Streptavidin proteins

• US 7,179,618 B2
• Filed: 11/01/2002
• Issued: 02/20/2007
• Est. Priority Date: 10/21/1991
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Sano, Takeshi, Cantor, Charles R., Glazer, Alexander N.
• Primary Examiner(s): Monshipouri; Maryam
• Application Number:
  US10/285,876
• Publication Number:
  US 20030215922A1
• Time in Patent Office: 1,572 Days
• Field of Search: 530/350, 435/69.1, 435/320.1, 435/252.3
• US Class Current: 435/69.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: C07K 14/825 Metallothioneins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/22 containing a Strep-tag

Abstract

Streptavidin-metallothionein chimeric proteins with biological recognition specificity in which the streptavidin moiety provides high affinity biotin binding and the metallothionein moiety provides a high affinity metal binding. The binding affinity of the streptavidin-metallothionein chimeric protein both for biotin and heavy metal ions allows specific incorporation into, conjugation with, or labelling of any biological material containing biotin with various heavy metal ions.

Claims

1. An isolated bifunctional recombinant streptavidin-metallothionein chimeric fusion protein comprising a streptavidin (Streptomyces avidinii) moiety which retains full biotin binding function, and a metallothioncin moiety which retains full metal ion binding function.

2. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety.

3. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety, wherein the peptide consists of the sequence set forth as SEQ ID NO:

4.

4. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO:

7) of mature streptavidin.

5. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1 made by a method comprising steps:

(a) introducing into a host cell a nucleic acid encoding the streptavidin-metallothionein chimeric fusion protein comprising a streptavidin moiety and a metallothionein moiety, and said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety;

(b) incubating said cell under conditions sufficient to express said fusion protein, wherein said conditions comprise a medium sufficiently minimal to substantially reduce proteolysis of the fusion protein;

(c) isolating said fusion protein, wherein said isolating step comprises 2-iminobiotin affinity chromatography performed at a pH of 10.5, and is performed at least in part in the presence of one or more proteinase inhibitors sufficient to reduce proteolysis and at least in part in the presence of dithiothreitol.

6. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 5, wherein said isolating step further comprises a renaturation step in the presence of one or more heavy metal ions which bind said metallothionein moiety.

7. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 5, wherein the nucleic acid comprises a pTSAMT-2 expression vector for the streptavidin-metallothionein chimeric protein, constructed from an expression vector pTSA-18F for streptavidin-containing chimeric proteins and a mouse metallothionein-1 cDNA.

8. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein the metallothionein moiety of the fusion protein is complexed with one or more heavy metal ions.

9. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein the streptavidin moiety of the fusion protein is bound to a biotinylated material.

10. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein the streptavidin moiety of the fusion protein is bound to a biotinylated antibody.

11. The bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, wherein the streptavidin moiety of the fusion protein is bound to a biotinylated nucleic acid.

12. A plurality of bifunctional recombinant streptavidin-metallothionein chimeric fusion proteins of claim 1, wherein each of the fusion proteins is complexed with a different one or more heavy metal ions, such that each of the fusion proteins may be discriminately detected.

13. A plurality of bifunctional recombinant streptavidin-metallothionein chimeric fusion proteins of claim 9, wherein each of the fusion proteins is complexed with a different one or more heavy metal ions, such that each of the fusion proteins may be discriminately detected.

14. A plurality of bifunctional recombinant streptavidin-metallothionein chimeric fusion proteins of claim 10, wherein each of the fusion proteins is complexed with a different one or more heavy metal ions, such that each of the fusion proteins may be discriminately detected.

15. A plurality of bifunctional recombinant streptavidin-metallothionein chimeric fusion proteins of claim 11, wherein each of the fusion proteins is complexed with a different one or more heavy metal ions, such that each of the fusion proteins may be discriminately detected.

16. A method of making the bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1, said method comprising steps:

(a) introducing into a host cell a nucleic acid encoding the streptavidin-metallothionein chimeric fusion protein comprising a streptavidin moiety and a metallothionein moiety, and said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety;

(b) incubating said cell under conditions sufficient to express said fusion protein, wherein said conditions comprise a medium sufficiently minimal to substantially reduce proteolysis of the fusion protein;

(c) isolating said fusion protein, wherein said isolating step comprises 2-iminobiotin affinity chromatography performed at a pH of 10.5, and is performed at least in part in the presence of one or more proteinase inhibitors sufficient to reduce proteolysis and at least in part in the presence of dithiothreitol.

17. A method according to claim 16, wherein said isolating step further comprises a renaturation step in the presence of one or more heavy metal ions which bind the metallothionein moiety of the fusion protein.

18. A method for specific labeling of a biotinylated biological material comprising one or more biotin molecules, the method comprising the step of:

contacting said material with the bifunctional recombinant streptavidin-metallothionein chimeric fusion protein of claim 1 under conditions wherein the fusion protein specifically binds the biotin molecules.

19. The method of claim 18, wherein the metallothionein moiety of the fusion protein is complexed with one or more heavy metal ions.

20. The method of claim 18, wherein the biological material is an antibody.

21. The method of claim 18, wherein the biological material is a nucleic acid.

22. A method for specifically multi-labeling a plurality of different biological materials each comprising one or more biotin molecules, the method comprising the step of:

contacting said material with a plurality of bifunctional recombinant streptavidin-metallothionein chimeric fusion proteins of claim 1 under conditions wherein the fusion proteins specifically bind the biotin molecules, wherein each of the fusion proteins is complexed with a different one or more heavy metal ions, such that each of the fusion proteins may be discriminately detected.

23. The method of claim 22, wherein the biological materials are antibodies.

24. The method of claim 22, wherein the biological materials are nucleic acids.