Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-10-06
2001-10-23
Nashed, Nashaat T. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023200, C424S094100, C435S252320, C435S320100
Reexamination Certificate
active
06307038
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polypeptide expression systems requiring cleavage of a precursor product, and to proteases for use in such systems. The present invention further relates to a novel polypeptide capable of reducing dichloroindophenol and oxidized glutathione, DNA encoding the novel polypeptide, vectors containing such DNA, hosts transformed with such vectors, and pharmaceutical compositions containing the polypeptide. In addition, the present invention also relates to monoclonal antibodies against this polypeptide, and a process for isolating and purifying the polypeptide using such an antibody.
PRIOR ART
The Potyviruses are a group of viruses each of which have a single-stranded, RNA genome of approximately 10,000 bases and which infects plants such as the family Solanaceae. The Potyvirus genome is characterized by possessing one extremely long open reading frame, or ORF, [Dougherty, W. G. and Hiebert, E. (1980), Virology 101: 466-474.; Allison, R. et al. (1986), Virology 154: 9-20]. In order to express the individual proteins encoded within the ORF, the translated polyprotein is digested by two types of protease, both of which are also encoded within the ORF [Dougherty, W. G. and Carrington, J. C. (1988), Ann. Rev. Phytopath. 26: 23-143].
Tobacco etch virus (TEV) is a member of the Potyvirus family, and this virus produces nuclear inclusions which can be stained with trypan blue in the infected cell. The nuclear inclusions apparently consist of two kinds of protein, one of which has proven to be a viral protease, and which has been designated Nuclear Inclusion a, or NIa [J. Virol., 61: 2540-2548 (1987)].
The Nuclear Inclusion a proteases of the Potyviruses recognize and cleave a peptide sequence which includes one of Gln-Gly, Gln-Ser and Gln-Ala, and it is believed that this sequence is hexameric and occurs at the C-terminal end of the relevant NIa within the polyprotein. Cleavage is between the two residues making up the dimers shown above.
The complete genomic sequences of TEV and tobacco vein mottling virus (TVMV), another member of the Potyvirus family, have been determined, and homology searching has allowed the location of the NIa's of these viruses within their respective genomes [Virology, 154: 9-20 (1986); Nucleic Acid Res., 14: 5417-5430 (1986)].
Clover Yellow Vein Virus, or CYVV, is also a Potyvirus. So far, only the gene occurring at the 3′ end of the CYVV genome, together with the coat protein it encodes, has been sequenced [Uyeda, I. et al. (1991), Intervirol. 32: 234-245]. The structure of NIa region of the genome has not previously been elucidated, nor has the corresponding NIa been isolated.
The production of exogenous proteins by expression systems can be straightforward, using techniques well known in the art. However, there are many polypeptides which cannot easily be expressed in an exogenous system. The problem may be that the polypeptide cannot be expressed in large amounts, and this cannot usually be corrected merely by placing a regulatory gene upstream. Alternatively, it may be that post-transcriptional events required to obtain the mature form do not take place, or take place incorrectly.
For example, many eukaryotic polypeptides are initially translated with an N-terminal methionine which is subsequently deleted to obtain the mature form. This processing cannot take place in prokaryotes, so that alternative means of obtaining expression have had to be found. One such technique involves fusing the desired exogenous protein with maltose-binding protein or glutathione S-transferase, for example, purifying the expressed fusion protein and then cleaving with a protease, such as Factor Xa, enterokinase, or thrombin. The main drawback of this cumbersome method is that it requires two purification steps, which results in a substantial loss of the end product.
U.S. Pat. No. 5,162,601 discloses the use of TEV protease in the manufacture of a polyprotein having linker sequences between each of the proteins it is desired to express, such as human tPA. However, this patent only discloses the cloning of a multigene encoding this polyprotein into a host. There is no disclosure of expression or purification of the proteolytically cleaved end product.
Oxygen for metabolic energy is generally provided in the form of oxidizing agents in the cellular environment. The activated form in which the oxygen is used is generally as a free radical, such as superoxide (O
2
−
), peroxide (H
2
O
2
) or a hydroxy radical (OH
−
), all of which are reduced to form water (H
2
O) after use. Oxygen gas, itself, is highly oxidizing, but the term “activated oxygen”, as used herein, relates to oxygen and oxygen-containing molecules which have greater oxidizing potential than atmospheric oxygen. The most potent form of activated oxygen is the free radical, which is a molecule or atom having one or more unpaired electrons.
Free radicals are typically unstable and, if not properly controlled, can denature lipids, proteins and nucleic acids. Consequently, although activated oxygen is essential to life, it is also a potential health hazard, and must be very closely controlled. Even in vanishingly small amounts, activated oxygen can cause disorders, due to high reactivity. As a result, living organisms are unable to survive unless they are equipped with a defense mechanism against activated oxygen.
In the cellular environment, the locations, amounts and times of generation of activated oxygen must be carefully balanced against the cell's ability to neutralize the associated danger. This ability is typically provided by a defense mechanism that uses its own antioxidants or antioxidation enzymes. In the context of the present invention, an “antioxidant” is the generic name for a naturally occurring substance which is able to prevent or inhibit the auto-oxidation of lipids, for example. The term “antioxidation enzyme” is used generically for an enzyme which catalyzes a reaction which eliminates activated oxygen, the term “antioxidative action” being construed accordingly.
Excessive amounts of activated oxygen are produced in a number of abnormal circumstances, such as when a person is stressed, is taking drugs, smokes, undergoes surgery, has an organ transplant or if he suffers ischemia through a cerebral or myocardial infarction. These large amounts are more than the control systems of the body can eliminate, so that the excess of activated oxygen can cause further damage to the body, seriously impairing normal cells. The resulting, so-called oxidative stress is responsible for a great many disease conditions.
To take arteriosclerosis as an example, the occurrence of low specific gravity lipoproteins which have been oxidized by activated oxygen is considered to be one of the causes of the disease [Steinberg, D. (1983,) Arteriosclerosis 3, 283-301]. Oxidative stress is also considered to be intimately involved with cause and effect in the mechanisms associated with the occurrence, metabolic abnormalities and vascular complications of diabetes [Kondo, M. ed., “Approaches from Modern Medicine (4) Free Radicals”, Medical View Pub., pp. 138-146].
Activated oxygen is also implicated in other pathological states and conditions, such as; ischemic disorders (reperfusion disorders, ischemic heart disease, cerebroischemia, ischemic enteritis and the like), edema, vascular hyperpermeability, inflammation, gastric mucosa disorders, acute pancreatitis, Crohn's disease, ulcerative colitis, liver disorders, Paraquat's disease, pulmonary emphysema, chemocarcinogenesis, carcinogenic metastasis, adult respiratory distress syndrome, disseminated intravascular coagulation (DIC), cataracts, premature retinopathy, auto-immune diseases, porphyremia, hemolytic diseases, Mediterranean anemia, Parkinson's disease, Alzheimer's disease, epilepsy, ultraviolet radiation disorders, radioactive disorders, frostbite and burns.
Several defence mechanisms exist both inside an
Kawashima Ichiro
Koishi Ryuta
Serizawa Nobufusa
Takahashi Tohru
Frishauf, Holtz Goodman, Langer & Chick, P.C.
Fronda Christian L
Nashed Nashaat T.
Sankyo Company Limited
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