Patent
Described herein is a variant of wild type Gaussia luciferase that catalyzes glow-type emission kinetics suited for high-throughput functional screening applications. Polypeptides, functional fragments, variants, and nucleic acids that encode the enhanced luciferase are further described. One such polypeptide corresponds to wild type Gaussia luciferase with a substitution mutation of I for M at position 43 of the mature peptide. Methods of use, assay systems and kits that contain the polypeptides and/or nucleic acids are further described.
1. An isolated nucleic acid fragment comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, with a substitution mutation at the position 43, or a functional fragment thereof, wherein the polypeptide catalyzes a stable light emission output in the presence of a detergent, as compared to a comparable polypeptide which has a Methionine at position 43.
2. The isolated nucleic acid fragment of claim 1 , wherein the detergent is Triton X-100, present in the amount of 0.1%.
3. The isolated nucleic acid fragment of claim 1 , wherein the substitution mutation is a conservative mutation
4. The isolated nucleic acid fragment of claim 3 , wherein the conservative substitution mutation is the substitution of Tyrosine, Leucine and Isoleucine.
5. The isolated nucleic acid fragment of claim 4 , wherein the substitution mutation is an Isoleucine for Methionine.
6. The isolated nucleic acid fragment of claim 1 , wherein the nucleotide sequence corresponds to nucleotides 52-555 of SEQ ID NO: 4, or to nucleotides 52-555 of SEQ ID NO: 5, with a nucleotide change in codon 60 that results in an amino acid substitution mutation.
7. The isolated nucleic acid fragment of claim 6 , wherein the substitution mutation is a conservative substitution mutation for Methionine.
8. The isolated nucleic acid fragment of claim 7 , wherein the conservative substitution mutation is a substitution of Tyrosine, Leucine or Isoleucine.
9. The isolated nucleic acid fragment of claim 7 , wherein the conservative substitution mutation is a substitution of Isoleucine.
10. The isolated nucleic acid fragment of claim 1 , which further comprises and is operatively linked to a nucleic acid fragment encoding an additional functional polypeptide sequence.
11. The isolated nucleic acid fragment of claim 10 , wherein the additional functional polypeptide sequence is selected from the group consisting of an affinity tag, an enzyme, and a visible marker.
12. The isolated nucleic acid fragment of claim 1 , which further comprises and is operatively linked to a regulatory response element.
13. A cell comprising an expression vector comprising the nucleic acid fragment of claim 1 .
14. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, with a substitution mutation at the position 43 , or a functional fragment thereof, wherein the polypeptide catalyzes a stable light emission output in the presence of a detergent, as compared to a comparable polypeptide which has a methionine at position 43.
15. The polypeptide or functional fragment thereof of claim 14 , wherein the substitution mutation is a conservative mutation.
16. The polypeptide or functional fragment thereof of claim 14 , wherein the conservative substitution mutation is the substitution of Tyrosine, Leucine and Isoleucine.
17. The polypeptide or functional fragment thereof of claim 14 , wherein the substitution mutation is an Isoleucine for Methionine.
18. The polypeptide or functional fragment thereof of claim 14 , which further comprises an additional functional polypeptide sequence.
19. The polypeptide or functional fragment thereof of claim 18 , wherein the additional functional polypeptide sequence is selected from the group consisting of an affinity tag, an enzyme, and a visible marker.
20. The polypeptide or functional fragment thereof of claim 14 , wherein the polypeptide or functional fragment is substantially pure.