IE59498B1

IE59498B1 - Human manganese superoxide dismutase analog, plasmid for its expression and method of recovering it in enzymatically active form

Info

Publication number: IE59498B1
Application number: IE285186A
Authority: IE
Original assignee: Bio Technology General Corp
Priority date: 1985-11-22
Filing date: 1986-10-29
Publication date: 1994-03-09
Also published as: ATA308986A; IL106449A; IL106449A0; CH676990A5; JP2609449B2; SE8604884D0; FI864756A7; FR2590591B1; SE8604884L; IT8667865A0; DE3639725C2; GB2183658A; GB8627294D0; FI90353B; HK75195A; AT400443B; AU607897B2; PT83792A; DK534686D0; NL8602960A

Abstract

A double-stranded cDNA molecule which includes DNA encoding human manganese super oxide dismutase has been created. The sequence of one strand of a double- stranded DNA molecule which encodes human manganese superoxide dismutase has been discovered. Such mole- cules may be introduced in procaryotic, e.g., bacteri- al, or eukaryotic, e.g. , yeast or mammalian, cells and the resulting cells cultured or grown under suitable conditions sa as to produce human manganese superoxide dismutase or analogs thereof which may then be recov- ered. Human MnSOD or analogs thereof may be used to catalyze the reduction of superoxide radicals, reduce reperfusion injury, prolong the survival time of iso- lated organs, or treat inflammations. The invention also concerns a method of producing enzy- matically active human manganese superoxide dismutase or an analog thereof in a bacterial cell which contains and is capable of expressing a DNA sequence encoding the superoxide dismutase by maintaining the bacterial cell under suitable conditions and in a suitable pro- duction medium. The production medium is supplemented with an amount of Mn++ so that the concentration of Mn++ in the medium is greater than about 2 ppm. This invention also concerns a method of recovering purified enzymatically active manganese super oxide dismutase from bacterial cells.

Description

This invention relates to an analog of human manganese superoxide dismutase and to the expression and recovery of enzymatically active forms of that analog.

Throughout this specification, various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are referred to in this application in order to more fully describe the state of art as known to those skilled therein as of the date of the invention described and claimed herein.

Superoxide dismutase (SOD) and the phenomenon of oxygen free radicals (0^-) was discovered in 1968 by McCord and Fridovich (1) . Superoxide radicals and other highly reactive oxygen species are produced in every respiring cell as by-products of oxidative metabolism, and they have been shown to cause extensive damage to a wide variety of macromolecules and cellular components (for review see 2,3). A group of metalloproteins known as superoxide dismutases catalyze the oxidation-reduction reaction 20^- + 2H* -h2°2 + °2 an<3 thus provide a defense mechanism against oxygen toxicity.

There are several known forms of SOD containing different metals and different proteins. Metals present in SOD include iron, manganese, copper and zinc. All of the known forms of SOD catalyze the same reaction.

These enzymes are found in several evolutionary groups.

Superoxide dismutases containing iron are found primarily in prokaryotic cells. Superoxide dismutases containing copper and zinc has been found in virtually all eukaryotic organisms (4). Superoxide dismutases containing manganese have been found in organisms ranging from microorganisms to man.

Since every biological macromolecule can serve as a target for the damaging action of the abundant superoxide radical, interest has evolved in the thera15 peutic potential of SOD. The scientific literature suggests that SOD may be useful in a wide range of clinical applications. These include prevention of oncogenesis and of tumor promotion, and reduction of the cytotoxic and cardiotoxic effects of anticancer drugs (10) , protection of ischemic tissues (12) and protection of spermatozoa (13). In addition, there is interest in studying the effect of SOD on the aging process (14).

The exploration of the therapeutic potential of human SOD has been limited mainly due to its limited availability.

Superoxide dismutase is also of interest because of its anti-inflammatory properties (11). Bovine-derived superoxide dismutase (orgotein) has been recognized to possess anti-inflammatory properties and is currently marketed in parts of Europe as a human pharmaceutical. It is also sold in the United States as a veterinary product, particularly for the treatment of inflamed tendons in horses. However, supplies of orgotein are limited. Prior techniques involving recovery from bovine or other animal cells have serious limitations and the orgotein so obtained may produce allergic reactions in humans because of its non-human origin.

Copper zinc superoxide dismutase (CuZn SOD) is the most studied and best characterized of the various forms of superoxide dismutase.

Human CuZn SOD is a dimeric metallo-protein composed of identical non-covalently linked subunits, each having a molecular weight of 16,000 dal tons and containing one atom of copper and one of zinc (5). Each subunit is composed of 153 amino acids whose sequence has been established (6,7).

The cDNA encoding human CuZn superoxide dismutase has been cloned (8). The complete sequence of the cloned DNA has also been determined (9). Moreover, expression vectors containing DNA encoding superoxide dismutase for the production and recovery of superoxide dismutase in bacteria have been described (24,25). The expression of a superoxide dismutase DNA and the production of SOD in yeast has also been disclosed (26) .

Recently, the CuZn SOD gene locus on human chromosome 21 has been characterized (27) and recent developments relating to CuZn superoxide dismutase have been summarized (28) .

Much less is known about manganese superoxide dismutase (MnSOD). The MnSOD of E. coli K-12 has recently been cloned and mapped (22). Barra et al. disclose a 196 amino acid sequence for the MnSOD polypeptide isolated from human liver cells (19). Prior art disclosures differ, however, concerning the structure cf the MnSOD molecule, particularly whether it has two or four identical polypeptide subunits (19,23). It is clear, hcw3 ever, that the MnSOD polypeptide and the CuZn SOD polypeptide are not homologous (19) . The amino acid sequence homologies of MnSODs and FeSOD from various sources have also been compared (18).

Baret et al. disclose in a rat model that the half life of human MnSOD is substantially longer than the halflife of human copper SOD; they also disclose that in the rat model, human MnSOD and rat copper SOD are not effective as anti-inflammatory agents whereas bovine 13 copper SOD and human copper SOD are fully active (20).

