Thermostable luciferases and methods of production
AssigneePromega Corporation
InventorsKeith V. Wood, Mary P. Hall, Monika G. Wood
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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.
Claims99
A thermostable beetle luciferase that retains at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein the thermostable beetle luciferase has at least one amino acid substitution at an amino acid residue corresponding to residue 2, 27, 91, 144, 183, 220, 262, 353, 356, 386, 399, 412, 413 or 499 of SEQ ID NO:37, or a combination thereof.
A method to prepare thermostable luciferases, comprising:
- a) selecting one or more isolated polynucleotide sequences encoding thermostable luciferases which have one or more other desired enzymological properties from a first population of polynucleotide sequences obtained from a first isolated polynucleotide sequence encoding a luciferase subjected to conditions that yield nucleotide mutations, wherein the one or more isolated polynucleotide sequences encode thermostable luciferases which have increased thermostability and one or more enhanced desired enzymological properties relative to the first isolated polynucleotide sequence;
- b) mutating the selected isolated polynucleotide sequence to yield a second population of polynucleotide sequences;
- and
- c) repeating step a) and step b) so as to yield a further polynucleotide sequence encoding a thermostable luciferase comprising a plurality of amino acid substitutions and having at least one enhanced enzymological desired property relative to the luciferase encoded by the first polynucleotide sequence.
A method to prepare an enzyme having enhanced enzymological properties, comprising:
- a) selecting one or more isolated polynucleotide sequences encoding an enzyme which has at least one enhanced enzymological property from a first population of polynucleotide sequences obtained from a first isolated polynucleotide sequence encoding an enzyme subjected to conditions that yield nucleotide mutations, wherein the enzyme encoded by the one or more selected isolated polynucleotide sequences has at least one enhanced enzymological property relative to the enzyme encoded by the first isolated polynucleotide sequence;
- b) mutating the selected isolated polynucleotide sequence to yield a second population of polynucleotide sequences, wherein the selected isolated polynucleotide sequence is subjected to oligonucleotide mediated mutagenesis with at least one oligonucleotide comprising a codon that encodes a consensus amino acid which is not present in the first polynucleotide sequence;
- and
- c) repeating step a) and step b) so as to yield a further polynucleotide sequence encoding an enzyme having at least one enhanced enzymological property and comprising a plurality of amino acid substitutions relative to the enzyme encoded by the first polynucleotide sequence.
A method to prepare an enzyme that is resistance to an inhibitor, comprising:
- a) selecting one or more isolated polynucleotide sequences encoding an enzyme which is resistance to an inhibitor from a first population of polynucleotide sequences obtained from a first isolated polynucleotide sequence encoding an enzyme subjected to conditions that yield nucleotide mutations, wherein the enzyme encoded by the one or more selected isolated polynucleotide sequences has increased resistance to an inhibitor relative to the enzyme encoded by the first isolated polynucleotide sequence;
- b) mutating the selected isolated polynucleotide sequence to yield a second population of polynucleotide sequences;
- and
- c) repeating step a) and step b) so as to yield a further polynucleotide sequence encoding an enzyme that is resistant to an inhibitor and comprises a plurality of amino acid substitutions relative to the enzyme encoded by the first polynucleotide sequence.
An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a thermostable beetle luciferase that retains at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein the thermostable beetle luciferase has a plurality of amino acid substitutions at residues corresponding to residue 2, 27, 91, 144, 173, 183, 204, 220, 221, 262, 294, 353, 354, 356, 386, 394, 399, 412, 413 or 499 of SEQ ID NO:37, wherein the plurality of substitutions is associated with enhanced thermostability relative to a corresponding beetle luciferase without the plurality of substitutions.
An isolated nucleic acid molecule encoding a thermostable beetle luciferase that retains at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., wherein the thermostable beetle luciferase has a plurality of amino acid substitutions relative to a corresponding beetle luciferase which does not retain at least 50% activity in aqueous solution for at least 2 hours at a temperature of about 50° C., 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 Luciola cruciata luciferase, Luciola lateralis luciferase, Pyrophorus plagiophthalamus luciferase, Pyrocoelia miyako luciferase, Lampyris noctiluca luciferase, Photuris pennsylvanica luciferase, Phengodes sp. luciferase and Photinus pyralis luciferase.
Dependencies are inferred from the claim text.