ARACHNOCAMPA LUCIFERASES

• US 20110015095A1
• Filed: 08/18/2006
• Published: 01/20/2011
• Est. Priority Date: 08/19/2005
• Status: Active Grant

• Current Assignee: Helen Dacres, Mira Maria Dumancic, Stephen Charles Trowell, Virginia Leitch
• Original Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Inventors: Leitch, Virginia, Dumancic, Mira Maria, Trowell, Stephen Charles, Dacres, Helen
• Granted Patent: US 8,227,572 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Abstract

Nucleotide and amino acid sequences of luciferase peptides that are encoded by genes within the genome of Arachnocampa (Diptera) are disclosed. Specifically provided are functional ATP-dependent luciferases that catalyze luminescence reactions with emission spectra within the blue portion of the spectrum. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and active subsequences of the enzyme peptides, and methods of identifying modulators and substrates of the luciferase peptides. Methods of assays, including multiple reporter assays utilizing at least two ATP-dependent luciferases are provided.

Claims

1. An isolated peptide that displays luciferase activity, wherein

a. the luciferase activity comprises catalysis of a luminescence reaction dependent upon ATP;

and

b. the luminescence reaction produces an emission spectrum with a maximum emission intensity at wavelengths less than or equal to 530±

5 nm; and

c. the peptide has an amino acid sequence that, when aligned with that of SEQ ID NO;

4, differs from that of SEQ ID NO;

4 by at least one change in amino acid sequence selected from the group consisting of deletion of R218, H245N, G315S, L342S, and T343S.

2. The isolated peptide of claim 1 comprising an amino acid sequence that, when aligned with that of SEQ ID NO:

4, further differs from that of SEQ ID NO;

4 by at least one additional change in amino acid sequence selected from the group consisting of A22Y, Y53A, L63T, E83D, F88Y, F89Y, P91I, A103C, Y109W, E113D, V139I, G160P, S198T, H212Q, a deletion of R218, D224S, a deletion of P225, G228D, P233K, P242Q, F243Y, H245N, L253M, Y255R, F273Y, Y280F, S298K, L300E, E311 R, G315S, P318T, L319V, G339F, L342S, T343S, D375H, K380A, E389F, T408S, W417Y, L418V, D427N, L441I, I442V, K443M, Y444V, K445D, Q448A, A452T, L458I, V485L, V486T, G490R, V506L, R513K, V516C, T527A, and a deletion of all residues from and including K549 to the carboxy terminus.

3. The isolated peptide of claim 1 comprising a fragment of an amino acid sequence of at least 10 contiguous amino acids selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

6, SEQ ID NO;

8, and SEQ ID NO;

10.

4. The isolated peptide of claim 1 comprising an amino acid sequence a variant of an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

6, SEQ ID NO;

8, and SEQ ID NO;

10, wherein the variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule with a sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7 and SEQ ID NO;

9 or a complement thereof.

5. The isolated peptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

6, SEQ ID NO;

8, and SEQ ID NO;

10.

16. The method of claim 14, wherein the first reporter enzyme comprises a peptide of claim 1.

6. An isolated peptide having an amino acid sequence that shares at least 70 percent identity with the amino acid sequence shown in SEQ ID NO:

2.

7. An antibody that selectively binds to a peptide of claim 6.

8. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the peptide of claim 6.

9. A gene chip comprising a nucleic acid molecule of claim 8.

10. A transgenic cell comprising a nucleic acid molecule of claim 8.

11. A nucleic acid vector comprising a nucleic acid molecule of claim 8.

12. A host cell containing the vector of claim 11.

13. A method of monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:

(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule according to claim 8, and further comprising a nucleic acid molecule encoding a nucleotide sequence or product of interest;

wherein the nucleic acid molecule according to claim 8 and the sequence or product of interest are co-expressed; and

(b) detecting the presence of the expression of the nucleic acid molecule according to claim 8, thereby monitoring expression of the sequence or product of interest.

14. A method of measuring the activity of at least two reporter enzymes in an aliquot of a sample comprising assaying for activity of a first reporter enzyme by measuring the light signal produced by catalysis of a luminescence reaction by the first reporter enzyme, and assaying for activity of at least a second reporter enzyme by measuring the light signal produced by catalysis of a luminescence reaction by at least a second reporter enzyme, wherein at least one of the first or second reporter enzymes displays luciferase activity comprising catalysis of a luminescence reaction dependent upon ATP and that produces an emission spectrum with a maximum emission intensity at wavelengths less than or equal to 530±

5 nm.

15. The method of claim 14, wherein both of at least two reporter enzymes are ATP dependent luciferases.

17. The method of claim 14, wherein the first reporter enzyme comprises a peptide of claim 5.

18. The method of claim 14, wherein the second reporter enzyme catalyzes a luminescence reaction and the luminescence reaction produces an emission spectrum with a maximum emission intensity at wavelengths greater than 530 nm.

19. The method of claim 14, wherein the assays are performed on the same aliquot of sample.

20. The method of claim 14, wherein the assays proceed simultaneously.