McCord et al. disclose that naturally occurring human manganese superoxide dismutase protects human phagocytosing polymorphonuclear (PMN) leukocytes from superoxide free radicals better than bovine or porcine CuZn superoxide dismutase in in vitro tests (21) .

The present invention concerns the preparation of a cDNA molecule encoding an analog of human manganese superoxide dismutase polypeptide. It is also directed to inserting this cDNA into efficient bacterial expression vectors, to producing human MnSOD polypeptide analog and enzyme in bacteria, to recovering the bacterially produced human MnSOD polypeptide analog or enzyme. This invention is also * directed to the human MnSOD polypeptide analogs so recovered and their uses.

This invention further provides a method for producing enzymatically active analog of human MnSOD in bacteria, as well as a method for recovering and purifying such enzymatically active human MnSOD analog.

The present invention also relates to using human manganese superoxide dismutase analogs to catalyze the reduction of superoxide radicals to hydrogen peroxide and molecular oxygen. In particular, the present invention concerns using bacterially produced MnSOD analogs to reduce reperfusion injury following ischemia and prolong the survival period of excised isolated organs.

In accordance with a first aspect of the invention, therefore, there is provided a plasmid for expression of an enzymatically active human manganese superoxide dismutase analog wherein the analog consists essentially of at least two polypeptides each comprising 199 amino acids, the sequence of each polypeptide having methionine at its N-terminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig. 1A and continuing to the lysine encoded by nucleotides 706-708 of Fig. IB which is the COOH terminus of the polypeptide. The invention provides also host cells which may be procaryotic or eucaryotic, into which such a plasmid has been introduced.

According to a further aspect of the invention, there is provided also an enzymatically active polypeptide analog of human manganese superoxide dismutase comprising 199 amino acids, the sequence of which has methionine at its N-terminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig. 1A and continuing to the lysine encoded by nucleotides 706-708 of Fig. IB which is the COOH terminus of the polypeptide.

Preferred features and other aspects of the invention are described in greater detail below.

A DNA molecule which includes cDNA encoding the human manganese superoxide dismutase polypeptide has been isolated from a human T-cell cDNA library, and the nucleotide sequence of a double-stranded DNA molecule which encodes human manganese superoxide dismutase polypeptide has been discovered. The sequence of one strand encoding the polypeptide is shown in Fig. 1 from nucleotide 115 downstream to nucleotide 708 inclusive. The sequence of one strand encoding the polypeptide analog to the present invention has a sequence at nucleotides 112-114 to encode methionine as the N-terminus and the sequence of nucleotides from 115 to 708 as shown in Fig. l. Other sequences encoding the analog may be substantially similar to the strand encoding the polypeptide. The nucleotide sequence of one strand of a double stranded DNA molecule which encodes a twentyfour (24) amino acid prepeptide is also shown in Fig. 1, from nucleotides number 43 through 114, inclusive.

The double-stranded cDNA molecule or any other double-stranded DNA molecule which contains a nucleotide strand having the sequence encoding the analog of human manganese superoxide dismutase polypeptide or mutant thereof may be incorporated into a cloning vehicle such as a plasmid or virus. Either DNA molecule may be introduced into a cell, either procaryotic, e.g., bacterial, or eukaryotic, e.g., yeast or mammalian, using known methods, including but not limited to methods involving cloning vehicles containing either molecule.

Preferably the cDNA or DNA encoding the analog of human manganese superoxide dismutase polypeptide or mutant thereof is incorporated into a plasmid, e.g. , pMSE-4 or pMS RB4, and then introduced into a suitable host cell where the DNA can be expressed and the human manganese superoxide dismutase (hMnSOD) polypeptide analog or mutant thereof produced. Preferred host cells include Escherichia coli, in particular £,_sail A4255 and coli A1645. The plasmid pMSE-4 in E. coli strain A4255 5 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250. The plasmid pMSARB4 may be obtained as shown in FIG. 4 and described in the Description of the Figures.

Cells into which such DNA molecules have been introduced may be cultured or grown in accordance with methods known to those skilled in the art under suitable conditions permitting transcription of the DNA into mRNA and expression of the mRNA as protein. The re15 suiting manganese superoxide dismutase protein may then be recovered.

Veterinary and pharmaceutical compositions containing human MnSOD analog or mutants thereof and suitable carriers may also be prepared. This human manganese superoxide dismutase or analogs or mutants may be used to catalyze the following reaction: 202- + 2H ,+ and thereby reduce cell injury caused by superoxide radicals.

More particularly, these enzymes or analogs or mutants thereof may be used to reduce injury caused by reperfusion following ischemia, increase the survival time of excised isolated organs, or treat inflammations.

This invention provides also a method of producing enzymatically active human manganese superoxide dismutase analog or mutant thereof in a bacterial cell. The bacterial cell contains and is capable of expressing a DNA sequence encoding the human manganese superoxide dismutase analog or mutant thereof. The method comprises maintaining the bacterial cell under suitable conditions and in a suitable production medium. The production medium is supplemented with an amount of Mn++ so that the concentration of Mn++ available to the cell in the medium is greater than 2 ppm.

In a preferred embodiment of the invention the bacterial cell is an Escherichia coli cell containing a plasmid which contains a DNA sequence encoding for the human manganese superoxide dismutase polypeptide e.g. pMSE-4 or pMS RB4 'in E. col i strain A4255. The concentration of Mn++ in the production medium ranges from 50 to 1500 ppm, with concentrations of 150 and 750 ppm being preferred.

This invention also concerns a method of manganese superoxide dismutase analog bacterial cells which contain the same, first treated to recover a protein fraction containing proteins present in the cells including human manganese superoxide dismutase analog or mutant thereof and then the protein fraction is treated to recover human manganese superoxide dismutase analog or mutant thereof. In a preferred embodiment of the invention, the cells are first treated to separate soluble proteins from insoluble proteins and cell wall debris and the soluble proteins are recovered. The soluble proteins are then treated to separate, e.g. recovering from The cells are ίΟ precipitate, a fraction of the soluble proteins containing the hMnSOD analog or mutant thereof and the fraction containing the hMnSOD analog or mutant is recovered. The recovered fraction of soluble proteins is then treated to separately recover the human manganese superoxide dismutase analog.

