferases