Design and construction of dimeric concanavalin a mutants

• US 20070207498A1
• Filed: 08/30/2006
• Published: 09/06/2007
• Est. Priority Date: 02/24/2005
• Status: Abandoned Application

• Current Assignee: Sensory Technologies LLC (DSI Video Systems LLC), Sensor Technologies, LLC
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Palmieri, Stephen, Matzinger, David, Bulseco, Dylan
• Application Number:
  US11/513,003
• Publication Number:
  US 20070207498A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C07K 14/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Embodiments of the invention provide for compositions comprising purified polypeptides such as purified Concanavalin A (ConA) mutants. In addition, embodiments provide for polypeptides and nucleic acids encoding those polypeptides, such as mutant ConA with reduced dimer-dimer interactions compared to wild type ConA. Some embodiments also provide for sensors comprising the polypeptides disclosed herein. The embodiments also provide an improved method of producing recombinant mutant ConA.

Claims

1. A purified mutant Concanavalin A (ConA) protein comprising the amino acid sequence of SEQ ID NO:

16, wherein said sequence comprises a substitution at amino acid residue 58 and a substitution at one or more of amino acid residue 118, amino acid residue 121, and amino acid residue 192, said purified mutant Con A having reduced dimer-dimer affinity compared to a corresponding wild type ConA protein.

2. The purified mutant ConA protein of claim 1, wherein an amino acid residue selected from the group consisting of asparagine, cysteine, proline, and glycine is substituted for the aspartic acid residue at position 58 of SEQ ID NO:

16.

3. The purified mutant ConA protein of claim 2, wherein an asparagine is substituted for the aspartic acid residue at position 58 of SEQ ID NO:

16.

4. The purified mutant ConA protein of claim 1, wherein an amino acid residue selected from the group of asparagine, cysteine, proline, glutamine, tyrosine, and glycine is substituted for the amino acid residue at one or more of position 118, 121, and 192 of SEQ ID NO:

16.

5. The purified mutant ConA protein of claim 1, wherein at least one of said substitutions replaces a naturally occurring amino acid residue with cysteine.

6. The purified mutant ConA protein of claim 1, wherein the protein comprises at least three substitutions.

7. The purified mutant ConA protein of claim 1, wherein the protein comprises at least four substitutions.

8. The purified mutant ConA protein of claim 1, said protein comprising a substitution at amino acid residue 58, amino acid residue 118, amino acid residue 121, and amino acid residue 192 of SEQ ID NO:

16.

9. The purified mutant ConA protein of claim 8, wherein a cysteine is substituted for the asparagine residue at position 118, a cysteine is substituted for the histidine residue at position 121, and a glutamine is substituted for the glutamic acid residue at position 192 of SEQ ID NO:

16.

10. The purified mutant ConA protein of claim 9, wherein an asparagine is substituted for the aspartic acid residue at position 58 of SEQ ID NO:

16.

11. The purified mutant ConA protein of claim 1, wherein the protein is substantially a dimer.

12. The purified mutant ConA protein of claim 1, wherein the protein is at least about 95% pure.

13. The purified mutant ConA protein of claim 1, wherein the protein exhibits glycoconjugate binding.

14. The purified mutant ConA protein of claim 1, wherein the protein further comprises a detectable label.

15. The purified mutant ConA protein of claim 14, wherein the label is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme, a proximity-based signal generating label moiety, a homogeneous time resolved fluorescence (HTRF) component, and a luminescent oxygen channeling assay (LOCI) component.

16. A device capable of sensing a change in an amount of an analyte, the device comprising the purified mutant ConA protein of claim 1.

17. The device of claim 16, wherein at least a portion of the device is implantable.

18. The device of claim 16, wherein fluorescence can be used to detect the change in the amount of the analyte.

19. The device of claim 16, wherein the analyte comprises a carbohydrate selected from the group consisting of monosaccharides, disaccharides, polysaccharides or a combination thereof.

20. The device of claim 19, wherein the carbohydrate comprises glucose.

23. A method of evaluating a carbohydrate in a sample comprising:

contacting the sample with a specific binding pair that comprises

(i) the purified mutant ConA protein of claim 1, and

(ii) a glycoconjugate, wherein the purified mutant ConA and glycoconjugate reversibly bind to each other; and

determining the extent to which carbohydrate present in the sample displaces glycoconjugate bound to the purified mutant ConA and reversibly binds to the purified mutant ConA.

24. The method of claim 23, wherein at least one of the purified mutant ConA protein and the glycoconjugate has a detectable label.

25. The method of claim 23, wherein the sample is selected from the group consisting of urine, blood, plasma, saliva, intracellular fluid, interstitial fluid, homogenized cells, and a cell extract.

26. The method of claim 23, wherein the glycoconjugate comprises a carbohydrate selected from the group consisting of monosaccharides, disaccharides, polysaccharides or a combination thereof.

27. The method of claim 26, wherein the carbohydrate comprises glucose.

21. A purified mutant Concanavalin A (Con A) molecule, wherein the molecule comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:

6, 8, 10, 12, 14, 18, 20, 22, 24, and 26, or biologically active variants thereof.

22. A purified, isolated nucleic acid selected from the group consisting of SEQ ID NOs:

5, 7, 9, 11, 13, 17, 19, 21, 23, and 25.