A more preferred embodiment of the invention concerns a method of recovering human manganese superoxide dismutase analog or mutant thereof from bacterial cells which contain human manganese superoxide dismutase analog or mutant thereof. The method involves first isolating the bacterial cells from the production medium and suspending them in suitable solution having a pH of about 7.0 to 8.0. The cells are then disrupted and centrifuged and the resulting supernatant is heated for about 30 to 120 minutes at a temperature between 55 and 65°C, preferably for 45-75 minutes at 58-62°C and more preferably for 1 hour at 60°C and then cooled to below 10°C, preferably to 4°C. Any precipitate which forms is to be removed e.g. by centrifugation, and the cooled supernatant is dialyzed against an appropriate buffer e.g. 2 mM potassium phosphate buffer having a pH of about 7.8. Preferably, the dialysis is by ultrafiltration using a filtration membrane smaller than 3OK. Simultaneously with or after dialysis the cooled supernatant optionally may be concentrated to an appropriate convenient volume e.g. 0.03 of its original volume. The retentate is then eluted on an anion exchange chromatography column with an appropriate buffered solution e.g. a solution of at least 20 mM potassium phosphate buffer having a pH of about 7.8. The fractions of eluent containing superoxide dismutase are collected, pooled and dialyzed against about 40 mM potassium aceίΐ tate, pH 5.5. The dialyzed pooled fractions are then eluted through a cation exchange chromatography column having a linear gradient of about 40 to about 200 mM potassium acetate and a pH of 5.5. The peak fractions containing the superoxide dismutase are collected and pooled. Optionally the pooled peak fractions may then be dialyzed against an appropriate solution e.g. water or a buffer solution of about 10 mM potassium phosphate buffer having a pH of about 7.8.

The invention also concerns purified enzymatically active human manganese superoxide dismutase analog e.g. met-hMnSOD, or mutants produced by the methods of this invention.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. The Sequence of human MnSOD cDNA FIG. 1 shows the nucleotide sequence of one strand of a double-stranded DNA molecule encoding the human manganese superoxide dismutase as well as the 198 amino acid sequence of human MnSOD corresponding to the DNA sequence. FIG. 1 also shows the nucleotide sequence of one strand of a double stranded DNA molecule encoding a prepeptide to the mature human MnSOD consisting of twenty-four amino acids and the amino acid sequence corresponding to that DNA sequence. Also shown are parts of the 5' and 3' untranslated sequences.

FIG. 2. Construction of pMSE-4; Human MnSOD Expression Plasmid Plasmid pMS8-4, containing MnSOD on an EcoRI (R1) insert, was digested to completion with Ndel and Nar I restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in FIG. 2. The resulting plasmid, pMS8-NN contains the coding region for the mature MnSOD, preceded by an ATG initiation codon. The above plasmid was digested with £££RI, ends were filled in with Klenow fragment of Polymerase I and further cleaved with Ndel. The small fragment harboring the MnSOD gene was inserted into pSODa 13 which was treated with Ndel and Stul. pSODa 13 may be obtained as described in U.S. Patent No. 4,742,004. This generated plasmid pMSE-4 containing the MnSOD coding region preceded by the ell ribosomal binding site and under the control of λ P promoter.

L Plasmid pMSE-4 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250.

FIG. 3 Effect of Mn++ Concentration on the Activity of SOD.Produced in E. Coli The chart in FIG. 3 shows the correlation between the specific activity in units/mg of recombinant soluble MnSOD produced by E. coli strain A4255 containing plasmid pMSE-4 under both non-induction (32°C) and induction (42°C) conditions, and the concentration of Mn+ (parts per million) in the growth medium.

FIG. 4 Construction of pMSARB4:_Human MnSOD Expression Plasmid © Tet expression vector, pARB, was generated from pSODB^T-ll by complete digestion with EcoRI followed by partial cleavage with BamHI restriction enzymes. pSOD^T-ll has been deposited with the American Type Culture Collection (ATCC) under Accession No. 53468. The digested plasmid was ligated with synthetic oligomer '- AATTCCCGGGTCTAGATCT - 3' 3'- GGGCCCAGATCTAGACTAG - 5' resulting in pARB containing the λ PL promoter.

The EcoRI fragment of MnSOD expression plasmid pMSE-4, containing ell ribosomal binding site and the complete coding sequence for the mature enzyme, was inserted into the unique EcoRI site of pARB. The resulting plasmid, pMS£RB4, contains the MnSOD gene under control of l P_ and ell RBS and confers resistance to tetracycline.

A double-stranded DNA molecule which includes cDNA encoding human manganese superoxide dismutase polypeptide or an analog or mutant thereof has been isolated from a human T-cell cDNA library. The nucleotide sequence of a double-stranded DNA molecule which encodes human manganese superoxide dismutase polypeptide or an analog or mutant thereof has been discovered. The sequence of one strand of DNA molecule encoding the human manganese superoxide dismutase polypeptide or analog thereof is shown in Fig. 1 and includes nucleotides numbers 115 to 708 inclusive. The sequence of one strand encoding hMnSOD analog or mutant is substantially similar to the strand encoding the hMnSOD polypeptide. The nucleotide sequence of the prepeptide of human manganese superoxide dismutase is also shown in Fig. 1. Nucleotides numbers 43 through 114 inclusive code for this prepeptide.

The methods of preparing the cDNA and of determining the sequence of DNA encoding the human manganese superoxide dismutase polypeptide, analog or mutant thereof are known to those skilled in the art and are described more fully hereinafter. Moreover, now that the DNA sequence which encodes the human manganese superoxide dismutase has been discovered, known synthetic methods can be employed to prepare DNA molecules containing portions of this sequence.

