Activation of bioluminescence by structural complementation

Patent metadata

Field	Value
Publication number	US-9869670-B2
Application number	US-201615073249-A
Country	US
Kind code	B2
Filing date	Mar 17, 2016
Priority date	Mar 15, 2013
Publication date	Jan 16, 2018
Grant date	Jan 16, 2018

Abstract

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.

First claim

The invention claimed is:

1. A system comprising
a. a cell expressing a first fusion comprising:
i. a target protein of interest, and
ii. a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; and

b. a second fusion comprising:
i. an antibody binding moiety, and
ii. a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a bioluminescent signal produced by the system in the presence of a substrate is substantially increased when the polypeptide contacts the peptide when compared to a bioluminescent signal produced in the presence of the substrate but in the absence of contact between the polypeptide and peptide.

2. The system of claim 1 , wherein the target protein of interest is a membrane protein.

3. The system of claim 1 , wherein the antibody binding moiety is Protein G or Protein A, Protein L, or Protein M.

4. The system of claim 1 , wherein the peptide amino acid sequence is selected from the peptides of Table 1.

5. The system of claim 1 , wherein the polypeptide amino acid sequence is selected from the peptides of Table 2.

6. The system of claim 1 , further comprising a substrate for a bioluminescent complex produced by the peptide and polypeptide.

7. The system of claim 1 , wherein the substrate is furimazine, coelenterazine, or a coelenterazine derivative.

8. The system of claim 1 , wherein the second fusion further comprises a pH sensitive dye.

9. An immunoassay method comprising:
a. expressing in a cell a first fusion comprising: (i) a target protein of interest, and (ii) a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the peptide contacts a polypeptide consisting of SEQ ID NO: 440;
b. adding to the cell:
i. a second fusion comprising: (i) an antibody binding moiety, and (ii) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced in the presence of the substrate when the polypeptide contacts a peptide consisting of SEQ ID NO: 2; and
ii. a test antibody

c. adding a substrate for a bioluminescent complex produced by contact between the peptide and polypeptide, wherein said contact is mediated by binding of the antibody binding moiety to the test antibody if the test antibody binds to the target protein of interest; and
d. detecting bioluminescence generated if the antibody binds to the target protein of interest.

10. The method of claim 9 , wherein the target protein of interest is a membrane protein.

11. The method of claim 9 , wherein the antibody binding moiety is Protein G or Protein A, Protein L, or Protein M.

12. The method of claim 9 , wherein the peptide amino acid sequence is selected from the peptides of Table 1.

13. The method of claim 9 , wherein the polypeptide amino acid sequence is selected from the peptides of Table 2.

14. The method of claim 9 , wherein the substrate is furimazine, coelenterazine, or a coelenterazine derivative.

15. The method of claim 9 , wherein the bioluminescence detected is indicative of the concentration and affinity of the test antibody.

16. The method of claim 9 , wherein the second fusion further comprises a pH sensitive dye for detection of antibody-mediated internalization.

17. The method of claim 16 , further comprising detecting fluorescence if the second fusion binds to target protein of interest through an antibody and is internalized into the cell.

18. The method of claim 16 , further comprising detecting BRET from bioluminescence generated when the antibody binding moiety of the second fusion binds to the antibody, which binds to target protein of interest, and is internalized into the cell.

19. A kit comprising:
a. a cell expressing a first fusion comprising: (i) a target protein of interest, and (ii) and a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the peptide contacts a polypeptide consisting of SEQ ID NO: 440;
b. a second fusion comprising: (i) an antibody binding moiety, and (ii) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the polypeptide contacts a peptide consisting of SEQ ID NO: 2; and
c. a substrate for a bioluminescent complex produced by the peptide and polypeptide.

20. The kit of claim 19 , wherein the target protein of interest is a membrane protein.

21. The kit of claim 19 , wherein the antibody binding moiety is Protein G or Protein A, Protein L or Protein M.

22. The kit of claim 19 , wherein the peptide amino acid sequence is selected from the peptides of Table 1.

23. The kit of claim 19 , wherein the polypeptide amino acid sequence is selected from the peptides of Table 2.

24. The kit of claim 19 , wherein the substrate is furimazine, coelenterazine, or a coelenterazine derivative.

25. The kit of claim 19 , wherein the second fusion further comprises a pH sensitive dye.

Assignees

• Promega Corp

Inventors

• Dixon Andrew S
• Encell Lance
• Hall Mary
• Wood Keith
• Wood Monika
• Schwinn Marie
• Binkowski Brock F
• Zegzouti Hicham
• Nath Nidhi
• Mondal Subhanjan
• Goueli Said
• Meisenheimer Poncho
• Kirkland Thomas
• Unch James
• Pulukkunat Dileep K
• Robers Matthew
• Dart Melanie
• Machleidt Thomas

Classifications

• C12Q1/66

  involving luciferase · CPC title

• C07K7/08 Primary

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• C07K14/43509

  from crustaceans · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• G01N33/533 Primary

  with fluorescent label · CPC title