Glucose sensor and uses thereof

• US 20070219346A1
• Filed: 11/27/2006
• Published: 09/20/2007
• Est. Priority Date: 04/22/2002
• Status: Abandoned Application

• Current Assignee: McGill University
• Original Assignee: McGill University
• Inventors: Trifiro, Mark
• Application Number:
  US11/605,173
• Publication Number:
  US 20070219346A1
• Time in Patent Office: Days
• US Class Current: 530/308
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Y 207/01001 Hexokinase (2.7.1.1)

Abstract

The present invention provides a glucokinase protein in which the catalytic activity has been disabled in order to enable its use as a glucose sensor. The catalytically disabled glucokinase protein can be used as the glucose sensor in hand-held glucose monitors and in implantable glucose monitoring devices. The glucose sensor can also be incorporated into biomedical devices for the continuous monitoring of glucose and administration of insulin.

Claims

1. An isolated or purified recombinant human glucokinase polypeptide having decreased catalytic activity but a substantially identical ability to bind glucose relative to a corresponding wild-type human glucokinase. polypeptide, wherein

said corresponding wild-type human glucokinase polypeptide has an amino acid sequence as set forth in GenBank Accession No. AAA52562, GenBank Accession No. AAA51824, GenBank Accession No. NP_—

000153, GenBank Accession No. P35557;

GenBank Accession No. AAB97681, GenBank Accession No. NP_—

277042;

GenBank Accession No. AAB97682, GenBank Accession No. NP_—

277043;

GenBank Accession No. AAB59563 or SEQ ID NO;

2, or is encoded by a nucleotide sequence as set forth in GenBank Accession No. M90299, GenBank Accession No. M88011, GenBank Accession No. NM_—

000162, GenBank Accession No. NM_—

033507;

GenBank Accession No. NM_—

033508;

GenBank Accession No. M69051, GenBank Accession No. AH005826 or SEQ ID NO;

1;

said recombinant human glucokinase polypeptide comprises an amino acid sequence that differs from the sequence of the corresponding wild-type human glucokinase polypeptide by a mutation at an amino acid residue corresponding to Asp 78, Ser 151, Asp 205, Arg 85, Lys 169, Gly 81, Lys 169, Thr 82, Asn83, Thr228, Ser 411, Lys 296, Ser 336, Thr332, Glu 290, Thr268, Glu256, Asn204, Asn231 or Lys269 of said corresponding wild-type human glucokinase polypeptide; and

said recombinant human glucokinase polypeptide further comprises at least one affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface.

2. The isolated or purified recombinant human glucokinase polypeptide of claim 1, wherein said recombinant human glucokinase polypeptide has the amino acid sequence of SEQ ID NO:

2 or is encoded by the nucleotide sequence of SEQ ID NO;

1.

3. The isolated or purified recombinant human glucokinase polypeptide of claim 1, wherein said affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a metal-binding motif or an affinity tag.

4. The isolated or purified recombinant human glucokinase polypeptide of claim 3, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a glutathione-S-transferase (GST) tag, a polyhistidine tag, avidin, streptavidin or biotin.

5. The isolated or purified recombinant human glucokinase polypeptide of claim 4, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a hexa-histidine tag.

6. The isolated or purified recombinant human glucokinase polypeptide of claim 1, wherein said solid surface is part of a biosensor.

7. The isolated or purified recombinant human glucokinase polypeptide of claim 1, wherein said decreased catalytic activity is between about 10 and about 10 000-fold less than the catalytic activity of the corresponding wild-type human glucokinase polypeptide.

14. The isolated or purified recombinant human glucokinase polypeptide of claim 1, wherein:

said recombinant human glucokinase polypeptide has the amino acid sequence of SEQ ID NO;

2 or is encoded by the nucleotide sequence of SEQ ID NO;

1;

said mutation is Asp205Ala; and

affinity tag or reactive group tag for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a glutathione-S-transferase (GST) tag or a polyhistidine tag.

16. The isolated or purified recombinant human glucokinase polypeptide of claim 14, wherein said solid surface is part of a biosensor.

8. An isolated or purified recombinant human glucokinase polypeptide having decreased catalytic activity but a substantially identical ability to bind glucose relative to a corresponding wild-type human glucokinase polypeptide, wherein:

said corresponding wild-type human glucokinase polypeptide has an amino acid sequence as set forth in GenBank Accession No. AAA52562, GenBank Accession No. AAA51824, GenBank Accession No. NP_—

000153, GenBank Accession No. P35557;

GenBank Accession No. AAB97681, GenBank Accession No. NP_—

277042;

GenBank Accession No. AAB97682, GenBank Accession No. NP_—

277043;

GenBank Accession No. AAB59563 or SEQ ID NO;

2, or is encoded by a nucleotide sequence as set forth in GenBank Accession No. M90299, GenBank Accession No. M88011, GenBank Accession No. NM_—

000162, GenBank Accession No. NM_—

033507, GenBank Accession No. NM_—

033508;

GenBank Accession No. M69051;

GenBank Accession No. AH005826 or SEQ ID NO;

1;

said recombinant human glucokinase polypeptide comprises an amino acid sequence that differs from the sequence of the corresponding wild-type human glucokinase polypeptide by a mutation at the amino acid residue corresponding to Asp 205 of said corresponding wild-type human glucokinase polypeptide; and

said recombinant human glucokinase polypeptide further comprises at least one affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface.

