Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1991-10-21
2002-05-21
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S252330, C435S320100, C530S402000, C530S350000, C530S404000, C530S413000, C536S024100
Reexamination Certificate
active
06391590
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of Invention
This invention concerns streptavidin-metallothionein chimeric proteins which possess biological recognition specificity.
RELATED DISCLOSURES
Recombinant streptavidin-metallothionein chimeric proteins containing various metal ions are molecules which have a great potential in preventive and therapeutic medicine in both humans and animals as well as for diagnostic use. While each individual molecule, i.e., streptavidin and metallothionein have been known and described previously, their composite molecule has never before been constructed.
Biochemistry of metallothionein, particularly its amino acid sequence in various species, its metal binding sites, metal thiolate clusters and spatial structures are described in
Biochemistry,
27:509 (1988).
Ann. Rev. Biochem.,
55:913 (1986) is directed to the gene structure, organization, amplification and transcriptional regulations, and describes also some genetic engineering applications, such as for example, metallothionein-rat growth hormone and metallothionein-human growth hormone genes expressed in transgenic mice, or conferring resistance to copper toxicity via the CUP 1 copper-metallothionein gene.
Streptavidin is a protein very closely related to a protein avidin which provides a very stable noncovalent complex with vitamin D-biotin. Avidin itself is a very highly specialized protein that is only rarely expressed. Streptavidin, on the other hand, is readily expressed in
Streptomyces
species particularly in
Streptomyces avidinii.
Streptavidin specifically binds a water soluble vitamin D-biotin (vitamin H). Similarly to avidin, it binds rapidly and almost irreversibly to any molecule which contains unhindered biotin with a remarkably high affinity. Streptavidin, contrary to avidin, is carbohydrate free and thus more suitable, for example, for X-ray crystallographic studies or for various other detection techniques. The comparative properties of avidins and streptavidins are described in
Methods in Enzymology,
184:51 (1990). Isolation and properties of streptavidin, as well as its preparation, are described in
Ibid.,
at page 80.
Expression of a cloned streptavidin gene in
Escherichia coli
is described in
Proc. Natl. Acad. Sci.,
87:142 (1990) and the cooperativity in the biotin binding to streptavidin is described in
J. Biol. Chem.,
265:3369 (1990).
SUMMARY
One aspect of the current invention is a recombinant streptavidin-metallothionein chimeric protein having biological recognition specificity.
Another aspect of the current invention is an expression system for the cloned streptavidin gene which expresses streptavidin in
Escherichia coli
and allows the expression of a streptavidin-metallothionein chimeric protein.
Another aspect of the current invention is an expression vector pTSAMT-2 constructed by inserting the mouse metallothionein-I cDNA into an expression vector for streptavidin-containing chimeric proteins pTSA-I8F.
Another aspect of the current invention is the expression of the gene fusion of streptavidin with metallothionein using T7 expression system.
Still another aspect of the current invention is the method for binding streptavidin-metallothionein chimeric protein with various metal ions.
Still yet another aspect of the current invention is incorporation of the metal-containing streptavidin-metallothionein chimeric protein into biological materials containing unhindered biotin.
Yet another aspect of the current invention is the method for introducing heavy metal ions into the tissue, removing the heavy metal ions from the tissue or labeling the tissue with heavy metal ions.
Still another aspect of the current invention is the use of the streptavidin-metallothionein chimeric protein for imaging of tumors, for radiotherapeutics, for labeling of biological materials, for detection of biological molecules present at very low levels and for simultaneous multi-mass labeling of short DNA molecules allowing determination of a number of DNA sequences.
REFERENCES:
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patent: 4839293 (1989-06-01), Cantor et al.
patent: 5196510 (1993-03-01), Rodwell et al.
patent: WO8602077 (1986-04-01), None
patent: WO8809344 (1988-12-01), None
patent: WO8909393 (1989-10-01), None
patent: 9006323 (1990-06-01), None
Weber et al. Structural origins of high-affinity biotin binding to streptavidin. Science, (Jan. 6, 1989) 243 (4887) 85-8.*
Hiller et al. Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin. Biochem. J., (Sep. 1, 1991) 278 (Pt 2) 573-85.*
Kurzban et al. The quaternary structure of streptavidin in urea J. Biol. Chem. (Aug., 1991), 266(22), 14470-7.*
Hendrickson et al. Proc. Natl. Acad. Sci. vol. 86, pp. 2190-2194, Apr. 1989.*
Rudolph, R. “Renaturation of recombinant, disulfide-bonded proteins from inclusion bodies,” In, Modern Methods of Protein and Nucleic Acid Research, 1990, pp. 149-171.*
Lowenadler et al “A gene fusion system . . . ”Gene 58: 87-97 (1987).*
Rhodes et al “A Yeast-Escherichia coliShuttle Vector . . . ”Plasmid 23: 159-162 (Mar. 1990).*
Arnold et al “A family of high-copy-number plasmid vectors . . . ” Gene 70: 171-179 (1988).*
Romeyer et al., “Bioaccumulation of heavy metals inEscherichia coli. . . ”,J. Biotech. 8: 207-220, 1988.*
Butt et al., “Ubiquitin fusion augments the yield of cloned gene products . . . ”,PNAS 86: 2540-2544, Apr. 1989.*
Argarana, C.E.; Kuntz, I.D.; Birken, S.; Axel, R.; Cantor, C.R. Molecular Cloning and Nucleotide Sequence of the Streptavidin Gene,Nuc. Acids Res., 14:1871 (1986).
Sano, T., Cantor, C.R. Expression of a Cloned Streptavidin Gene inEscherichia coli, Proc. Nat. Acad. Sci., 87:142-146 (1990).
Sano, T., Cantor, C.R. Expression Vectors for Streptavidin-containing Chimeric Proteins,Bioch. Biophys. Res. Comm., 176:571-577 (1991).
Hamer, D.H., Metallothionein,Ann. Rev. Biochem., 55:913-51 (1986).
Sano, T., Cantor, C.R., Cooperative Biotin Binding by Streptavidin,J. Biol. Chem., 265: 3369-3373 (1990).
Bayer, E.A., Ben-Hur H., Wichek, M., Isolation and Properties of Streptavidin,Meth. Enzym., 184: 80-89 (1990).
Green, M.N., Avidin and Streptavidin,Meth. Enzym., 184: 51-67 (1990).
Kagi, J.H.R.; Schaffer, A. Biochemistry of Metallothionein,Biochemistry, 27:8509-8515 (1988).
Cantor Charles R.
Glazer Alexander N.
Sano Takeshi
Osman Richard Aron
Romeo David S.
The Regents of the University of California
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