Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2001-08-24
2003-10-07
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S212000, C435S226000, C530S412000, C530S416000, C530S417000, C530S856000, C514S002600
Reexamination Certificate
active
06630139
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the purification and use of a novel family of fibrinogenolytic proteases. Specifically, this invention relates to a fibrinogenolytic protease which possesses a strong beta-fibrinogenolytic activity and does not cause formation of fibrin clots, known effects associated with thrombin and thrombin-like proteases of snake venom. In addition, the fibrinogenolytic proteases of the present invention exhibits anti-clotting and antihypertensive effects on experimental animals.
2. Description of the Related Art
Venoms from various snake species alter the haemostatic and blood coagulation systems of human victims or experimental animals in a complex manner. Different venoms contain multiple components which behave as either pro- or anti-coagulants that directly or indirectly induce or inhibit fibrinogen and/or platelet aggregation and related complex biochemical processes, resulting in common clinical complications of blood clotting or uncontrolled hemorrhage by envenomation of snakebites [Ouyang, C. (1957) J. Formosan Med. Assoc. 56, 435-448; Meaume, J. (1966) Toxicon 4, 25-58; and Kini, R. M., and Evans, H. J. (1990) Toxicon 28, 1387-1422.] These apparently contradicting activities have been attributed to the presence of fibrinogenolytic or fibrinogen clotting enzymes in snake venoms [Brinkhous, K. M., and Smith, S. V. (1988) in Hematology, Haemostasis and Animal Venoms (Pirkle, H. and Markland, F. S., Jr., Eds.) Vol. 7, pp. 363-375, Marcel Dekker, New York; Stocker, K. F. (1990) in Medical Use of Snake Venom Proteins (Stocker, K. F., Ed.) pp. 97-160, CRC Press Boston, Mass.; and Tu, A. T. (1982) In Rattlesnake Venoms: Their Actions and Treatment (Tu, A. T., Ed.), pp. 247-312, Marcel Dekker, New York.] On the other hand, platelet-aggregating enzymes in venom generally lack fibrinogenolytic activity, but can directly aggregate platelets in platelet-rich plasma [Serrano, S. M. T., Mentele, R., Sampaio, C. A. M., and Fink, E. (1995) Biochemistry 34, 7186-7193.] Current interest is directed to some fibrinolytic proteinases including metalloproteinases and thrombin-like enzymes because of their potential clinical application in the treatment of vascular thrombotic diseases [Markland, F. S. Jr. (1998) Thromb. Haemost. 79, 668-674.]
It is well known that snake venoms contain complex mixtures of pharmacologically active peptides and proteins. Reptilian venoms, particularly those obtained from the snake families of Crotalidae and Viperidae, are also shown to possess many different fibrinogenolytic proteases which may initiate or affect blood coagulation process associated with snakebites [Tu, A. T. (1982) In Rattlesnake Venoms: Their Actions and Treatment (Tu, A. T., Ed.), pp. 247-312, Marcel Dekker, New York.] Different researchers reported disparate proteases from venoms of various crotalid snakes. They included crotalase, a thrombin-like enzyme isolated from the American-Eastern diamondback rattlesnake (
Crotalus adamanteus
) [Markland, F. S., and Damus, P. S. (1971) J. Biol. Chem. 246, 6460-6473], hemorrhagic toxins, anticoagulant proteases and kallikrein-like enzymes from the American-Western diamondback rattlesnake
Crotalus atrox
[Pandya, B. V., and Budzynski, A. Z. (1984) Biochemistry 23, 460-470; Bjarnason, J. B., Barish, A., Direnzo, G. S., Campbell, R., and Fox, J. W. (1983) J. Biol. Chem. 258, 12566-12573.] Applicant has previously evaluated the venom components from
Crotalus atrox
and found that all fractions isolated from the anion-exchange chromatography showed varying extents of specific proteolytic activity against alpha-and/or beta-chains of fibrinogen molecules [Chiou, S. -H., Hung, C. -C., and Lin, C. -W. (1992) Biochem. International 26, 105-112; Chiou, S. -H., Hung, C. -C., and Huang, K. -F. (1992) Biochem. Biophys. Res. Commun. 187, 389-396.] Concurrently, studies on the toxin components from Taiwan habu (
Trimeresurus mucrosquamatus
) [Ouyang, C., and Teng, C. M. (1976) Biochim. Biophys. Acta 420, 298-308; Huang, K. -F., Hung, C. -C., and Chiou, S. -H. (1993) Biochem. Mol. Biol. International 31, 1041-1050; and Hung, C. -C., Huang, K. -F., and Chiou, S. -H. (1994) Biochem. Biophys. Res. Commun. 205, 1707-1715.], a major and abundant crotalid species in Taiwan, indicated several kinds of fibrinogenases present in this phylogenetically related species to those American rattlesnakes.
