Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1994-05-03
2001-11-13
Sayala, Chhaya D. (Department: 1761)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S350000
Reexamination Certificate
active
06316602
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the discovery of non-toxic beta taipoxin as a cell growth factor and a potent mitogen from poisonous snake venom. Said composition consists of a polypetide of beta taipoxin having molecular weight approximately 14,000 daltons and is free of toxic effects.
BACKGROUND OF THE INVENTION
Taipoxin as a whole intact molecule isolated from the venom of the Australian taipan
Oxyuranus s. scutellatus
is the most lethal neurotoxin. The whole molecule of taipoxin is a complex, composed of three subunits designated as alpha, beta and gamma, having molecular weight 45,6000 daltons. Taipoxin can not be isolated by ion-exchange chromatography, since ion exchangers tend to dissociate the active toxin complex. The major lethality of taipoxin is due to the very basic alpha subunit. The molecular weights of alpha, beta and gamma subunits of taipoxin are 13,750, 13,473 and 18,354 daltons respectively.
To date numerous growth factors have been isolated from various sources, and have been characterized. To name a few, epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF) and platelet derived growth factor (PDGF).
It is an object of this invention that the beta taipoxin as a cell growth factor can be used to grow cells in serum free medium. Routinely, cells are grown in the presence of 5 to 20% fetal bovine serum (FBS) for research or production. The purification of products derived from cells grown in serum containing medium is a cumbersome task and furthermore, fetal bovine serum is the most expensive ingredient of the medium. A mitogen like cell growth factor will provide serum free environment with adequate cell proliferation.
A further object of this invention is to provide non-toxic beta taipoxin having mitogenic activity as a composition for the promotion of rapid bum and wound healing. In the case of cuts, surgical incisions, and abrasions to lessen the risk of infection and to shorten recovery time.
SUMMARY OF THE INVENTION
Cell growth factor has been isolated as the beta subunit of taipoxin by fractionating
Oxyuranus s. scutellatus
snake venom by high pressure liquid chromatography. After testing for mitogenic activity in all fractions, the mitogenic activity was revealed in one of the fractions. Cell growth factor is a beta taipoxin, which is a non-toxic fraction of snake venom and is a potent mitogen. Cell growth factor has been isolated from the venom of the poisonous snake species
Oxyuranus s. scutellatus.
The concentration of 0.1 &mgr;/ml of the beta taipoxin non-toxic subunit becomes a cell growth factor and in serum free medium it gives growth equivalent to medium containing 10% serum for a wide range of cells. Cell growth factor is a beta taipoxin peptide having a molecular weight 14,000 daltons.
In vivo experiments proved that beta taipoxin as a cell growth factor acts as a potent mitogen. The cut portion of mouse hip skin healed much faster when cell growth factor was applied as compared to the control counter part. Cell growth factor helped heal a chronic foot wound of a friend. Cell growth factor is claimed as wound healer and its use may be extended to treat burns.
DETAILED DESCRIPTION OF THE INVENTION
The cell growth factor consists essentially of a peptide of which first 15 amino acid sequence is: Asp-Leu-Val-Glu-Phe-Gly-Lys-Met-Ile-Glu-Cys-Ala-Ile-Arg-Asn, which is exactly similar to beta taipoxin. For convenience this sequence is referred herein as SEQ ID No: 1. It is believed that any peptide having the partial amino acid sequence SEQ ID No: 1 exhibits substantial utility as a cell growth promoter, a potent mitogen, regardless whether it is synthesized or derived from natural sources. By the term, cell growth factor, we mean a substance whose presence produces a substantial mitogenic effect on various types of cells, and that the mitogenic effect is indicated, by an increase in cell growth or by a decreased duplication time of various types of cells.
Preferably, the peptide cell growth factor contains the first fifteen amino acids at N-terminal as given by SEQ ID No: 1, and has a molecular weight of about 14,000 daltons revealed by electrophoresis which is similar to beta taipoxin. In addition, cell growth factor is water soluble and stable at 4° C. storage for its biological activity. Cell growth factor is stable at room temperature, 74° C. for several weeks and its biological mitogenic activity is not altered by exposing it to ultra violet light overnight.
Cell growth factor, beta taipoxin, may be obtained essentially as a fraction of venom, from species of poisonous snake. Cell growth factor is preferably obtained from the venom of a species of Australian taipan snake, particularly the species
Oxyuranus s. scutellatus.
The non-toxic beta taipoxin which is an active cell growth factor is obtained by separating the peptide fraction by, high pressure liquid chromatography, using ion exchange chromatography.
Fractionation of Venom: The active cell growth factor, a non-toxic beta taipoxin is preferably separated from fresh frozen venom, although lyophilized whole venom may also be used. The liquid venom is diluted 1:1 with 0,01 M phosphate buffer saline (PBS) and preferably centrifuged to sediment insoluble debris, which can also be removed by filtration. Typically, the diluted and centrifuged 50 mg venom is loaded on high pressure liquid chromatography, from Toso Co. Japan and the ion exchange column from Polymer Laboratories UK, maintained at 20° C. temperature. A plurality of fractions according to the relative ionic charge are eluted preferably, using gradient Trizma 2-amino-2-(hydroxymethyl)propane-1,3-diol-HCl buffer pH 7.3. The Toso high pressure liquid chromatography automatically mixes 1.0 molar Trizma-HCl buffer with water to yield gradient Trizma-HCl buffer from 0.01 molar to 1.0 molar. Any suitable gradient buffer may be used and Trizma-HCl buffer having pH 6.0 to 8.0 can be used.
REFERENCES:
Harris et al., “Protein purification methods”, IRL Press, pp. 175-215, 1989.*
Siigur et al., Comp. Biochem. Physiol., vol. 83B(3), pp 621-25, 1986.*
Banks et al., Biochem J., vol. 108, pp. 157 to 158, 1968.*
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Scopes, “Protein Purification”, Third Edition, Springer-Verlag pp. 154-158, 1993.*
Fohlman et al., Eur. J. Biochem., vol. 68, pp. 457-469 1976.*
Lind, Eur. J. Biochem, vol. 128(1), p. 71-75, 1982.*
J. Fohlman, D. Eaker, E. Karlsson and S. Thesleff, Taipoxin, an Extremely Potent Presynaptic Neurotoxin from the Venom of the Australian Snake Taipan, Eur. J. Biochem 68, 457-469 (1976).
P. Lind, Amino-Acid Sequence of the B1 Isosubunit of Taipoxin . . . Eur. J. Biochem, 128; 71-75 (1982).
G. S. Schultz et al., Epithelial Wound Healing Enhanced by Transforming Growth Factor, Science, 235, 350-352, (1987).
A. Wells, J.B. Welsh, C.S. Lazar, H.S. Wiley, G.N. Gili & M.G. Rosenfeld, Ligand-Induced Transformation by a Noninternalizing Epidermal Growth Factor Receptor, Science, 247, 962-964, (1990).
A. Buckley, J.M. Davidson, C.D. Kamerath, T.B. Wolf & S.C. Wooward, Release of Epidermal Growth Factor Accelerates Repair, Proc. Natl Acad Sci. USA 82, 7340-7344, (1985).
J.F. Rubin et al., Purification & Characterization of a Newly Identified Growth Factor Specific for Epithelial Cells, Proc. Natl. Acad. Sci. USA 86, 802-806 (1989).
G.L. Brown et al., Enhancement of Epidermal Regeneration by Biosynthetic Epidermal Growth Factor, J.Exp. Med. The Rockefeller University Press, 163, 1319-1324, (1986).
Casperson John R
Sayala Chhaya D.
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