Biosensor

• US 6,277,627 B1
• Filed: 12/31/1998
• Issued: 08/21/2001
• Est. Priority Date: 12/31/1997
• Status: Expired due to Term

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Hellinga, Homme W.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US09/223,775
• Time in Patent Office: 964 Days
• Field of Search: 204/400, 204/403, 422/82.05, 422/82.08, 435/7.2, 435/14, 435/172.1, 435/287.1, 435/287.2, 435/288.7, 435/808, 435/817, 436/172, 436/518, 436/805
• US Class Current: 435/287.1
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

The present invention relates to a glucose biosensor comprising a genetically engineered Glucose Binding Protein (GBP). In a specific embodiment, the invention relates to a GBP engineered to include mutations that allow site specific introduction of environmentally sensitive reporter groups. The signal of these prosthetic groups changes linearly with the degree of glucose binding. Thus, the glucose sensor of the invention can be used, for example, for detection of glucose in blood or industrial fermentation processes.

Claims

1. A glucose biosensor comprising a glucose binding protein (GBP) and a reporter group that transduces a detectable signal, wherein said reporter group is attached to said GBP so that a signal transduced by said reporter group when said GBP is bound to glucose differs from a signal transduced by said reporter group when said GBP is not bound to glucose.

2. The biosensor according to claim 1 wherein said reporter group is attached to said GBP at a site distant from a glucose binding site of said GBP.

3. The biosensor according to claim 1 wherein said reporter group is attached to a glucose binding site of said GBP.

4. The biosensor according to claim 1 wherein said reporter group is a fluorophore.

5. The biosensor according to claim 1 wherein said reporter group is a redox cofactor.

6. The biosensor according to claim 1 wherein said GBP has a binding constant for glucose in the range of 0.8 μ

M to 20 mM.

7. The biosensor according to claim 6 wherein said GBP has a binding constant for glucose in the range of 1 mM to 20 mM.

8. The biosensor according to claim 1 wherein said GBP is a mutant bacterial protein.

9. The biosensor according to claim 8 wherein said GBP has a binding constant for glucose in the range of 0.8 μ

M to 20 mM.

10. The biosensor according to claim 9 wherein said GBP has a binding constant for glucose in the range of 1 mM to 20 mM.

11. The biosensor according to claim 9 wherein said mutant protein is a mutant E coli protein.

12. The biosensor according to claim 11 wherein alanines are present at positions 154 and 183.

13. The biosensor according to claim 12 wherein said reporter group is a fluorophore.

14. The biosensor according to claim 12 wherein said reporter group is attached to a cysteine residue at position 255.