Microarrays of cellulose binding chimeric proteins and methods of use thereof

• US 20050118729A1
• Filed: 09/03/2004
• Published: 06/02/2005
• Est. Priority Date: 03/07/2002
• Status: Abandoned Application

• Current Assignee: Zephyr Proteomix Ltd.
• Original Assignee: Zephyr Proteomix Ltd.
• Inventors: Morag, Ely
• Application Number:
  US10/933,404
• Publication Number:
  US 20050118729A1
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: C07K 14/33 from Clostridium (G)
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides microarrays of proteins comprising a cellulose binding region as a means for binding to a cellulase substrate such as cellulose. The cellulose binding region may further contain biologically active exogenous moities introduced therein while maintaining the ability to bind cellulase substrates with high affinity. In addition, methods of construction of the microarrays and using the microarrays are disclosed. The microarrays of the invention are particularly useful for displaying peptide libraries, random or rationally designed, and for high throughput screening for ligands, epitopes or ligand binding sites.

Claims

1. An array comprising a plurality of chimeric proteins, each protein comprising a cellulose binding region, wherein the cellulose binding region is devoid of cellulase activity;

the proteins being immobilized onto discrete locations on a solid surface comprising a polymer capable of binding the cellulose binding region;

with each location having at least one the protein displayed within the location.

2. The array according to claim 1, wherein the polymer is a homopolymer or a heteropolymer comprising polysaccharides selected from the group consisting of cellulose acetate, microcrystalline cellulose, lignin, starch, and xylane.

3. The array according to claim 1 wherein the cellulose binding region is derived from a protein other than a member of a polysaccharidase complex.

4. The array according to claim 3, wherein the cellulose binding region is derived from Clostridium Thermocellum.

5. The array according to claim 1, wherein the cellulose binding region comprises the amino acid sequence of SEQ ID NO 4.

6. The array according to claim 5, wherein the cellulose binding region further comprises at least one additional amino acid sequence of SEQ ID NO 5 or SEQ ID NO 6.

7. The array according to claim 1, wherein the cellulose binding region is encoded by a polynucleotide sequence comprising SEQ ID NO 1.

8. The array according to claim 7, wherein the polynucleotide sequence further comprises at least one additional polynucleotide sequence of SEQ ID NO 2 or SEQ ID NO 3.

9. The array according to claim 1, wherein the cellulose binding region further contains at least one exogenous peptide introduced therein, wherein the protein comprising the cellulose binding region is immobilized onto the solid surface such that the at least one exogenous peptide is exposed to the external environment in an accessible orientation.

10. The array according to claim 9, wherein the at least one exogenous peptide comprises a sequence of 5 to 50 amino acids.

11. The array according to claim 1, each protein further comprising at least one biologically active moiety fused thereto to form a fusion protein that is immobilized onto the solid surface such that each biologically active moiety is exposed to an external environment in an accessible orientation.

12. The array according to claim 11, wherein each protein further comprises at least two biologically active moieties.

13. The array according to claim 12, wherein the biologically active moiety is an enzyme, peptide, polypeptide, antibody, antigen, antigenic epitope, polynucleotide, hormone, carbohydrate, lipid, phospholipid, or biotinylated probe.

14. The array according to claim 12, wherein the at least two biologically active moieties are the same or different from each other.

15. The array according to claim 1, wherein the array is spatially addressable.

16. The array according to claim 1, wherein the array is one- or two-dimensional.

17. The array according to claim 1, consisting essentially of a plurality of genetically modified proteins each comprising a cellulose binding region and cellulose.

18. The array according to claim 1, wherein each protein comprises a detectable label.

19. The array according to claim 18, wherein the amount of the detectable label is proportional to the amount of protein.

