Luciferase enzymes for use with thermostable luciferins in bioluminescent assays
AssigneePromega Corporation
InventorsMichael Killoran, Ce Shi, Mary Hall, Lance P. Encell, Thomas Kirkland
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Abstract
Provided herein are enhanced luciferase enzymes for use with thermostable luciferin analogs for bioluminescent assays. In particular, the present disclosure provides compositions, assays, and methods for performing a bioluminescent assay using enhanced, high-activity luciferase enzymes compatible with thermostable luciferins, such as 5,5-disubstituted luciferin analogs.
Claims26
A luciferase polypeptide capable of producing an increased bioluminescent signal in the presence of a 5,5-disubstituted luciferin compared to a bioluminescent signal produced by a luciferase polypeptide of SEQ ID NO:
- 1 in the presence of the 5,5-disubstituted luciferin, wherein the luciferase polypeptide is not naturally occurring and comprises at least 90% sequence identity with the amino acid sequence of SEQ ID NO:
- 2 and less than 100% sequence identity with the amino acid sequence of SEQ ID NO:
- 1, wherein the polypeptide comprises amino acid substitution H244W or H244G relative to the amino acid sequence of SEQ ID NO:
- 1.
A reagent composition for a bioluminescent assay, the composition comprising:
- a luciferase polypeptide capable of producing an increased bioluminescent signal in the presence of a 5,5-disubstituted luciferin compared to a bioluminescent signal produced by a luciferase polypeptide of SEQ ID NO:
- 1 in the presence of the 5,5-disubstituted luciferin, wherein the polypeptide comprises at least 90% sequence identity with the amino acid sequence of SEQ ID NO:
- 2 and less than 100% sequence identity with the amino acid sequence of SEQ ID NO:
- 1, and wherein the polypeptide comprises amino acid substitution H244W or H244G relative to the amino acid sequence of SEQ ID NO:
- 1;
- and a substrate for the luciferase polypeptide.
A kit comprising:
- a luciferase polypeptide capable of producing an increased bioluminescent signal in the presence of a 5,5-disubstituted luciferin compared to a bioluminescent signal produced by a luciferase polypeptide of SEQ ID NO:
- 1 in the presence of the 5,5-disubstituted luciferin, wherein the polypeptide comprises at least 90% sequence identity with the amino acid sequence of SEQ ID NO:
- 2 and less than 100% sequence identity with the amino acid sequence of SEQ ID NO:
- 1, and wherein the polypeptide comprises amino acid substitution H244W or H244G relative to the amino acid sequence of SEQ ID NO:
- 1;
- and a luciferin.
Dependencies are inferred from the claim text.