9. The isolated or purified recombinant human glucokinase polypeptide of claim 8, wherein said recombinant human glucokinase polypeptide has the amino acid sequence of SEQ ID NO:

2 or is encoded by the nucleotide sequence of SEQ ID NO;

1.

10. The isolated or purified recombinant human glucokinase polypeptide of claim 8 wherein said mutation is Asp205Ala.

11. The isolated or purified recombinant human glucokinase polypeptide of claim 8, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a metal-binding motif or an affinity tag.

12. The isolated or purified recombinant human glucokinase polypeptide of claim 11, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a glutathione-S-transferase (GST) tag, a polyhistidine tag, avidin, streptavidin or biotin.

13. The isolated or purified recombinant human glucokinase polypeptide of claim 12, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a hexa-histidine tag.

15. The isolated or purified recombinant human glucokinase polypeptide of claim 8, wherein said solid surface is part of a biosensor.

17. The isolated or purified recombinant human glucokinase polypeptide of claim 8, wherein said decreased catalytic activity is between about 10 and about 10 000-fold less than the catalytic activity of the corresponding wild-type human glucokinase polypeptide.

18. An isolated or purified recombinant human glucokinase polypeptide having decreased catalytic activity but a substantially identical ability to bind glucose relative to a corresponding wild-type human glucokinase. polypeptide, wherein:

said corresponding wild-type human glucokinase polypeptide has an amino acid sequence as set forth in GenBank Accession No. AAA52562, GenBank Accession No. AAA51824, GenBank Accession No. NP_—

000153, GenBank Accession No. P35557;

GenBank Accession No. AAB97681, GenBank Accession No. NP_—

277042;

GenBank Accession No. AAB97682, GenBank Accession No. NP_—

277043;

GenBank Accession No. AAB59563 or SEQ ID NO;

2, or is encoded by a nucleotide sequence as set forth in GenBank Accession No. M90299, GenBank Accession No. M88011, GenBank Accession No. NM_—

000162, GenBank Accession No. NM_—

033507;

GenBank Accession No. NM_—

033508;

GenBank Accession No. M69051, GenBank Accession No. AH005826 or SEQ ID NO;

1;

said recombinant human glucokinase polypeptide comprises an amino acid sequence that differs from the sequence of the corresponding wild-type human glucokinase polypeptide by a mutation at the amino acid residue corresponding to Ser336 of said corresponding wild-type human glucokinase polypeptide; and

said recombinant human glucokinase polypeptide further comprises at least one affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface.

19. The isolated or purified recombinant human glucokinase polypeptide of claim 18, wherein said recombinant human glucokinase polypeptide has the amino acid sequence of SEQ ID NO:

2 or is encoded by the nucleotide sequence of SEQ ID NO;

1.

20. The isolated or purified recombinant human glucokinase polypeptide of claim 18 wherein said mutation is Ser336Val, Ser336Leu or Ser336Ile.

21. The isolated or purified recombinant human glucokinase polypeptide of claim 18, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a metal-binding motif or an affinity tag.

22. The isolated or purified recombinant human glucokinase polypeptide of claim 21, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase. polypeptide onto a solid surface is a glutathione-S-transferase (GST) tag, a polyhistidine tag, avidin, streptavidin or biotin.

23. The isolated or purified recombinant human glucokinase polypeptide of claim 22, wherein affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a hexa-histidine tag.

24. The isolated or purified recombinant human glucokinase polypeptide of claim 18, wherein:

said recombinant human glucokinase polypeptide has the amino acid sequence of SEQ ID NO;

2 or is encoded by the nucleotide sequence of SEQ ID NO;

1;

said mutation is Ser336Val, Ser336Leu or Ser336Ile; and

said affinity tag or reactive group for immobilizing said recombinant human glucokinase polypeptide onto a solid surface is a glutathione-S-transferase (GST) tag or a polyhistidine tag.

25. The isolated or purified recombinant human glucokinase polypeptide of claim 18, wherein said solid surface is part of a biosensor.

26. The isolated or purified recombinant human glucokinase polypeptide of claim 24, wherein said solid surface is part of a biosensor.

27. The isolated or purified recombinant human glucokinase polypeptide of claim 18, wherein said decreased catalytic activity is between about 10 and about 10 000-fold less than the catalytic activity of the corresponding wild-type human glucokinase polypeptide.