Recombinant streptavidin-metallothionein chimeric protein having biological recognition specificity

• US 6,391,590 B1
• Filed: 10/21/1991
• Issued: 05/21/2002
• 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): Romeo, David S.
• Application Number:
  US07/780,717
• Time in Patent Office: 3,865 Days
• Field of Search: 530/345, 530/402, 530/350, 530/413, 530/404, 435/69.7, 435/252.33, 435/320.1, 435/172.3, 435/69.1, 536/24.1
• US Class Current: 435/69.7
• 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. A method of making a recombinant bifunctional streptavidin-metallothionein chimeric protein, said method comprising steps:

(a) introducing into a host cell a nucleic acid encoding a bifunctional fusion protein comprising a streptavidin moiety and a metallothionein moiety, wherein said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO;

7) of mature streptavidin;

(b) incubating said cell under conditions sufficient to express said fusion protein;

(c) isolating said fusion protein.

2. A method according to claim 1, wherein said isolating step comprises a renaturation step in the presence of a heavy metal ion which binds said metallothionein moiety.

3. A method according to claim 1, wherein said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety.

4. A method according to claim 1, wherein said conditions comprise a medium sufficiently minimal to substantially reduce proteolysis of the expressed chimeric protein.

5. A method according to claim 1, wherein said isolating step is performed at least in part in the presence of one or more proteinase inhibitors sufficient to reduce proteolysis.

6. A method according to claim 1, wherein said isolating step is performed at least in part in the presence of dithiothreitol.

7. A method according to claim 1, wherein said isolating step comprises 2-iminobiotin affinity chromatography performed at least in part at a pH of 10.5.

8. A method of making a bifunctional recombinant streptavidin-metallothionein chimeric protein, said method comprising steps:

(a) introducing into a host cell a nucleic acid encoding a fusion protein comprising a streptavidin moiety and a metallothionein moiety, wherein said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO;

     7) of mature streptavidin, 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 expressed chimeric protein;

(c) isolating said fusion protein, said isolating step 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, wherein said isolating step comprises 2-iminobiotin affinity chromatography performed at a pH of 10.5.

9. A method according to claim 8, wherein said isolating step a renaturation step in the presence of a heavy metal ion which binds said metallothionein moiety.

10. A bifunctional recombinant streptavidin-metallothionein chimeric protein, said protein made by a method comprising steps:

(a) introducing into a host cell a nucleic acid encoding a bifunctional fusion protein comprising a streptavidin moiety and a metallothionein moiety, wherein said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO;

     7) of mature streptavidin;

(b) incubating said cell under conditions sufficient to express said fusion protein;

(c) isolating said fusion protein.

11. A recombinant streptavidin-metallothionein chimeric protein according to claim 10, wherein said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety.

12. A recombinant streptavidin-metallothionein chimeric protein according to claim 10, wherein said conditions comprise a medium sufficiently minimal to substantially reduce proteolysis of the expressed chimeric protein.

13. A recombinant streptavidin-metallothionein chimeric protein according to claim 10, wherein said isolating step is performed at least in part in the presence of one or more proteinase inhibitors sufficient to reduce proteolysis.

14. A recombinant streptavidin-metallothionein chimeric protein according to claim 10, wherein said isolating step performed at least in part in the presence of dithiothreitol.

15. A recombinant streptavidin-metallothionein chimeric protein according to claim 10, wherein said isolating step comprises a renaturation step in the presence of a heavy metal ion which binds said metallothionein moiety.

16. A bifunctional recombinant streptavidin-metallothionein chimeric protein, said protein made by a method comprising steps:

(a) introducing into a host cell a nucleic acid encoding a fusion protein comprising a streptavidin moiety and a metallothionein moiety, wherein said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO;

7) of mature streptavidin, and said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety;

(b) incubating sad cell under conditions sufficient to express said fusion protein;

(c) isolating said fusion protein, said isolating step 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 recombinant streptavidin-metallothionein chimeric protein according to claim 16, wherein said isolating step comprises 2-iminobiotin affinity chromatography performed at least in part at a pH of 10.5.

18. A bifunctional recombinant streptavidin-metallothionein chimeric protein, wherein said protein comprises a streptavidin moiety and a metallothionein moiety, and said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO:

7) of mature streptavidin.

19. A bifunctional recombinant streptavidin-metallothionein chimeric protein comprising a streptavidin moiety and a metallothionein moiety, wherein said streptavidin moiety consists of residues 16 to 133 (SEQ ID NO:

7) of mature streptavidin, and said fusion protein additionally comprises a peptide between said streptavidin moiety and said metallothionein moiety.

20. An expression vector comprising a truncated streptavidin gene encoding a streptavidin moiety wherein said streptavidin moiety consists of residues 16 to 133 of mature streptavidin (SEQ ID NO:

7).

21. An expression vector according to claim 20, wherein said truncated streptavidin gene is under the transcriptional control of a T7 promoter.

22. An expression vector according to claim 20, wherein said truncated streptavidin gene is joined to a polylinker comprising a cloning site.

23. An expression vector according to claim 20, comprising a gene fusion of said truncated streptavidin gene and a target protein gene.

24. An expression vector according to claim 20, comprising a gene fusion of said truncated streptavidin gene and a target protein gene, wherein said target protein gene encodes metallothionein.

25. A bifunctional recombinant streptavidin chimeric protein, wherein said protein comprises a streptavidin moiety consisting of residues 16 to 133 (SEQ ID NO:

7) of mature streptavidin.

26. A chimeric protein comprising a functional streptavidin moiety consisting of residues 16 to 133 (SEQ ID NO:

7) of mature streptavidin.

27. A functional streptavidin consisting of residues 16 to 133 (SEQ ID NO:

7) of mature streptavidin.