Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1995-01-19
2001-12-18
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C536S023100, C536S023200
Reexamination Certificate
active
06331421
ABSTRACT:
DESCRIPTION OF SPECIFIC EMBODIMENTS
1. Field of the Invention
Superoxide dismutase (“SOD”) is in fact a variety of different enzymes found in most living organisms. One function in mammals is to destroy superoxide, a material naturally produced during phagocytosis. The superoxide dismutases are characterized in families based on the metal associated with the enzyme, where the metals vary amongst iron, manganese, copper and copper-zinc. Superoxide dismutase, e.g., from bovine liver, has found clinical use, particularly as an anti-inflammatory agent in mammals including humans. Other utilities include scavenging superoxide anions due to exposure of a host to various superoxide-inducing agents, e.g. radiation, paraquat, etc.; prophylaxis or therapy for certain degenerative diseases, e.g., emphysema; food preservation; and the like.
It is therefore important that stable supplies of physiologically acceptable superoxide dismutase be made available, particularly for use in vivo as an anti-inflammatory agent or for other therapeutic purposes. For human application it would be preferable to employ the homologous enzyme to prevent or minimize possible immune response. By employing recombinant DNA techniques, there is the opportunity to produce products efficiently, which have the desired biological activities of superoxide dismutase, such as immunological and enzymatic activities.
2. Description of the Prior Art
The amino acid sequence of human erythrocyte Cu—Zn superoxide dismutase is described in Jabusch et al.,
Biochemistry
(1980) 19:2310-2316 and Barra et al.,
FEBS Letters
(1980) 120:53-55. Bovine erythrocyte Cu—Zn SOD is described by Steinman et al.,
J. Biol. Chem
. (1974) 249:7326-7338. A SOD-1 cDNA clone is described by Lieman-Hurwitz et al.,
Proc. Natl. Acad. Sci. USA
(1982) 79:2808-2811. Concerning the effect on efficiency of translation of varying the untranslated region upstream from the initiation codon, see Gheysen et al.,
Gene
(1982) 17:55-63; Thummel et al.,
J. Virol
. (1981) 37:683-697; and Matteucci and Heyneker,
Nucl. Acids Res
. (1983) 11:3113-3121.
SUMMARY OF THE INVENTION
Efficient production of polypeptides demonstrating the biological activity of human Cu—Zn superoxide dismutase is demonstrated by the preparation of cDNA of the major portion of the structural gene, linking to a mixture of adapters providing for varying sequences extending from the ribosomal binding site to degenerate nucleotides in the coding region, and insertion of the complete gene with its translational signals into an expression vector. Transformation of microorganisms results in efficient production of a competent polypeptide demonstrating biological activity of human Cu—Zn superoxide dismutase. The gene may be further used for combining with secretory and processing signals for secretion in an appropriate host.
Novel protocols are provided for enhancing expression of a polypeptide involving the use of mixtures of adapters having varying sequences flanking the initiation site for translation, i.e., in the region between the ribosomal binding site and translational initiation site and in the initial several 5′-codons of the polypeptide, where permitted by redundancy constraints of the genetic code.
Polypeptides acetylated at their N-terminus and methods for producing such acetylated polypeptides are also provided. By providing a particular acetylation signal sequence at the 5′-end of the structural gene for a desired polypeptide, the N-terminal amino acid will be acetylated when the gene is expressed in yeast. The acetylation signal sequence encodes for at least the first two N-terminal amino acids, where the first amino acid is either alanine or glycine, and the second amino acid is a polar amino acid, usually being threonine, serine or aspartate. Acetylation of human superoxide dismutase produced in yeast is demonstrated when the first two amino acids are alanine and threonine, respectively.
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Hallewell Robert A.
Mullenbach Guy T.
Alexander Lisa E.
Blackburn Robert B.
Chiron Corporation
Nashed Nashaat T.
Robins Roberta L.
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