Enzyme assay for mutant firefly luciferase
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United States of America Patent
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Jul 24, 2001
Grant Date -
N/A
app pub date -
Aug 25, 1999
filing date -
Apr 11, 1997
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Abstract
Enzymes and methods suitable for assaying ATP, and specific application for such assays are described and claimed. In particular, there is described a recombinant mutant luciferase having a mutation for example, in the amino-acid corresponding to amino acid residue number 245 in Photinus pyralis, is such that the K.sub.m for ATP of the luciferase is increased e.g. five-fold with respect to that of the corresponding non-mutated enzyme such that it is of the order of 500 .mu.m-1 mM. Also disclosed are luciferases having additional mutations conferring improved thermostability or altered wavelength of emitted light. Recombinant polynucleotides, vectors and host cells are also disclosed, as are methods of assaying the amount of ATP in a material (e.g. cells) optionally in real-time. Also disclosed are test-kits for in vitro assays.
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