Conventional cloning vehicles such as plasmids, e.g., pBR322, viruses or bacteriophages, e.g., λ, can be modified or engineered using known methods so as to produce novel cloning vehicles which contain cDNA encoding human manganese superoxide dismutase Similarpolypeptide analogs or mutants thereof. ly, such cloning vehicles can be modified or engineered so that they contain DNA molecules, one strand of which includes a segment having the sequence shown in Fig. 1 for human manganese superoxide dismutase polypeptide or segments substantially similar thereto. The DNA molecule inserted may be made by various methods including enzymatic or chemical synthesis.

The resulting cloning vehicles are chemical entities which do not occur in nature and may only be created by the modern technology commonly described as recombinant DNA technology. Preferably the cloning vehicle is a plasmid, e.g. pMSE-4 or pMS RB4. These cloning vehicles may be introduced in cells, either procaryotic, e.g., bacterial (Escherichia_£2l_i, B.subtilis. etc.) or eukaryotic, e.g., yeast or mammalian, using techniques known to those skilled in the art, such as transformation, transfection and the like. The cells into which the cloning vehicles are introduced will thus contain cDNA encoding human manganese superoxide dismutase polypeptide analog or mutant thereof if the cDNA was present in the cloning vehicle or will contain DNA which includes a strand, all or a portion of which has the sequence for human MnSOD polypeptide shown in Fig. 1 or sequence substantially similar thereto if such DNA was present in the cloning vehicle.

Escherichia coli are preferred host cells for the cloning vehicles of this invention. A presently preferred auxotrophic strain of E. coli is Ά1645 which has been deposited with the American Type Culture Collection in Rockville, Maryland, U.S.A. containing plasmid pApoE-Ex2, under ATCC Accession No. 397 87. All deposits with the American Type Culture Collection re•j υ ferred to in this application were made pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

A1645 was obtained from A1637 by selection for Gal+ (ability to ferment galactose) as well as loss of tetracycline resistance. It still contains elements of phage χ . Its phenotype is C600 r"m+ gal+ thr" leu" lacZ~ bl (xcI857 δΗΙ ABamHl N+) .

A1637 was obtained from C600 by inserting transposon containing tetracycline resistance gene into the galactose operon as well as elements of phage λ including those elements responsible for cl repressor synthesis. C600 is available from the American Type Culture Collection, as ATCC Accession No. 23724.

Prototrophic strains of Escherichia coli which enable high level polypeptide expression even when grown in a minimal media are even more preferred as hosts for expression of genes encoding manganese superoxide dismutase. One presently preferred prototrophic strain is A4255. Strain A4255 containing the plasmid pMSE-4 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250.

The resulting cells into which DNA encoding human manganese superoxide dismutase polypeptide analog or mutant thereof has been introduced may be treated, e.g. grown or cultured as appropriate under suitable conditions known to those skilled in the art, so that the DNA directs expression of the genetic information encoded by the DNA, e.g. directs expression of the hMnSOD polypeptide analog or mutant thereof, and the cell expresses the hMnSOD polypeptide analog or mutant thereof which may then be recovered. 1.7 As used throughout this specification, the term superoxide dismutase (SOD) means an enzyme or a polypeptide acting upon superoxide or oxygen-free radicals as receptors, or which catalyze the following dismutation reaction: 202- + 2H+ -O2 + H2O2 The term manganese superoxide dismutase (MnSOD) as used herein means any superoxide dismutase molecule containing the element manganese, in any of its chemical forms.

The term human manganese superoxide dismutase polypeptide as used herein means a polypeptide of 198 amino acids a portion of the amino acid sequence of which is shown in Fig. 1; the N-terminus of the sequence is the lysine encoded by nucleotides 115-117 of Fig. 1 and the COOH terminus of the sequence is the lysine encoded by nucleotides 706-708 of Fig. 1.

The term polypeptide manganese complex as used herein means a molecule which includes a human manganese superoxide dismutase polypeptide in a complex with manganese in any of its chemical forms and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.

The term human manganese superoxide dismutase as used herein means a molecule which includes at least two human manganese superoxide dismutase polypeptides in a complex with manganese in any of its chemical forms and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.

The term human manganese superoxide dismutase polypeptide analog" as used herein means a polypeptide which includes a human manganese superoxide dismutase polypeptide to the N-terminus (lysine) of which is attached methionine to form a new N-terminus.

The term polypeptide manganese complex analog as used herein means a molecule which includes a polypeptide manganese complex, the polypeptide portion of which includes one or more additional amino acids attached to it at either or both ends.

The term human manganese superoxide dismutase analog as used herein means a molecule that includes at least two polypeptides at least one of which is human manganese superoxide dismutase polypeptide analog, in a complex with manganese in any of its chemical forms, and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.

The term human manganese superoxide dismutase polypeptide mutant as used herein means a polypeptide having an amino acid sequence substantially identical to that of the human manganese superoxide dismutase polypeptide but differing from it by one or more amino acids.

The term polypeptide manganese complex mutant means a molecule which includes a human manganese superoxide dismutase polypeptide mutant in a complex with manganese in any of its chemical forms and which has the enzymatic activity of manganese superoxide dismutase.

The term human manganese superoxide dismutase mutant as used herein means a molecule which includes at least two polypeptides at least one of which polypeptides is a human manganese superoxide dismutase polypeptide mutant in a complex with manganese in any of its chemical forms and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.

The mutants of hMnSOD polypeptide and hMnSOD which are included as a part of this invention may be prepared by mutating the DNA .sequence shown in Fig. 1, the N-terminus of which sequence is the lysine encoded by nucleotides 115-117 and the COOH terminus of which sequence is encoded by nucleotides 7 06-708.

The DNA may be mutated by methods known to those of ordinary skill in the art, e.g. Bauer et al., Gene 37: 73-81 (1985). The mutated sequence may be inserted into suitable expression vectors as described herein, which are introduced into cells which are then treated so that the mutated DNA directs expression of the hMnSOD polypeptide mutants and the hMnSOD mutants.

The enzymatically active form of human manganese superoxide dismutase is believed to be a protein having at least two, and possibly four, identical subunits, each of which has approximately 198 amino acids in the sequence shown in Fig. 1 for human manganese superoxide dismutase, the N-terminus of the sequence being the lysine encoded by nucleotides 115-117 of Fig. 1 and the OOOH terminus of the sequence being the lysine eAcoded by nucleotides 7 06-708 of Fig. 1.

