Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence.
Luciferase from the North American firefly (Photinis pyralis) is a useful reporter gene in vivo, allowing noninvasive imaging of tumor growth, metastasis, gene transfer, drug treatment, and gene expression. Luciferase is heat labile with an in vitro halflife of approximately 3 min at 37 degrees C. We have characterized wild type and six thermostabilized mutant luciferases. In vitro, mutants showed half-lives between 2- and 25-fold higher than wild type. Luciferase transfected mammalian cells were used to determine in vivo half-lives following cycloheximide inhibition of de novo protein synthesis. This showed increased in vivo thermostability in both wild-type and mutant luciferases. This may be due to a variety of factors, including chaperone activity, as steady-state luciferase levels were reduced by geldanamycin, an Hsp90 inhibitor. Mice inoculated with tumor cells stably transfected with mutant or wild-type luciferases were imaged. Increased light production and sensitivity were observed in the tumors bearing thermostable luciferase. Thermostable proteins increase imaging sensitivity. Presumably, as more active protein accumulates, detection is possible from a smaller number of mutant transfected cells compared to wild-type transfected cells.
Baggett
Brenda
B
University of Arizona Cancer Center, Tucson, AZ 85724-5024, USA.
Roy
Rupali
R
Momen
Shafinaz
S
Morgan
Sherif
S
Tisi
Laurence
L
Morse
David
D
Gillies
Robert J
RJ
D000818:Animals; D045744:Cell Line, Tumor; D004795:Enzyme Stability / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D049409:Luciferases, Firefly / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D012333:RNA, Messenger; D011994:Recombinant Proteins; D049409:Luciferases, Firefly