Modified apoaequorin having increased bioluminescent activity

• US 5,360,728 A
• Filed: 12/01/1992
• Issued: 11/01/1994
• Est. Priority Date: 12/01/1992
• Status: Expired due to Term

• Current Assignee: Douglas Prasher, Virginia K. Eckenrode, Woods Hole Oceanographic Institution
• Original Assignee: Woods Hole Oceanographic Institution
• Inventors: Prasher, Douglas
• Primary Examiner(s): Walsh, Stephen G.
• Application Number:
  US07/982,650
• Time in Patent Office: 700 Days
• Field of Search: 530/350, 530/855, 435/189, 930/240
• US Class Current: 435/189
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: Y10S 530/855 Proteins from animals other than mammals or birds

Abstract

The present invention encompasses modified apoaequorin nucleotide and amino acid sequences capable of emitting light in the presence of luciferin and a light-triggering cation such as Ca²⁺, which possess bioluminescent activity greater than unmodified apoaequorin and methods of use therefore.

Claims

1. A composition comprising an isolated altered apoaequorin peptide, wherein at least the amino acid at amino acid position 124 in the amino acid sequence of natural apoaequorin is changed from aspartic acid to serine, said isolated altered apoaequorin, when combined with a luciferin and a light-triggering cation, having greater bioluminescent activity than natural apoaequorin combined with a luciferin and a light-triggering cation.

2. The composition of claim 1 wherein the altered apoaequorin is SEQ ID NO:

3.

3. A composition comprising an isolated altered apoaequorin peptide, wherein at least the amino acid at amino acid position 135 in the amino acid sequence of natural apoaequorin is changed from glutamic acid to serine, said isolated altered apoaequorin, when combined with a luciferin and a light-triggering cation, having greater bioluminescent activity than natural apoaequorin combined with a luciferin and a light-triggering cation.

4. The composition of claim 3 wherein the altered apoaequorin is SEQ ID NO:

4.

5. A composition comprising an isolated altered apoaequorin peptide, wherein at least the amino acid at amino acid position 129 in the amino acid sequence of natural apoaequorin is changed from glycine to alanine, said isolated altered apoaequorin, when combined with a luciferin and a light-triggering cation, having greater bioluminescent activity than natural apoaequorin combined with a luciferin and a light-triggering cation.

6. The composition of claim 5 wherein the altered apoaecluorin is SEQ ID NO:

5.

7. A composition comprising an isolated altered apoaequorin peptide, wherein at least the amino acid at position 117 of SEQ ID NO:

2 is altered from aspartic acid to serine, said isolated altered apoaequorin peptide, when combined with a luciferin and a light-triggering cation, having greater bioluminescent activity than natural apoaequorin combined with a luciferin and a light-triggering cation.

8. A composition comprising an isolated altered apoaequorin peptide, wherein at least the amino acid at position 128 of SEQ ID NO:

2 is altered from glutamic acid to serine, said isolated altered apoaecluorin peptide, when combined with a luciferin and a light-triggering cation, having greater bioluminescent activity than natural apoaequorin combined with a luciferin and a light-triggering cation.

9. A composition comprising an isolated altered apoaequorin peptide, wherein at least the amino acid at position 122 of SEQ ID NO:

2 is altered from glycine to alanine, said isolated altered apoaequorin peptide, when combined with a luciferin and a light-triggering cation, having greater bioluminescent activity than natural apoaequorin combined with a luciferin and a light-triggering cation.