Promega Corporation

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

Lumora Limited

James A H Murray

David James Squirrell

Christopher Robin Lowe

Laurence Carlo Tisi

Peter J White

Rachel Louise Price

Melenie Jane Murphy

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.

[object Object]

A method for determining the amount of template nucleic acid present in a sample comprising the steps of: I) bringing into association with the sample all the components necessary for nucleic acid amplification, and all the components necessary for a bioluminescence assay for nucleic acid amplification and subsequently: ii) performing the nucleic acid amplification, iii) monitoring the intensity of light output from the bioluminescence assay; and iv) determining the amount of template nucleic acid present in the sample.

Method for determining the amount of template nucleic acid present in a sample

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.

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Cambridge, United Kingdom

James A H Murray