Polypeptides, systems, and methods useful for detecting glucose

• US 8,465,981 B2
• Filed: 08/06/2008
• Issued: 06/18/2013
• Est. Priority Date: 08/06/2007
• Status: Expired due to Fees

• Current Assignee: University Of Kentucky Research Foundation (University of Kentucky)
• Original Assignee: University Of Kentucky Research Foundation (University of Kentucky)
• Inventors: Daunert, Sylvia, Joel, Smita, Rowe, Laura, Turner, Kendrick
• Primary Examiner(s): Wallenhorst, Maureen
• Application Number:
  US12/672,403
• Publication Number:
  US 20110117661A1
• Time in Patent Office: 1,777 Days
• Field of Search: 436/56, 436/63, 436/86, 436/89, 436/95, 436/164, 436/166, 436/172, 422/82.05, 422/82.08, 435/6.1, 435/14, 530/350, 600/365
• US Class Current: 436/95
• CPC Class Codes: A61B 5/14532 for measuring glucose, e.g. by tissue impedance measurement
• CPC Class Codes: A61B 5/1459 invasive, e.g. introduced into the body by a catheter A61B5/1464 takes precedence
• CPC Class Codes: A61B 5/412 Detecting or monitoring sepsis
• CPC Class Codes: Y10T 436/13 Tracers or tags
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

The presently-disclosed subject matter provides biosensors for detecting molecules of interest. The biosensors include a polypeptide capable of selectively-binding glucose, wherein the polypeptide molecule is selected from: an unnatural analogue of wild type glucose binding protein; a fragment of wild type glucose binding protein; and an unnatural analogue fragment of wild type glucose binding protein.

Claims

1. A biosensor for detecting a molecule of interest, comprising:

an isolated polypeptide molecule capable of selectively-binding glucose, wherein the polypeptide molecule is selected from;

an unnatural analogue of the amino acid sequence of SEQ ID NO;

2; and

an unnatural analogue fragment of the amino acid sequence of SEQ ID NO;

2; and

a label associated with the polypeptide molecule;

wherein binding of glucose to the polypeptide molecule causes the label to generate a signal, such that the glucose can be detected.

2. The biosensor of claim 1, wherein the polypeptide molecule is an unnatural analogue fragment of the amino acid sequence of SEQ ID NO:

2.

3. The biosensor of claim 1, wherein the polypeptide molecule comprises the amino acid sequence of SEQ ID NO:

2, wherein one or more natural amino acids are replaced with one or more unnatural amino acids.

4. The biosensor of claim 1, wherein one or more natural amino acids of the unnatural analogue or unnatural analogue fragment are replaced with one or more unnatural amino acids as set forth in Table A.

5. The biosensor of claim 4, wherein one or more natural leucines are replaced with 5,5,5-trifluoroleucine.

6. The biosensor of claim 4, wherein one or more natural tryptophans are replaced with 5-fluorotryptophan.

7. The biosensor of claim 4, wherein one or more natural leucines are replaced with 5,5,5-trifluoro-DL-leucine.

8. The biosensor of claim 4, wherein one or more natural tryptophans are replaced with 5-fluoro-L-tryptophan.

9. The biosensor of claim 1, wherein the polypeptide molecule comprises the amino acid sequence of SEQ ID NO:

2, wherein one or more natural leucines are replaced with 5,5,5-trifluoroleucine.

10. The biosensor of claim 1, wherein the polypeptide molecule comprises the amino acid sequence of SEQ ID NO:

2, wherein one or more natural tryptophans are replaced with 5-fluorotryptophan.

11. The biosensor of claim 1, wherein the polypeptide molecule is stable over a wider range of temperatures relative to wild type GBP (wtGBP).

12. The biosensor of claim 1, wherein the polypeptide molecule is stable at temperatures between about 55°

C. and about 75°

C.

13. The biosensor of claim 12, wherein the polypeptide molecule is an unnatural analogue or an unnatural analogue fragment, wherein one or more natural leucines are replaced with unnatural leucines.

14. The biosensor of claim 13, wherein the unnatural leucines are 5,5,5-trifluoroleucine.

15. The biosensor of claim 1, wherein the label is a fluorophore.

16. The biosensor of claim 15, wherein the polypeptide molecule comprises:

an unnatural analogue of the amino acid sequence of SEQ ID NO;

2;

or an unnatural analogue fragment of the amino acid sequence of SEQ ID NO;

2;

wherein the label is attached to an amino acid site of the polypeptide molecule selected from Asp 44, Val 68, Ala 71, Phe 90, Lys 92, Glu 93, Pro 94, Lys 97, Gly 109, Thr 110, Asp 111, Gly 151, Cys 152, Pro 153, Ala 155, Asp 184, Asp 212, Leu 255, Ala 258, Asn 260, Ala 262, Arg 292, Val 293, and Pro 294, wherein SEQ ID NO;

2 comprises amino acids numbered 14-296 of wild type GBP (wtGBP).

17. The biosensor of claim 16, wherein label is the fluorophore N-[2-(1-maleimidyl(ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC).

18. The biosensor of claim 16, wherein binding of glucose to the polypeptide molecule causes the label to generate a detectably altered fluorescence intensity of the fluorophore such that the glucose can be detected.

19. The biosensor of claim 18, wherein the fluorescence intensity can be correlated to the concentration of the glucose in a sample.

20. A method for detecting a glucose, comprising:

contacting the biosensor of claim 1 with a sample of interest;

detecting the signal; and

collecting and displaying the signal with a detection and data collection device, to thereby detect the glucose.

21. The method of claim 20, wherein the glucose is continuously detected.

22. An isolated polypeptide molecule capable of selectively-binding glucose, wherein the polypeptide molecule is selected from an unnatural analogue of the amino acid sequence of SEQ ID NO:

2; and

an unnatural analogue fragment of the amino acid sequence of SEQ ID NO;

2.