Genetically encoded biosensors

• US 10,684,282 B2
• Filed: 06/07/2018
• Issued: 06/16/2020
• Est. Priority Date: 10/07/2011
• Status: Active Grant

• Current Assignee: Howard Hughes Medical Institute
• Original Assignee: Howard Hughes Medical Institute
• Inventors: Marvin, Jonathan S., Looger, Loren
• Primary Examiner(s): Noakes, Suzanne M
• Application Number:
  US16/002,697
• Publication Number:
  US 20190376964A1
• Time in Patent Office: 740 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 14/43504 from invertebrates
• 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 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• 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 33/66 involving blood sugars, e.g. galactose
• 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 with at least 85% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

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 super fluorescent (SF) protein.

10. The recombinant peptide biosensor of claim 9, wherein the SF protein is selected from the group consisting of a green fluorescent protein, a yellow fluorescent protein, a red fluorescent protein, and a blue fluorescent protein.

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

12. The recombinant peptide biosensor of claim 1, wherein the recombinant peptide biosensor comprises an amino acid sequence with at least 90% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

13. The recombinant peptide biosensor of claim 1, wherein the recombinant peptide biosensor comprises an amino acid sequence with at least 95% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

14. The recombinant peptide biosensor of claim 1, wherein the recombinant peptide biosensor comprises an amino acid sequence with at least 99% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

15. The recombinant peptide biosensor of claim 1, wherein the recombinant peptide biosensor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

176-182.

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

22. A vector comprising the nucleic acid of claim 21.

23. A cell comprising the nucleic acid of claim 21.

24. A cell comprising the vector of claim 22.

25. A kit comprising the recombinant peptide biosensor of claim 1, the nucleic acid of claim 21, the vector of claim 22, the cell of claim 23, or the cell of claim 24.

16. A recombinant peptide biosensor comprising an amino acid sequence with at least 90% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182, wherein the recombinant peptide biosensor binds specifically to glutamate.

17. The recombinant peptide biosensor of claim 16, comprising a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182 comprising 10 or fewer conservative amino acid substitutions, wherein the recombinant peptide biosensor binds specifically to glutamate.

18. The recombinant peptide biosensor of claim 16, comprising a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

19. The recombinant peptide biosensor of claim 16, wherein the recombinant peptide biosensor comprises an amino acid sequence with at least 95% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

20. The recombinant peptide biosensor of claim 16, wherein the recombinant peptide biosensor comprises an amino acid sequence with at least 99% identity to a recombinant peptide biosensor selected from the group consisting of SEQ ID NOs:

176-182.

26. A method for detecting glutamate, the method comprising detecting a level of fluorescence emitted by a recombinant peptide biosensor, the peptide biosensor having an amino acid sequence having at least 85% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:

176-182, and correlating the level of fluorescence with the presence of glutamate.

27. The method of claim 26, wherein the recombinant peptide biosensor is expressed from a nucleic acid.

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

29. The method of claim 26, comprising correlating the level of fluorescence with a concentration of glutamate.

30. The method of claim 29, 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.

31. The method of claim 26, wherein the method is performed in vitro.

32. The method of claim 26, wherein the recombinant peptide biosensor comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:

176-182.

33. The method of claim 26, wherein the recombinant peptide biosensor comprises an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:

176-182.

34. The method of claim 26, wherein the recombinant peptide biosensor comprises an amino acid sequence having at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:

176-182.

35. The method of claim 26, wherein the recombinant peptide biosensor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

176-182.