pH-insensitive glucose indicator protein

• US 8,741,591 B2
• Filed: 10/12/2010
• Issued: 06/03/2014
• Est. Priority Date: 10/09/2009
• Status: Expired due to Fees

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Ye, Kaiming, Jin, Sha
• Primary Examiner(s): HOBBS, LISA JOE
• Application Number:
  US12/902,725
• Publication Number:
  US 20110091919A1
• Time in Patent Office: 1,330 Days
• Field of Search: None
• US Class Current: 435/14
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• 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 33/84 involving inorganic compounds or pH

Abstract

The present invention encompasses a glucose indicator protein, a biosensor comprising one or more glucose indicator proteins, and methods of use thereof.

Claims

1. A pH insensitive glucose indicator protein (GIP) that has a glucose binding dissociation constant (K_d) between about 1 mM and about 20 mM comprising:

a. a glucose binding protein that changes conformation upon exposure to glucose, wherein said glucose binding protein comprises amino acids 24-333 of SEQ ID NO;

1 and has a threonine substituted for phenylalanine at position 39;

b. at least one pH insensitive fluorescence donor coupled to the glucose binding protein; and

c. at least one pH insensitive fluorescence acceptor coupled to the glucose binding protein, wherein the conformational changes of the glucose binding protein upon binding or releasing glucose produces a detectable and reversible signal change.

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

3. The GIP of claim 1, wherein the donor is enhanced cyan fluorescent protein (ECFP) and the acceptor is yellow fluorescent protein (YFP).

4. The GIP of claim 1, wherein the donor is fused to the N terminus of the glucose binding protein and the acceptor is fused to the C terminus of the glucose binding protein.

5. The GIP of claim 1, further comprising two donors.

6. A pH insensitive glucose indicator protein (GIP) that produces a detectable signal in the presence of from at least 1 mM to about 32 mM glucose comprising:

(a) a glucose binding protein that changes conformation upon exposure to glucose, wherein said glucose binding protein comprises amino acids 24-333 of SEQ ID NO;

1 and has a threonine substituted for phenylalanine at position 39;

(b) at least one pH insensitive fluorescence donor coupled to the glucose binding protein; and

(c) at least one pH insensitive fluorescence acceptor coupled to the glucose binding protein, wherein the conformational changes of the glucose binding protein upon binding or releasing glucose produces a detectable and reversible signal change.

7. The GIP of claim 6, wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.

8. The GIP of claim 6, wherein the donor is enhanced cyan fluorescent protein (ECFP) and the acceptor is yellow fluorescent protein (YFP).

9. The GIP of claim 6, wherein the donor is fused to the N terminus of the glucose binding protein and the acceptor is fused to the C terminus of the glucose binding protein.

10. The GIP of claim 6, further comprising two donors.