Human MnSOD analogs or mutants thereof may be prepared from cells into which DNA or cDNA encoding human manganese superoxide dismutase analogs or mutants have been introduced. This human MnSOD analog or mutant may be used to catalyze the dismutation or univalent reduction of the superoxide anion in the presence of protons to form hydrogen peroxide as shown in the following equation: 202- + 2H human MnSOD -> H2°2 + °2 Veterinary and pharmaceutical compositions may also be prepared which contain effective amounts of hMnSOD analog or mutant and a suitable carrier. Such carriers are well-known to those skilled in the art. The hMnSOD analog or mutant thereof may be administered directly or in the form of a composition to the animal or human subject, e.g., to treat a subject afflicted by inflammations or to reduce injury to the subject by oxygen-free radicals on reperfusion following ischemia or organ transplantation. The hMnSOD analog or mutant may also be added directly or in the form of a composition to the perfusion medium of an isolated organ, to reduce injury to an isolated organ by oxygen-free radicals on perfusion after excision, thus prolonging the survival period of the organ. Additionally, the hMnSOD analog or mutant thereof may be used to reduce neurological injury on reperfusion following ischemia and to treat bronchial pulmonary dysplasia.

A method of producing enzymatically active human manganese superoxide dismutase analog or mutant thereof in a bacterial cell has also been discovered. The bacterial cell contains and is capable of expressing a DNA sequence encoding the human manganese superoxide dismutase analog or mutant thereof. The method involves maintaining the bacterial cell under suitable conditions and in a suitable production medium. The production medium is supplemented with an amount cf Mn++ so that the concentration of Mn++ in the medium is greater than about 2 ppm.

The bacterial cell can be any bacterium in which a DNA sequence encoding human manganese superoxide dismutase analog has been introduced by recombinant DNA techniques. The bacterium must be capable of expressing the DNA sequence and producing the protein product. The suitable conditions and production medium will vary according to the species and strain of bacterium.

The bacterial cell may contain the DNA sequence encoding the superoxide dismutase analog in the body of a vector DNA molecule such as a plasmid. The vector or plasmid is constructed by recombinant DNA techniques to have the sequence encoding the SOD incorporated at a suitable position in the molecule.

In a preferred embodiment of the invention the bacterial cell is an Escherichia coli cell. A preferred auxo25 trophic strain of E. coli is A1645. A preferred prototrophic strain of E. coli is A4255 The E. coli cell of this invention contains a plasmid which encodes for human manganese superoxide dismutase analog or mutant thereof.

In a preferred embodiment of this invention, the bacterial cell contains the plasmid pMSE-4. A method of constructing this plasmid is described in the De22 scription of the Figures and the plasmid itself is described in Example 2. This plasmid has been deposited with the ATCC under Accession No. 43250.

In another preferred embodiment of this invention, the bacterial cell contains the plasmid pMS^B4. A method of constructing this plasmid is described in the Description of the Figures and the plasmid itself is described in Example 5. This plasmid may be constructed from pSOD^T-11 which has been deposited with the American Type Culture Collection under Accession No. 53 468.

In specific embodiments of the invention, an enzymatically active human manganese superoxide dismutase analog is produced by E. coli strain A4255 cell containing the plasmid pMSE-4 and by E. coli strain A4255 cell containing the plasmid pMS£RB4.

The suitable production medium for the bacterial cell can be any type of acceptable growth medium such as casein hydrolysate or LB (Luria Broth) medium, the latter being preferred. Suitable growth conditions will vary with the strain of E. coli and the plasmid it contains, for example E. coli A4255 containing plasmid pMSE-4 is induced at 42°C and maintained at that temperature from about 1 to about 5 hours. The suitable conditions of temperature, time, agitation and aeration for growing the inoculum and for growing the culture to a desired density before the production phase as well as for maintaining the culture in the production period may vary and are known to those of ordinary skill in the art.

The concentration of Mn++ ion in the medium that is necessary to produce enzymatically active MnSOD will vary with the type of medium used.

In LB-type growth media Mn++ concentrations of 150 ppm to 750 ppm have been found effective. It is preferred that in all complex types of growth mediums the concentration of Mn++ in the medium is from 50 to 1500 ppm.

The specific ingredients of the suitable stock, culture, inoculating and production mediums may vary and are known to those of ordinary skill in the art.

This invention also concerns a method of recovering human manganese superoxide dismutase analog or mutant thereof from bacterial cells which contain the same. The cells are first treated to recover a protein fraction containing proteins present in the cells including human manganese superoxide dismutase analog or mutant thereof and then the protein fraction is treated to recover human manqanese superoxide dismutase analog or mutant thereof.

In a preferred embodiment of the invention, the cells are first treated to separate soluble proteins from insoluble proteins and cell wall debris and the soluble proteins are then recovered. The soluble proteins so recovered are then treated to separate, e.g. precipitate, a fraction of the soluble proteins containing the human manganese superoxide dismutase analog or mutant thereof and the fraction is recovered. The fraction is then treated to separately recover the human manganese superoxide dismutase analog or mutant thereof.

The following is a description of a more preferred embodiment of the invention. First, the bacterial cells are isolated from the production medium and suspended in a suitable solution having a pH of 5 7.0 or 8.0. The cells are then disrupted and centrifuged. The resulting supernatant is heated for a period of 30 to 120 minutes at a temperature between 55 to 65°C, preferably for 45-75 minutes at 58 to 62°C and more preferably one hour at 10 60°C, and then cooled to below 10°C, preferably to about 4°C. Any precipitate which may form during cooling is removed, e.g. by centrifugation and then the cooled supernatant is dialyzed against an appropriate buffer. Preferably the cooled supernatant is dialyzed by ultrafiltration employing a filtration membrane smaller than 30K, most preferably 10K. Appropriate buffers include 2 mM potassium phosphate buffer having a pH of about 7.8. After or simultaneously with this dialysis the cooled supernatant may optionally be concentrated to an appropriate volume, e.g. 0.03 of the supernatant's original volume has been found to be convenient. The retentate is then eluted on an anion exchange chromatography column with an appropriate buffered solution, e.g., a solution at least 20 mM potassium phosphate buffer having a pH of about 7.8. The fractions of eluent containing superoxide dismutase are collected, pooled and dialyzed against about 40 mM potassium acetate, pH 5.5. The dialyzed pooled fractions are then eluted through a cation exchange chroma30 tography column having a linear gradient of 4 0 to 200 mM potassium acetate (KOAC) and a pH of 5.5.

