A Highly Sensitive Biosensor for ATP Using a Chimeric Firefly Luciferase.
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP. Our methodology can be used with a variety of samples, including human and bacterial cells, where measurements of ATP can be used as a biosensor for the detection of viable cells.
Connecticut College, New London, CT, United States. Electronic address: brbra@conncoll.edu.
Bruce R
BR
Branchini
Connecticut College, New London, CT, United States.
Tara L
TL
Southworth
D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D049408:Luminescent Agents / Q000032:analysis* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D015202:Protein Engineering / Q000379:methods
D049408:Luminescent Agents; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly
ATP; Bioluminescence; Chimera; Imaging; Luciferase; Luciola italica; PLG2; Reporter; luc2