Promega Corporation

The Secretary of State for Defence in Her Britannic Majesty's Government

The Secretary of State for Defence in Her Britannic Majesty's Government of the United Kingdom of Great Britain and Northern Ireland

The United States of America As Represented by the Secretary of the State of Defence

Peter J White

David James Squirrell

Christopher Robin Lowe

Rachel Louise Price

Melenie Jane Murphy

James A H Murray

Laurence Carlo Tisi

James Augustus Henry Murray

James Augusts Murray

Tara Louise Willey

A recombinant protein having luciferase activity and at least 60% similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 inluciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase. In general, the residue corresponding to 357 inluciferase is changed from an acidic amino acid to a non-acidic amino acid and preferably an uncharged polar amino acid such as tyrosine. Mutant luciferases in accordance with the invention can produce a large (50 nm) wavelength shift in emitted light and have good thermostability. The resultant colour shift can be reversed by addition of coenzyme A. These properties make the mutant particularly useful in a variety of assays.

Luciferase mutant

An isolated recombinant luciferase having luciferase activity. The recombinant luciferase has an amino acid sequence which differs from the wild-type luciferase fromor. In the sequence of the recombinant luciferase, the amino acid residue corresponding to phenylalanine 295 inwild-type luciferase or to leucine 297 inorwild-type luciferases, is mutated compared to the corresponding amino acid which appears in the corresponding wild-type luciferase sequence. The recombinant luciferase has increased thermostability compared to the corresponding wild-type luciferase.

Mutant luciferase

A protein having luciferase activity and at least 60% similarity to luciferase fromororwherein in the sequence of the enzyme, at least one of (a) the amino acid residue corresponding to residue 214 inluciferase; (b) the amino acid residue corresponding to residue 232 inluciferase; (c) the amino acid residue corresponding to residue 295 inluciferase; (d) the amino acid residue corresponding to acid 14 ofluciferase; (e) the amino acid residue corresponding to amino acid 35 ofluciferase; (f) the amino acid residue corresponding to amino acid residue 105 ofluciferase; (g) the amino acid residue corresponding to amino acid residue 234 ofluciferase; (h) the amino acid residue corresponding to amino acid residue 420 ofluciferase; (i) the amino acid residue corresponding to amino acid residue 310 ofluciferase; is different to the amino acid which appears in the corresponding wild type sequence and wherein the luciferase enzyme has increased thermostability as compared to an enzyme having the amino acid of the corresponding wild-type luciferase at this position.

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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,is such that the K,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 &mgr;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.

Enzyme assay for mutant firefly luciferase

Proteins are provided having luciferase activity with lower K,than wild-type luciferases by altering the amino acid residue at position 270 of the wild-type to an amino acid other than glutamate. Greater heat stability than wild-type luciferases while retaining the lower K,is provided by also replacing the glutamate equivalent to that at position 354 of,luciferase or 356 of Luciola luciferases with an alternative amino acid, particularly lysine and/or the amino acid residue at 215 of,and 217 of the Luciola species with a hydrophobic amino acid. DNA, vectors and cells that encode for and express the proteins are also provided as are test kits and reagents for carrying out luminescence assays using the proteins of the invention.

Mutant luciferases

Luciferases

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Cambridge, United Kingdom

Peter J White