The peak fractions containing the superoxide dismutase are collected and pooled. Optionally the pooled peak fractions may then be dialyzed against an appropriate solution, e.g. water or a buffer solution of about 10 mM potassium phosphate having a pH of about 7.8.

The invention also concerns purified, i.e. substantially free of other substances of human origin, human manganese superoxide dismutase analog or mutant thereof produced by the methods of this invention. In particular, it concerns a human manganese superoxide dismutase analog having at least two polypeptides, at least one of which polypeptides has the amino acid sequence shown in Fig. 1, the Nterminus of which sequence is the lysine encoded by nucleotides 115-117 of Fig. 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Fig. 1 plus an additional methnione residue at the N-terminus (Met-hMnSOD). A preferred embodiment of this invention concerns purified Met-hMnSOD having a specific activity of 3500 units/mg.

LWLELES The Examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit its scope in any way. The Examples do not include detailed descriptions for conventional methods employed in the construction of vectors, the insertion of genes encoding polypeptides into such vectors or the introduction of the resulting plasmids into hosts. The Examples also do not include detailed description for conventional methods employed for assaying the polypeptides produced by such host vector systems or determining the identity of such polypeptides by activity staining of isoelectric focusing (IEF) gels. Such methods are well-known to those .or ordinary skill in the art and are described in numerous publications including by way of example the following: T. Maniatis, E.F. Fritsch and J. Sombrook, Molecular Cloning;_A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1982).

J.M. McCord and I. Fridovich, J. Biol. Chem. 244:604955 (1969) .

C. Beauchamp and I. Fridovich, Anal. Biochem. 44:276-87 (1971) .

The word Coomassie used in the Examples is a Registered Trade Mark.

SXAMPLP-1 In order to identify MnSOD cDNA clones, mixed oligomer probes were synthesized according to the published amino acid sequence (18,19) . '-probe - 30 mer sequence from AA^5 - AAg4 (18,19) ' 3' TTG CAT AATTTGTGCCTT AATGTGTGGTTC T G T G G G 3' —pr obe - 32 mer sequence from AAj^-AA^ (18) ' 3' TCTGTTAOGTTTTCCCAGTTTATTAOGTTCCA G G G G The 5'-probe consisting of 30 nucleotides corresponds to amino acids 15 to 24 of mature MnSOD. The 3'-probe consisting of 32 nucleotides corresponds to amino acids 179 to 189 of mature MnSOD. The 5'-probe is a mixed probe consisting of 36 different sequences, as shown above. The 3'-probe is a mixed probe consisting of 16 different sequences as shown above. (when more than one nucleotide is shown at a given position, the DNA strand was synthesized with equimolar amounts of each of the shown nucleotides thus resulting in the mixed probe) .

The 5'-probe was employed to screen 300,000 plaques of a T-cell cDNA library cloned into the λ gt-10 vector. Hybridization to phage plaque replicas immobilized on nitrocellulose filters was performed according to standard procedures (Maniatis et al. supra) except that the hybridization was performed at 50°C in 8xSSC for 16 hrs. The filters were then washed at 50°C with 5xSSC and 0.1% SDS. Three positive plaques were iso5 lated and named Phi MS8, Phi MSI and Phi MS1J.

EcoRI digests of DNA from Phi MS8 and Phi MSI showed that they both have cDNA inserts approximately 800 bp long, which hybridize to both the 5’ and 3’ oligonucleotide probes. Phi MS1J carried only 450 bp cDNA insert which hybridized only to the 5’ end probe.

The EcoRI inserts of the three phage clones were subcloned into the EcoRI site of pBR322 thus yielding pMS8-4, pMSl-4 and pMSU, respectively. Restriction analysis and hybridization to the 5' and 3' ol igonu15 cleotide probes revealed similar patterns for both pMS8-4 and pMSl-4. The following restriction map showing the 5' -^*3' orientation has been deduced for both plasmids.

R1 PvuII PvuII Seal BamI Stul R1 siz ZL Ί. 100 200 300 400 500 600 700 800 bp The sequence of the cDNA insert of pMS8-4 is shown in Fig. 1. The predicted amino acid sequence differs from the published amino acid sequence (19) in that Glu appears instead of Gin in three (3) locations (AA 42, 88, 108) and an additional two amino acids, Gly and Trp appear between AAi23-l24* Sequence analysis of pMSl-4 and pMSU revealed that the three MnSOD clones were independently derived and confirmed these differences from the published amino acid sequence.

The sequence upstream of the N-terminal Lysine of mature MnSOD predicts a pre-peptide sequence of 24 amino acids. 3P £2&H£L£_2 Construction ..of pMS.E-_j;_AmpjL Human MnSOD Expression Plasmid The starting point for the construction of pMSE-4 is the plasmid pMS8-4 which was obtained as described in Example 1. Plasmid pMS8-4, containing human MnSOD cDNA on an EcoRI insert, was digested to completion with £dfi.I and Narl restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in Fig. 2. The resulting plasmid, pMS8-NN contains the coding region for the mature MnSOD, preceded by an ATG initiation codon. The above plasmid was digested with E coR I. ends were filled in with Klenow fragment of Polymerase I and further cleaved with £^£.1. The small fragment containing the MnSOD was inserted into pSOD 13 which treated with Ndel and Stul. pSOD 13 as described in U.S. 4,742,004. This generated containing the MnSOD preceded by gene was may Patent plasmid coding be obtained No. pMSE-4 region the ell ribosomal binding site and under the control of λ p promoter. Plasmid pMSE-4 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250. All methods utilized in the above processes are essentially the same as those described in Maniatis, supra.

