Claims
- 1. A purified nucleic acid construct comprising:
a gene cassette encoding at least one modified bioluminescent protein, said modified protein comprising at least one modification in its amino acid sequence relative to the sequence of an unmodified form of said protein, wherein said modification comprises the addition of a peptide sequence to the protein, said addition reducing a first duration of bioluminescence emitted by said modified bioluminescent protein relative to a second duration of bioluminescence emitted by said unmodified form of said protein.
- 2. The purified nucleic acid construct of claim 1, wherein said gene cassette encodes a luciferase protein.
- 3. The purified nucleic acid construct of claim 1, wherein said gene cassette encodes all proteins necessary for production of bioluminescence without addition of an exogenous substrate.
- 4. The purified nucleic acid construct of claim 3, wherein said nucleic acid construct comprises a lux CDABE cassette.
- 5. The purified nucleic acid construct of claim 2, wherein said luciferase protein comprises at least one a Lux protein selected from Lux A and Lux B.
- 6. The purified nucleic acid construct of claim 5, wherein said Lux protein comprises the amino acid sequence of a Lux protein isolated from bacteria selected from the group consisting of Photorhabdus luminescens, Vibrio fischeri and Vibrio harveyi.
- 7. The purified nucleic acid construct of claim 1, wherein the modified form of said bioluminescent protein comprises a peptide sequence that specifically binds to a protein associated with a proteolytic pathway.
- 8. The purified nucleic acid construct of claim 7, wherein said protein associated with a proteolytic pathway is a tail-specific protease.
- 9. The purified nucleic acid construct of claim 8, wherein the peptide sequence of the modified bioluminescent protein comprises SEQ ID NO:8.
- 10. The purified nucleic acid construct of claim 8, wherein the peptide sequence of the modified bioluminescent protein comprises SEQ ID NO:9.
- 11. The purified nucleic acid construct of claim 8, wherein the peptide sequence of the modified bioluminescent protein comprises SEQ ID NO:10.
- 12. The purified nucleic acid construct of claim 9, wherein the modified protein is at least one of LuxA or LuxB.
- 13. The purified nucleic acid construct of claim 10, wherein the modified protein is at least one of Lux A or Lux B.
- 14. The purified nucleic acid construct of claim 11, wherein the modified protein is at least one of Lux A or Lux B.
- 15. The purified nucleic acid construct of claim 7, wherein said protein associated with a proteolytic pathway mediates degradation of said modified bioluminescent protein via a ubiquitin-proteasome pathway.
- 16. The purified nucleic acid construct of claim 15, wherein said protein associated with a ubiquitin-proteasome pathway is SCF(Grr1).
- 17. The purified nucleic acid construct of claim 15, wherein the peptide sequence of said modified bioluminescent protein comprises a PEST-rich sequence.
- 18. The purified nucleic acid construct of claim 17, wherein said PEST-rich sequence comprises a PEST-rich carboxy terminus of G1 cyclin (Cln2).
- 19. A vector comprising a purified nucleic acid construct comprising a gene cassette encoding at least one modified bioluminescent protein, said modified protein comprising at least one modification in its amino acid sequence relative to the sequence of an unmodified form of said protein, said addition reducing a first duration of bioluminescence emitted by said modified bioluminescent protein relative to a second duration of bioluminescence emitted by said unmodified form of said protein.
- 20. The vector of claim 19, wherein said vector is a plasmid.
- 21. The vector of claim 19, wherein said vector is an expression vector suitable for driving expression in a cell type selected from a bacterial cell, a yeast cell and a mammalian cell.
- 22. A prokaryotic cell comprising the vector of claim 19.
- 23. The prokaryotic cell of claim 22, wherein said cell is a bacterial cell.
- 24. The prokaryotic cell of claim 22, wherein said vector iii said bacterial cell comprises the purified nucleic acid of claim 7 or 8.
- 25. A eukaryotic cell comprising the vector of claim 19.
- 26. The eukaryotic cell of claim 25, wherein said cell is a yeast cell or a mammalian cell.
- 27. The eukaryotic cell of claim 25, wherein said vector in said cell comprises the purified nucleic acid of claim 15.
- 28. The purified nucleic acid of claim 1, wherein the duration of bioluminescence is determined by comparing a time course of a first measure of bioluminescence emitted by said modified protein and a time course of a second measure of bioluminescence emitted by said unmodified protein.
- 29. The purified nucleic acid of claim 28, wherein said first measure is between about 100-fold and 1000-fold lower than said second measure.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority of U.S. provisional patent application No. 60/463,851, entitled “Destabilized Lux,” filed Apr. 18, 2003. The foregoing is herein incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with U.S. government support under grant number ROI 2580 127 awarded by the Department of Energy. The U.S. government may have certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60463851 |
Apr 2003 |
US |