Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Patent
1995-12-07
2000-02-15
Priebe, Scott D.
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
800 8, 800 4, 800 7, 800 23, 800 25, C12N 500, C12N 1500, A01K 6700, C12P 2100
Patent
active
060255404
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a mammalian expression system comprising a DNA sequence encoding human extra cellular superoxide dismutase (EC-SOD) or encoding a variant of said protein having the superoxide dismutating property of native extracellular superoxide dismutase by means of production in transgenic non-human mammals, and to a method of producing a transgenic mammal capable of expressing EC-SOD or a variant thereof. The present invention furthermore relates to a genomic DNA sequence advantageously used in the production of recombinant human EC-SOD or a variant thereof.
Organisms living in the presence of oxygen have been forced to develop a number of protective mechanisms against toxic oxygen reduction metabolites, such as superoxide radicals, which are formed in connection with a variety of biological oxidations. The protective factors include superoxide dismutases (SOD) which dismutate the superoxide radical and are found in relatively constant amounts in mammalian cells and tissue. The secretory extracellular superoxide dismutase (EC-SOD) is one of three different SOD isoenzymes present in mammals [1]. It is the major SOD isoenzyme in plasma, lymph and synovial fluid [2-5], but exists primarily in the interstitial space of tissues [6-8]. The other two isoenzymes are the dimeric CuZn-SOD, which is an intracellular cytosolic enzyme and the tetrameric Mn-SOD, which is found in the mitochondrial matrix [9-11]. The human EC-SOD cDNA has been isolated and sequenced [12] and the recombinant protein has been produced in mammalian cells (WO 87/01387).
The DNA sequence shown in SEQ ID NO:1 is the part of the cDNA sequence corresponding to amino acid residues 1-222. EC-SOD is a secretory protein and the complete cDNA also encodes an 18 amino acids long signal sequence which is absent in mature and recombinant EC-SOD. [12] As deduced from the cDNA sequence, the subunit molecular weight of the mature enzyme is 24,200. The exact size of the carbohydrate substituent is not known, but the apparent molecular weight on gel chromatography is 140-150 kDa. EC-SOD is a tetrameric Cu and Zn containing glycoprotein. On SDS-PAGE electrophoresis the sub-units display a molecular weight of 30-32 kDa. The sequence contains one glycosylation site Asn-89. The tetramers contain 4 Cu and 4 Zn atoms, in that each subunit binds one copper and one zink atom. The active site, which contains the metal atoms, is homologous to the active site of the intracellular CuZn SODs.
The part of the EC-SOD constituting amino acid sequence 1-96 is contemplated to be involved in the formation of oligomers of the polypeptide whereas the part of EC-SOD constituting amino acid sequence 97-193 is contemplated to comprise the active site of the enzyme.
The glycan of the native protein has not been subject for any thorough investigation but the mature enzyme binds to the lectins concanavalin A, wheat germ lectin and lentil lectin. A fundamental and distinguishing property of EC-SOD is its affinity for some glycosaminoglycans such as heparin and heparan sulfate [1, 3, 4, 16, 17]. The latter, which exists in the glycocalyx of cell surfaces and in the connective tissue matrix, is the important physiological ligand of EC-SOD. Because of this affinity, EC-SOD forms in the vasculature an equilibrium between the plasma phase and the glycocalyx of the endothelium [3, 4, 15, 18]. In tissues, virtually all EC-SOD exists anchored to heparan sulfate in the interstitial space and in the cell surfaces [8].
Upon intravenous injection, EC-SOD rapidly binds to the surface of the endothelium and it displays a long half-life (15-20 hours) in the vasculature [8, 15]. When administered parenterally, CuZn-SOD has been shown to exert a host of therapeutic actions [21-25]. The experience with EC-SOD is in comparison small [26-33] but this isoenzyme seems to be even more potent. The high efficacy is apparently related to the special pharmacokinetic properties of the enzyme.
The heparin-binding domain of the enzyme is located to the positively charged carboxyterminal end [1
REFERENCES:
Oury, Tim D. et al., "Cold-induced brain edema in mice: Involvement of extracellular superoxide dismutase and nitric acid." The Journal of Biological Chemistry, vol. 268, No. 21, pp. 15394-15398 (1993).
Oury, Tim D. et al., "Extracellular superoxide dismutase, nitric oxide, and central nervous system O.sub.2 toxicity." Proc. Natl. Acad. Sci., vol. 89, pp. 9715-9719 (Oct. 1992).
Hennighausen, Lothar, "The mammary gland as a bioreactor: Production of foreign proteins in milk." Protein Expression and Purification, vol. 1, pp. 3-8 (1990).
Cooper Iver P.
Priebe Scott D.
Wilson Michael C.
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