EXAMPLE 3 Expression of the Recombinant Human MnSOD Plasmid pMSE-4 was introduced into Escherichia col i strain A4255 using known methods. Then the E. coli strain 4255, containing pMSE-4, were grown at 32°c in Luria Broth (LB) medium containing 100 g/ml) of ampicillin until the Optical Density (OD) at 6 00 nm was 0.7. Induction was performed at 42°C. Samples taken at various time intervals were electrophoresed separated on sodium dodecyl sulfate - polyacrylamide gels electrophoresis (SDS-PAGE) . The gels showed increases in human MnSOD levels up to 120 minutes post-induction, at which stage the recombinant MnSOD protein comprised 27% of total cellular proteins as determined by scanning of Coomassie-blue stained gel. Sonication of samples for 90 sec. in a W-375 sonicator and partitioning of proteins to soluble (s) and non-soluble (p) fractions by. centrifugation at 10,000 g for 5 min. revealed that most of the recombinant MnSOD produced was non-soluble. The induced soluble protein fraction contained only slightly more SOD activity than the uninduced counterpart, as assayed by standard methods. See McCord et al., supra. Apparently a portion of the MnSOD found in the soluble fraction is inactive. This suggested that most of the human MnSOD produced under the conditions described in this Example is, in effect, inactive.

EXAMPLE 4 Effect of Mn~l'+ in Growth Media on MnSOD Solubility and Activity The addition of Mn++ in increasing concentrations up to 450 ppm to the growth media of E. coli A4 2 55, containing pMSE-4, prior to a 2 hr. induction at 42°C at OD600=0.7, had no adverse effect on the overall yield of human MnSOD. Analysis of sonicated protein fractions soluble (s) and non-soluble (p) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), showed increased solubilization of the recombinant protein with increased Mn++ concentrations (Table 1) . An assay of SOD activity (see McCord et al., supra) suggests a correlation between increased Mn++ concentrations in the growth media and increased solubility of the MnSOD with an apparent optimum at 150 ppm Mn++ concentration in the media (Fig. 3) . Furthermore increased Mn++ concentrations activated previously inactive soluble enzyme. Soluble protein fractions of induced cultures grown at these Mn++ levels show up to 60-fold increase in SOD activity over soluble protein fractions of non-induced cultures grown at these Mn++ levels. Activity staining of isoelectric focusing (IEF) gels (see Beauchamp et al, supra.) revealed that multi forms of the recombinant MnSOD were identical to those of native human liver MnSOD.

Results for human MnSOD production by E. coli A1645 containing pMSE-4 were similar to those described above. table 1 Mn++ Per cent Percent Specific (ppm) Soluble Soluble Activity human Mn human Mn units/mg SOD of SOD of Soluble Total human MnSOD Induced Soluble Bacterial Proteins Proteins 0 30.6 7.2 30 50 72.7 15.4 241 100 78.0 16.9 3 56 150 82.9 18.8 606 200 82.0 20.8 338 250 79.2 20.4 380 300 80.8 20.3 381 450 89.2 22.4 323 EXAMPLE 5 Construction of pMSARBl;-Tet1! Human MnSOD Expression Plasmid p Tet expression vector, PARB, was generated from pSODB ^T-ll by complete digestion with EcoRI followed by partial cleavage with BamHI restriction enzymes. pSOD e^T-11 has been deposited with the American Type Culture Collection under Accession No. 53468. The digested plasmid was ligated with synthetic oligomer '- AATTCCCGGGTCTAGATCT - 3' 3'- GGGCCCAGATCTAGACTAG - 5' resulting in pARB containing the λ PL promoter.

The EcoRI fragment of MnSOD expression plasmid pMSE-4, containing ell ribosomal binding site and the complete coding sequence for the mature enzyme, was inserted into the unique EcoRI site of pARB. The resulting plasmid, pMSARB4, contains the MnSOD gene under control of λ PL and ell RBS and confers resistance to tetracycline (Fig. 4).

EXAMPLE 6 Expression of Human MnSOD from pMSARB4 Plasmid pMSARB4 was introduced into Escherichia coli strain A4255, using known methods. Cultures were grown at 32°C in Luria Broth (LB) containing various concentrations of Mn++, until the Optical Density (OD) at 600 nm reached 6.7. Induction was performed at 42°C. Samples taken at various time intervals were electrophoresed on SDS-PAGE. hMnSOD level increased IQ with induction time up to 120 minutes, at which stage it comprised about 15% of total cellular proteins as determined by scanning of Coomassie Blue stained gel.

The induced MnSOD was soluble, regardless of Mn++ concentration in growth media. This is in contrast with n observations for the Amp plasmid pMSE-4. (See Example 4.) However, maximum SOD activity and expression level were dependent on Mn++ supplementation (Table 2) .

TABLE 2 MnSOD Expression in E.Coli A4255 (pMSARB4) u ppm Mn Percent Soluble hMnSOD of Soluble Bacterial Proteins Specific Activity Units/mg Soluble Proteins 42° 32° 42° 0 10.9 8.0 23 50 19.8 8.0 227 100 16.0 8.0 2 41 200 17.0 10.0 278 300 16.0 9.3 238 EXAMPLE. 7 Purification of Enzymatically Active Recombinant Human MnSOD E. coli strain A4255 harboring plasmid pMSARB4 was fermented in LB supplemented with 750 ppm Mn++, at 32°C to an OD600 of 17.0. Induction of human MnSOD expression was effected by a temperature shift to 42°C for 2 hours at which stage the culture reached OD600 of 43.0. Cells were harvested by centrifugation and resuspended in 0.2 original volume in 50 mM potassium phosphate buffer, pH 7.8 containing 250 mM NaCl. Bacteria were disrupted by a double passage through Dynomill, centrifuged and cell debris were discarded. The supernatant was heated for 1 hour at 60°c, cooled to 4°C and the cleared supernatant was concentrated to 0.03 original volume and dialyzed against 2 mM potassium phosphate buffer, pH 7.8, on a Pelicon ultra filtration unit equipped with a 10K membrane. The crude enzyme preparation was loaded onto a DE52 column, washed thoroughly with 2 mM potassium phosphate buffer, pH 7.8 and eluted with 20 mM potassium phosphate buffer, pH 7.8. Pooled fractions containing the enzyme were dialyzed against 40 mM potassium acetate, pH 5.5, loaded onto a CM52 column and eluted with a linear gradient of 40 200 mM potassium acetate, pH 5.5. Peak fractions containing human MnSOD were pooled, dialyzed against H2O, adjusted to 10 mM potassium phosphate buffer, pH 7.8 and frozen at -20°C.

