Fluorescent protein sensors for detection of analytes

• US 5,998,204 A
• Filed: 03/14/1997
• Issued: 12/07/1999
• Est. Priority Date: 03/14/1997
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tsien, Roger Y., Miyawaki, Atsushi
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US08/818,253
• Time in Patent Office: 998 Days
• Field of Search: 435/325, 435/252.3, 435/254.2, 435/320.1, 536/23.4, 536/23.5
• US Class Current: 435/325
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Fluorescent indicators including a binding protein moiety, a donor fluorescent protein moiety, and an acceptor fluorescent protein moiety are described. The binding protein moiety has an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte. The donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region. The donor moiety and the acceptor moiety exhibit fluorescence resonance energy transfer when the donor moiety is excited and the distance between the donor moiety and the acceptor moiety is small. The indicators can be used to measure analyte concentrations in samples, such as calcium ion concentrations in cells.

Claims

1. An isolated nucleic acid sequence which encodes a fluorescent indicator, the indicator comprising:

a binding protein moiety having an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte;

a donor fluorescent protein moiety covalently coupled to the binding protein moiety; and

an acceptor fluorescent protein moiety covalently coupled to the binding protein moiety,

wherein the donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region, altering fluorescence resonance energy transfer between the donor moiety and the acceptor moiety when the donor moiety is excited.

2. The nucleic acid of claim 1, wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.

3. The nucleic acid of claim 2, wherein the indicator further includes a target peptide moiety and a linker moiety that covalently couples the binding protein and the target peptide moiety and is a peptide moiety, and the binding protein moiety further includes a peptide-binding region for binding the target peptide moiety.

4. The nucleic acid of claim 3, wherein the donor fluorescent protein moiety is fused to the binding protein moiety and the acceptor fluorescent protein moiety is fused to the target peptide moiety.

5. The nucleic acid of claim 2, wherein the binding protein moiety is calmodulin, a calmodulin-related protein moiety, cGMP-dependent protein kinase, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, protein kinase C, inositol-1,4,5-triphosphate receptor, or recoverin.

6. The nucleic acid of claim 3, wherein the binding protein moiety is calmodulin or a calmodulin-related protein moiety.

7. The nucleic acid of claim 6, wherein the donor fluorescent protein moiety is P4-3, EBFP, or W1B, and the acceptor fluorescent protein moiety is S65T, EGFP, or 10c.

8. The nucleic acid of claim 6, wherein the target peptide moiety is a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofructokinase calcineurin, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cylclase, Bordetella pertussis adenylyl cyclase, Neuromodulin, S-pectrin, MARCKS, F52, β

-Adducin, HSP90a, HIV-1 gp160, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, or Model Peptide CBP2.

9. The nucleic acid of claim 6, wherein the target peptide moiety is M13.

10. The nucleic acid of claim 9, wherein the linker moiety is --Gly-Gly--.

11. The nucleic acid of claim 1, wherein the indicator further comprises a localization sequence.

12. An expression vector comprising the nucleic acid sequence of claim 1.

13. An expression vector comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator, the indicator comprising:

a binding protein moiety having an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte;

a donor fluorescent protein moiety fused to the binding protein moiety; and

an acceptor fluorescent protein moiety fused to the binding protein moiety,

wherein the donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region, altering fluorescence resonance energy transfer between the donor moiety and the acceptor moiety when the donor moiety is excited.

14. The expression vector of claim 13 adapted for function in a prokaryotic cell.

15. The expression vector of claim 13 adapted for function in a eukaryotic cell.

16. A host cell transfected with an expression vector comprising an expression control sequence operatively linked to a sequence coding for the expression of a fluorescent indicator, the indicator comprising:

a binding protein moiety having an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte;

a donor fluorescent protein moiety covalently coupled to the binding protein moiety; and

an acceptor fluorescent protein moiety covalently coupled to the binding protein moiety,

wherein the donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region, altering fluorescence resonance energy transfer between the donor moiety and the acceptor moiety when the donor moiety is excited.

17. The cell of claim 16, wherein the cell is a prokaryote.

18. The cell of claim 17, wherein the cell is E. coli.

19. The cell of claim 16, wherein the cell is a eukaryotic cell.

20. The cell of claim 19, wherein the cell is a yeast cell.

21. The cell of claim 19, wherein the cell is a mammalian cell.