20. A method for generating an array comprising a plurality of chimeric proteins comprising a cellulose binding region devoid of cellulase activity wherein the cellulose binding region further contains at least one exogenous peptide introduced therein, comprising the following steps:

(a) providing a polynucleotide encoding a cellulose binding region;

(b) introducing at least one oligonucleotide sequence encoding at least one exogenous peptide into the polynucleotide of (a) at a predetermined location;

(c) expressing the modified polynucleotide of (b) to obtain a protein comprising a cellulose binding region wherein the cellulose binding region further contains at least one exogenous peptide introduced therein;

(d) determining the binding efficiency of the protein of (c) to a cellulose polymer;

(e) attaching a plurality of proteins of (d) onto discrete locations on a solid support comprising a cellulose polymer to form an array of immobilized proteins; and

(f) determining the exposure of the at least one exogenous peptide contained within the proteins of (d) to the external environment at an accessible orientation.

21. The method according to claim 20 wherein the cellulose binding region is derived from a protein other than a member of a polysaccharidase complex.

22. The method according to claim 21, wherein the cellulose binding region is derived from Clostridium Thermocellum.

23. The method according to claim 20, wherein the cellulose binding region comprises the amino acid sequence of SEQ ID NO 4.

24. The method according to claim 23, wherein the cellulose binding region further comprises at least one additional amino acid sequence of SEQ ID NO 5 or SEQ ID NO 6.

25. The method according to claim 20, wherein the cellulose binding region is encoded by a polynucleotide sequence comprising SEQ ID NO 1.

26. The method according to claim 25, wherein the polynucleotide sequence further comprises at least one additional polynucleotide sequence of SEQ ID NO 2 or SEQ ID NO 3.

27. The method according to claim 20, wherein the at least one exogenous peptide comprises a sequence of 5 to 50 amino acids.

28. The method according to claim 20, wherein the array is spatially addressable.

29. The method according to claim 21, wherein each protein comprises a detectable label.

30. The method according to claim 29, wherein the amount of the detectable label is proportional to the amount of protein.

31. A method for the production of an array comprising a plurality of chimeric proteins comprising a cellulose binding region, wherein each chimeric protein comprises at least one biologically active moiety while maintaining the capacity to bind a cellulose polymer, comprising the steps of:

(a) providing a polynucleotide comprising a nucleic acid encoding a cellulose binding region devoid of cellulase activity fused to at least one biologically active moiety;

(b) expressing the polynucleotide of (b) to obtain a fusion protein;

(c) determining the binding efficiency of the fusion protein of (b) to a cellulose polymer;

(d) attaching a plurality of fusion proteins of (c) onto discrete locations over a solid support comprising a cellulose polymer to form an array of immobilized fusion proteins; and

(e) determining the exposure of the at least one biological active moiety to the external environment at an accessible orientation.

32. A method for the production of an array comprising a plurality of chimeric proteins comprising a cellulose binding region capable of binding cellulose polymers with high affinity, wherein each chimeric protein comprises at least one additional biologically active moiety covalently linked to the cellulose binding region while maintaining the capacity to bind a cellulose polymer with high affinity and wherein the at least one biologically active moiety is exposed to the external environment in an accessible orientation while the protein is attached to a solid support, comprising the steps of:

(a) providing a polynucleotide comprising a nucleic acid encoding a cellulose binding region devoid of cellulase activity;

(b) expressing the polynucleotide of (a) to obtain a protein comprising a cellulose binding region devoid of cellulase activity;

(c) covalently linking the protein of (b) to at least one biologically active moiety to obtain a chimeric protein;

(d) determining the binding efficiency of the chimeric protein of (c) to a cellulose polymer;

(e) attaching a plurality of chimeric proteins of (d) onto discrete locations over a solid support comprising a cellulose polymer to form an array of immobilized chimeric proteins; and

(f) determining the exposure of the at least one biological active moiety to the external environment at an accessible orientation.

33. The method according to claim 32, wherein the at least one biologically active moiety is obtained by modifying a C-terminus or an N-terminus sequence of the cellulose binding region, wherein the terminus has essentially no cellulose binding activity.

34. The method according to claim 32 wherein the cellulose binding region is derived from a protein other than a member of a polysaccharidase complex.

35. The method according to claim 34, wherein the cellulose binding region is derived from Clostridium Thermocellum.

36. The method according to claim 32, wherein the cellulose binding region comprises the amino acid sequence of SEQ ID NO 4.

