Kits and methods of detection using cellulose binding domain fusion proteins

• US 5,738,984 A
• Filed: 06/02/1995
• Issued: 04/14/1998
• Est. Priority Date: 04/14/1993
• Status: Expired due to Fees

• Current Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem)
• Original Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem)
• Inventors: Shoseyov, Oded
• Primary Examiner(s): Scheiner, Toni R.
• Assistant Examiner(s): UNGAR, SUSAN NMN
• Application Number:
  US08/460,458
• Time in Patent Office: 1,047 Days
• Field of Search: 436/827, 436/518, 435/6, 435/7.1, 435/7.5, 435/7.72, 435/7.92, 435/69.1, 435/69.3, 435/69.7, 435/4, 435/179, 435/7.4, 435/7.9
• US Class Current: 435/4
• CPC Class Codes: A01N 25/10 Macromolecular compounds
• CPC Class Codes: A01N 63/50 Isolated enzymes; Isolated proteins peptides A01N37/46
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/33 from Clostridium (G)
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 2319/705 containing a protein-A fusion
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 5/0025 Culture media for plant cell or plant tissue culture
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: G01N 33/548 Carbohydrates, e.g. dextran
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: Y10S 436/827 Lectins
• CPC Class Codes: Y10S 436/828 Protein A
• CPC Class Codes: Y10S 530/82 Proteins from microorganisms
• CPC Class Codes: Y10S 530/861 involving IgG3, IgG4, IgA, or IgY

Abstract

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Claims

1. A diagnostic kit for the detection of a substance of interest comprising:

(a) a cellulose binding domain (CBD) fusion product comprising (i) a CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2 and which is capable of binding to cellulose with high affinity and substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding a substance of interest;

(b) a detectable label; and

(c) cellulose.

2. The diagnostic kit of claim 1 in which said cellulose is replaced by chitin.

3. The diagnostic kit of claim 1 or 2 an which said second protein is Protein A.

4. The diagnostic kit of claim 1 or 2 in which said second protein is a heat shock protein (HSP) antibody;

HSP-related protein, peptide or antigenic portion thereof.

5. The diagnostic kit of claim 1 or 2 in which said CBD fusion product further comprises a ligand affinity bound to said second protein, said ligand capable of binding said substance of interest.

6. The diagnostic kit of claim 5, in which said ligand is an antibody.

7. An immunoassay method of detecting the presence of a substance of interest in a test sample comprising:

(a) incubating a test sample, which may contain a substance of interest, with a sufficient amount of a cellulose binding domain (CBD fusion product comprising (i) a CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2 and which is capable of binding to cellulose with high affinity and substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding said substance of interest, under conditions that allow for the binding of said substance of interest to said second protein of said CBD fusion product;

(b) adding an amount of cellulose effective to bind the amount of said CBD fusion product used in step (a) to provide an insoluble cellulose-CBD fusion product binding complex;

(c) separating said insoluble cellulose-CBD fusion product binding complex from unbound components;

(d) incubating said insoluble cellulose-CBD fusion product binding complex with a sufficient amount of a detectable label, said label capable of binding to said substance of interest; and

(e) separating said insoluble cellulose-CBD fusion product binding complex of step (d) from unbound components and determining the presence or absence of said label, to provide an indication of the presence or absence of said substance of interest in said test sample.

12. The method of claim 7, 8 or 9 in which said test sample comprises a bodily fluid.

13. The method of claim 7 or 9 in which said cellulose is replaced by chitin.

15. The method of claim 7, 8 or 9 in which said substance of interest is a protein or peptide and said second protein is an antibody against said protein or peptide.

16. The method of claim 7, 8 or 9 in which said substance of interest is an antibody and said second protein is Protein A.

17. The method of claim 7 or 9 in which said substance of interest comprises a biotinylated probe bound to a protein, peptide, hormone, nucleic acid or other probe-targetable molecule and said second protein is streptavidin.

18. The method of claim 7, 8 or 9 in which said label comprises a radioisotope, a fluorescent molecule or an enzyme.

22. The method of claim 7 in which step (d) is performed by incubating said insoluble cellulose-CBD fusion product binding complex with a sufficient amount of a detectable label comprising a labeled substance of interest, said label capable of binding to any second protein of said CBD fusion product which remains unbound to said substance of interest, and in which step (e) is performed by separating said insoluble cellulose-CBD fusion product binding complex of step (d) from unbound components and comparing the signal observed from said test sample relative to the signal observed from a control sample.

