Genetically encoded biosensors

• US 10,060,920 B2
• Filed: 02/26/2018
• Issued: 08/28/2018
• Est. Priority Date: 10/07/2011
• Status: Active Grant

• Current Assignee: Brigham and Women's Hospital Incorporated, Howard Hughes Medical Institute
• Original Assignee: Brigham and Women's Hospital Incorporated, Howard Hughes Medical Institute
• Inventors: Marvin, Jonathan S., Looger, Loren, Lee, Richard T., Schreiter, Eric
• Primary Examiner(s): Noakes, Suzanne Marie
• Application Number:
  US15/904,574
• Publication Number:
  US 20180209972A1
• Time in Patent Office: 183 Days
• Field of Search: None
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/24 containing a MBP (maltose binding protein)-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 2458/00 Labels used in chemical analysis of biological material
• CPC Class Codes: G01N 33/557 using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6812 Assays for specific amino acids

Abstract

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

Claims

1. A recombinant peptide biosensor comprising an analyte-binding framework portion and a signaling portion, wherein the signaling portion is present within the framework portion at a site or amino acid position that undergoes a conformational change upon interaction of the framework portion with a defined, specific, or selected analyte, wherein the recombinant peptide biosensor comprises an amino acid sequence having at least 85% identity to a sequence selected from the group consisting of SEQ ID NO:

62 and 63.

2. The recombinant peptide biosensor of claim 1, wherein the signaling portion is allosterically regulated by the framework portion such that signaling from the signaling portion is altered upon interaction of the framework portion with the analyte.

3. The recombinant peptide biosensor of claim 1, wherein signaling by the signaling portion detectably increases upon interaction of the framework portion with the analyte.

4. The recombinant peptide biosensor of claim 1, wherein signaling by the signaling portion detectably decreases upon interaction of the framework portion with the analyte.

5. The recombinant peptide biosensor of claim 1, wherein signaling by the signaling portion is proportional to the level of interaction between the framework portion and the analyte.

6. The recombinant peptide biosensor of claim 1, wherein the framework portion has a first structure in the absence of an analyte and a second structure, that is detectably distinct from the first structure, in the presence of the analyte.

7. The recombinant peptide biosensor of claim 6, wherein the conformational change between the first structure and the second structure allosterically regulates the signaling portion.

8. The recombinant peptide biosensor of claim 1, wherein the framework portion is a periplasmic binding protein (PBP) or a variant of a PBP.

9. The recombinant peptide biosensor of claim 1, wherein the signaling portion is a circularly permuted fluorescent protein (cpFP).

10. The recombinant peptide biosensor of claim 1, wherein the analyte-binding framework portion binds specifically to glutamate.

13. A nucleic acid encoding the recombinant peptide biosensor of claim 1.

15. A vector comprising the nucleic acid of claim 13.

17. A cell comprising the nucleic acid of claim 13.

19. A cell comprising the vector of claim 15.

21. A method for detecting glutamate, the method comprising detecting a level of fluorescence emitted by the recombinant peptide biosensor of claim 1, and correlating the level of fluorescence with the presence of glutamate.

22. The method of claim 21, wherein the recombinant peptide biosensor is expressed from a nucleic acid.

23. The method of claim 21, comprising contacting the recombinant peptide biosensor with a sample comprising glutamate.

24. The method of claim 21, comprising correlating the level of fluorescence with a concentration of glutamate.

25. The method of claim 24, comprising comparing the level of fluorescence with a level of fluorescence emitted by the recombinant peptide biosensor in the presence of a sample comprising a known concentration or range of concentrations of glutamate.

26. A method for detecting glutamate, the method comprising detecting a level of fluorescence emitted by a recombinant peptide biosensor expressed from the nucleic acid of claim 13 and correlating the level of fluorescence with the presence of glutamate.

27. The method of claim 26, comprising contacting the recombinant peptide biosensor with a sample comprising the glutamate.

28. The method of claim 27, comprising correlating the level of fluorescence with a concentration of the glutamate.

29. The method of claim 28, comprising comparing the level of fluorescence with a level of fluorescence emitted by the recombinant peptide biosensor in the presence of a sample comprising a known concentration or range of concentrations of the glutamate.

11. A recombinant peptide biosensor comprising an amino acid sequence with at least 95% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NO:

62 and 63, wherein the recombinant peptide biosensor binds specifically to glutamate.

12. The recombinant peptide biosensor of claim 11, comprising a recombinant peptide biosensor selected from the group consisting of SEQ ID NO:

62 and 63.

14. A nucleic acid encoding the recombinant peptide biosensor of claim 11.

16. A vector comprising the nucleic acid of claim 14.

18. A cell comprising the nucleic acid of claim 14.

20. A cell comprising the vector of claim 16.