Concerning the pharmacological action of Formosan snake venoms on blood coagulation, it was reported early in 1921-1925 that the crude venoms of two crotalid snake species,
Agkistrodon acutus
and
Trimeresurus gramineus,
had a coagulant action on whole blood and plasma, while the venom of another species
Trimeresurus mucrosquamatus
of the same family showed an inhibitory action Ouyang, C. (1957) J. Formosan Med. Assoc. 56, 435-448. The inhibitory action on blood coagulation was believed to be caused mostly by destruction of fibrinogen in the case of the venom of
Trimeresurus mucrosquamatus.
As one object of the present invention, applicant discloses herein a family of fibrinogenases isolated from Taiwan habu, which show a specific fibrinogen-degrading activity without being associated with any activity causing fibrin clot formation. In addition, the type of fibrinogenases posesses an unexpectedly strong kallikrein-like hypotensive activity on experimental rats and may find their clinical applications in hypertension therapy.
Another object of the present invention is to develop a new expression process to produce snake venom proteins, including those identified fibrinogenases of the present invention. Expression and purification of recombinant proteins from host organisms are often a critical and time-consuming task in achieving the goal of obtaining pure and large quantities of proteins from recombinant sources [Uhlen, M., and Moks, T. (1990) Methods Enzymol. 185, 129-143.] Facile removal of contaminant expression proteins is essential to accurate characterisation of functional properties of a cloned protein. The most common solution to this problem is to engineer the expressed protein product so as to contain additional amino acid residues which give a unique property to the protein of interest that can be exploited for purification purposes. Such a strategy would greatly increase the availability of recombinant proteins for further structural and functional study. The protein with those additional amino acid residues can generally bind to transition metal ions, thereby allowing the protein to be purified using immobilized metal ion affinity chromatography (“IMAC”) [Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Nature 258, 598-599.] In this method a specific chelating peptide can be cloned onto the amino terminus (“N-terminus”) of a recombinant protein to serve as a purification tag or handle [Smith, M. C., Furman, T. C., Ingolia, T. D., and Pidgeon, C. (1988) J. Biol. Chem. 263, 7211-7215] and subsequently purified by using IMAC.
Recent advance in recombinant DNA technology has allowed in vitro fusion of genes or gene fragments in a simple and predictable manner. There are several reasons to use gene fusion for expression of recombinant proteins in heterologous hosts. In particular, a more reliable and reproducible method to obtain a native protein might be to use in vitro cleavage of the fusion protein, as compared to in vivo removal of the formyl-methionine or cleavage of a signal peptide, which in both cases may yield a heterogeneous N-terminus. Many expression vectors currently used to encode, encoding a protease cleavage site that allows release of carboxyl-terminal (“C-terminal”) fusion partners from fusion proteins without leaving unwanted amino-terminal (“N-terminal”) amino acid residues behind. In snake venom protein expression system, removal of the upstream fusion partners has been conducted with one of the three different proteases, including thrombin (Maed
Academia Sinica
Cohen & Pontani, Lieberman & Pavane
Prouty Rebecca E.
Swope Sheridan L.
LandOfFree
Fibrinogenolytic proteases with thrombolytic and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Fibrinogenolytic proteases with thrombolytic and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Fibrinogenolytic proteases with thrombolytic and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3141687