37. The method according to claim 36, wherein the cellulose binding region further comprises at least one additional amino acid sequence selected of SEQ ID NO 5 or SEQ ID NO 6.

38. The method according to claim 32, wherein the cellulose binding region is encoded by a polynucleotide sequence comprising SEQ ID NO 1.

39. The method according to claim 38, wherein the polynucleotide sequence further comprises at least one additional polynucleotide sequence of SEQ ID NO 2 or SEQ ID NO 3.

40. The method according to claim 32, wherein each chimeric protein comprises at least two biologically active moieties.

41. The method according to claim 40, wherein the at least two biologically active moieties are the same or different from each other.

42. The method according to claim 32, wherein the array is spatially addressable.

43. The method according to claim 32, wherein each protein comprising a detectable label.

44. The method according to claim 43, wherein the amount of the detectable label is proportional to the amount of protein.

45. The method according to claim 32, wherein the biologically active moiety is selected from the group consisting of enzymes, peptides, polypeptides, antibodies, antigens, antigenic epitopes, polynucleotides, hormones, carbohydrates, lipids, phospholipids, and biotinylated probes.

46. A method for screening a plurality of biologically active moieties for interactions with one or more components of a fluid sample comprising the steps of:

providing a plurality of chimeric proteins immobilized at discrete locations over a solid surface, wherein the surface comprises a cellulose polymer and with the chimeric proteins comprising a cellulose binding region which is devoid of cellulase activity and at least one exogenous peptide introduced therein or at least one biologically active moiety fused or covalently linked thereto thus forming an addressable array of the chimeric proteins at discrete locations such that the at least one exogenous peptide or biologically active moiety is exposed in an accessible orientation;

contacting the array with a sample to be analyzed;

detecting binding interactions between the array and the sample to be analyzed; and

determining the loci of interaction.

47. The method according to claim 46, wherein the cellulose binding region of each chimeric protein contains at least one exogenous peptide introduced therein to form an addressable array of locations such that each exogenous peptide is exposed in an accessible orientation to a fluid sample in an external environment.

48. The method according to claim 46, wherein the cellulose binding region is derived from a protein other than a member of a polysaccharidase complex.

49. The method according to claim 48, wherein the cellulose binding region is derived from Clostridium Thermocellum.

50. The method according to claim 46, wherein the cellulose binding region comprises the amino acid sequence SEQ ID NO 4.

51. The method according to claim 50, wherein the cellulose binding region further comprises at least one additional amino acid sequence selected from the group consisting of SEQ ID NO 5 and SEQ ID NO 6.

52. The method according to claim 46, wherein the cellulose binding region is encoded by a polynucleotide sequence comprising SEQ ID NO 1.

53. The method according to claim 52, wherein the polynucleotide sequence further comprises at least one additional polynucleotide sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 3.

54. The method according to claim 47, wherein the at least one exogenous peptide comprises a sequence of 5 to 50 amino acids.

55. The method according to claim 46, wherein the array is spatially addressable.

56. The method according to claim 46, wherein each protein comprises a detectable label.

57. The method according to claim 56, wherein the amount of the detectable label is proportional to the amount of protein.

58. The method according to claim 46, wherein the cellulose binding region of each chimeric protein includes at least one biologically active moiety fused or covalently linked thereto, and which further comprises forming an addressable array of the chimeric proteins at discrete locations, each having at least one biologically active moiety displayed at each the location such that the at least one biologically active moiety is exposable in an accessible orientation to a fluid sample in an external environment.

59. The method according to claim 58, wherein the at least one biologically active moiety is obtained by modifying a C-terminus or an N-terminus sequence of the cellulose binding region, wherein the terminus has essentially no cellulose binding activity.

60. The method according to claim 58, wherein each protein comprises at least two biologically active moieties.

61. The method according to claim 60, wherein the at least two biologically active moieties are the same or different from each other.

62. The method according to claim 58, wherein the biologically active moiety is selected from the group consisting of enzymes, peptides, polypeptides, antibodies, antigens, antigenic epitopes, polynucleotides, hormones, carbohydrates, lipids, phospholipids, and biotinylated probes.