Promega Corporation

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

Melenie Jane Murphy

David James Squirrell

Rachel Louise Price

Peter J White

Christopher Robin Lowe

Laurence Carlo Tisi

James A H Murray

Peter John White

Tara Louise Willey

Rachel Louise Leslie

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.

[object Object]

A method for detecting the presence of a lysed eukaryotic cell in a sample, said method comprising: (i) adding adenosine diphosphate (ADP) to said sample under conditions which allows the conversion of ADP to adenosine triphosphate (ATP) by cellular adenylate kinase, (ii) detecting ATP in said sample and relating that to the presence of adenylate kinase and thus to the presence of lysed cells. The method is useful in detecting the cell lysis, for example when screening for drugs which are required to cause lysis, for example for use in tumor therapy. However, in addition, cells may, in a preliminary step, be lysed and the contents quantitated in order to establish for example the health of condition of the cells or to detect the presence of cells in sample such as milk or urine, for diagnostic purposes.

Cell assay, method and reagents

Growing community of inventors

Salisbury, United Kingdom

Melenie Jane Murphy