Thermostable luciferases and methods of production
AssigneePromega Corporation
InventorsKeith V. Wood, Mary P. Hall, Monika G. Wood
Life of this patent
Estimating the term…
Abstract
Luciferase enzymes with greatly increased thermostability, e.g., at least half lives of 2 hours at 50° C., cDNAs encoding the novel luciferases, and hosts transformed to express the luciferases, are disclosed. Methods of producing the luciferases include recursive mutagenesis. The luciferases are used in conventional methods, some employing kits.
Claims35
A thermostable beetle luciferase variant comprising amino acid substitutions at amino acids corresponding to residues 2, 91, 183, 220, 221, 249, 262, 294, 353, 354, 386, 399, 542, 543, 544, or 545 of SEQ ID NO:37.
An isolated nucleic acid molecule encoding a thermostable beetle luciferase comprising at least one amino acid substitution at an amino acid corresponding to residue 2, 27, 91, 144, 173, 183, 204, 220, 221, 262, 294, 354, 356, 386, 394, 399, 412, 413 or 499 of SEQ ID NO:37, or a combination thereof.
A thermostable beetle luciferase variant comprising a plurality of amino acid substitutions relative to a corresponding beetle luciferase, wherein at least three substitutions are associated with increased thermostability, and wherein at least one substitution is to a consensus amino acid identified by comparing a plurality of wild-type beetle luciferases selected from the group consisting of Ludola cruciata luciferase, Luciola lateralis luciferase, Pyrophorus plagiophthalamus luciferase, Pyrocoelia miyako luciferase, Lampyris noctiluca luciferase, Photuris pennsylvanica luciferase, Phengodes luciferase and Photinus pyralis luciferase.
Dependencies are inferred from the claim text.