23. The method of claim 7 in which said CBD fusion product is included in a dip stick.

8. A method of detecting the presence of a substance of interest in a test sample comprising:

(a) contacting a test sample, which may contain a substance of interest, with an insoluble matrix capable of immobilizing said substance of interest;

(b) incubating said insoluble matrix with a sufficient amount of a CBD fusion product comprising (i) a cellulose binding domain (CBD) the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2 and which is capable of binding to cellulose with high affinity and substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding said immobilized substance of interest, under conditions that allow for the binding of said immobilized substance of interest to said second protein of said CBD fusion product;

(c) separating said insoluble matrix of step (b) from unbound components;

(d) incubating said insoluble matrix of step (c) with a detectable label capable of binding said substance of interest or said CBD fusion product under conditions that allow for the binding of said label to said substance of interest or said CBD fusion product; and

(e) separating said insoluble matrix of step (d) from unbound components and determining the presence or absence of said label, to provide an indication of the presence or absence of said substance of interest in said test sample.

9. The method of claim 8 which further comprises (i) contacting said insoluble matrix of step (c) with a sufficient amount of cellulose under conditions that allow for the binding of said cellulose to said CBD fusion product to form a cellulose-CBD fusion product binding complex, and (ii) separating said insoluble matrix of step (i) from unbound components, including unbound cellulose.

10. The method of claim 9 in which said label is capable of binding said substance of interest or said cellulose-CBD fusion protein binding complex.

11. The method of claim 10 in which said label is capable of binding to the cellulose of said cellulose-CBD fusion protein binding complex.

14. The method of claim 8 or 9 in which said insoluble matrix is an electrophoresis gel blot.

19. The method of claim 11 in which said label comprises a CBD fusion product comprising CBD and alkaline phosphatase.

20. The method of claim 11 in which said label comprises a CBD fusion product comprising CBD and horse radish peroxidase.

21. The method of claim 19 or 20 which further comprises adding a sufficient amount of a substrate for said enzyme, which substrate is converted by said enzyme to a detectable compound.

24. A dip stick useful in detecting a substance of interest in a test sample comprising a cellulose binding domain (CBD) fusion product, said CBD fusion product comprising (i) a CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO:

2 and which is capable of binding to cellulose with high affinity and substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding a substance of interest.

25. The dip stick of claim 24 in which said second protein is selected from the group consisting of Protein A;

HSP protein;

HSP antibody;

cross-reactive HSP-related protein or peptide or an antigenic portion thereof;

an enzyme; and

hormone.

36. The dip stick of claim 24 in which said second protein is an antigen or an antibody.

26. A signal amplification kit comprising

(a) a first cellulose binding domain (CBD) fusion product comprising:

(i) a CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2, and

(ii) a second protein capable of binding a chimeric probe, said chimeric probe further capable of binding a protein of interest;

(b) a chimeric probe; and

(c) a second CBD fusion product comprising (i) a CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2, and (ii) an enzyme capable of acting on a substrate to produce a detectable compound.

27. The signal amplification kit of claim 26 further comprising the substrate for said enzyme of said second CBD fusion product.

31. The signal amplification kit of claim 26, 28 or 29 which further comprises cellulose or chitin.

32. The signal amplification kit of claim 26 in which said probe is biotinylated and said second protein is streptavidin.

33. The signal amplification kit of claim 26 in which said enzyme of said second fusion product is selected from the group consisting of alkaline phosphatase and horse radish peroxidase.

35. The signal amplification kit of claim 31 in which the cellulose is pebble-milled cellulose.

28. A signal amplification kit comprising:

(a) a cellulose binding domain (CBD) fusion product comprising

(i) a CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2, and

(ii) a second protein capable of binding a chimeric probe, said probe further capable of binding a substance of interest; and

(b) labeled CBD, said CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2.

34. The signal amplification kit of claim 28 or 30 in which said labeled CBD comprises CBD and a radioisotope, a fluorescent molecule or an enzyme.

29. A signal amplification kit comprising:

(a) a cellulose binding domain (CBD) fusion comprising;

(i) a CBD, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO;

2, and

(ii) a second protein capable of binding a third protein, said third protein further capable of binding a protein of interest; and

(b) a third protein.

30. The signal amplification kit of claim 29 further comprising labeled CBD, said CBD the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO:

2.