Recombinant human MnSOD obtained was more than 99% pure, with a specific activity of about 3500 units/mg. The overall yield of the purification procedure was about 30% (Table 3) .

Sequencing of the purified enzyme shows the presence of an additional methionine at the N-terminal amino acid as compared with the known human MnSOD (19) .

Analysis for metal content by atomic absorption re5 vealed about 0.77 atoms Mn per enzyme subunit. This is in accordance with published data (23).

TABLE 3 Purification of Recombinant Human Mn-^SOD Total Proteins Yield Specific Acitivity Step gin gmSOD % uni ts/mg Dynomill supernatant 100.0 11.9 100.0 417 60°C supernatant 24.0 8.2 68.9 1197 Pel icon retentate 20.0 7.5 63.0 1350 DE52 eluate 7.3 5.7 48.0 2732 CM52 eluate 4.2 4.2 35.3 3500 * Values for enzyme purified from 15 L fermentation.

REFERENCES 1. McCord, J.M. and Fridovich, I., J. Biol. Chem. 244: 6049-55 (1969) . 2. Fridovich, I. in Advances in Inorganic. Biochemistry . eds. Eichhorn, G.L. and Marzilli, L.G. (Elsevier/North Holland, New York) , pp. 67-90 (1979) . 3. Freeman, B.A. and Crapo, J.D., Laboratory Investion 47:412-26 (1982). 4. Steinman, H.M. in Superoxide Dismutase. ed. Oberley, L.W. (CRC Press, Florida) , pp. 11-68 (1982) .

. Hartz, J.W. and Deutsch, H.F. , J. Biol. Chem. 247:7043-50 (1972). 6. Jabusch, J.R., Farb, D.L., Kerschensteiner, D. A. and Deutsch, H. F., Biochemistry 19:2310-16 (1980) . 7. Barra, D., Martini, F., Bannister, J.v., Schinina, M.W., Rotilio, w. H., Bannister, W.H. and Bossa, F., FEBS Letters 120:53-56 (1980). 8. Lieman-Hurwitz, J., Dafni, N., Lavie, V. and Groner, Y. , Proc. Natl. Acad. Sci. USA 79:280811 (1982). 9. Sherman, L., Dafni, N., Lieman-Hurwitz, J. and Groner, Y., Proc. Natl. Acad. Sci. USA 80:546569 (1983). Αί . Oberley, L.W. and Buettner, G.R. , Cancer Research 39:1141-49 (1979). 11. Huber, W. and Menander-Huber, K.B., Clinics in Rheum. Dis. 6:465-98 (1980) . 12. McCord, J.M. and Roy, R.S. , Can. J. Physiol. Pharma. 60:1346-52 (1982). 13. Alvarez, J.G. and Storey, B.T., Biol. Re prod. 28:1129-36 (1983). 14. Talmasoff, J.M. , Ono, T. and Cutler, R.G. , PEQC^Natl^-Asa.iL-Sci^-.USA 77:2777-81 (1980).

. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem. 193:26 5-7 5 (1951) . 16. Weser, U. and Hartmann, H.J. , FEBS Letters 17:78-80 (1971). 17. Jewett, S.LO., Latrenta, G. S. and Beck, C.M. , Arc. Biochem. Biophys. 215:116-128 (1982). 18. Harris, J.I. and Steinman, H.M., Superoxide and Superoxide Dismutase. Michel son, A.M., McCord, J.M. and Fridovich, I. eds., Academic Press, London, pp. 225-230 (1977). 19. Barra, D., Schinina, M.E., Simmaco, M., Bannister, J.V. , Bannister, W.H., Rotilio, G. and Bossa, F. , J. Biol. Chem. 259:12595-601 (October 25, 1984) . Baret, A., Jadot, G., and Michelson, A.M. , Biochemical Pharmacology 33:2755-60 (September 1, 1984). 21. McCord, J.M. and Salin, M.L., Movement. Metabolism and Bactericidal Mechanisms of Phagocytes. Ross, A., Patriarca, P.L., Romeo, D. (eds) pp. 257-264 (1977). 22. Touati, D. , Journal of Bacteriology 155:1078-87 (1983) . 23. McCord, J.M., Boyle, J.A., Day, Jr., E.D., Rizzolo, L.J. and Salin, M.L., Superoxide and Superoxide_Dismutase, Mi ch a el son, A.M., McCord, J.M., and Fridovich, I. (eds) Academic Press, London pp. 129-138 (1977). 24. European Patent Publication No. 0131843 Al, published January 23, 1985, corresponding to European Patent Application No. 84107717.5, filed July 3, U.S. Serial No. 1984, which claims priority of 514,188, filed July 15, 1983. 25. Hallewell, et al.. Nucleic Acids Res. 5, (1985) . 26. Patent Application No. corresponding to Europe an Patent Application No. 84111416.8, filed September 25, 1984, which claims priority of U.S. Serial No. 538,607, filed October 3, 1983 , and U.S. Serial No. 609,412, filed May 11, 1984. 27. EMBO Journal, Vol. 4, No. 1, pp. 77-84 (January 1985). 28.

Abstracts of the Fourth International Conference on Superoxide and Superoxide Dismutase, Rome, Italy, September 1-6, 1985.

Claims

CLAIMS:

1. A plasmid for expression of an enzymatically active human manganese superoxide dismutase analog wherein the analog consists essentially of at least two polypeptides each comprising 199 amino acids, the sequence of each polypeptide having methionine at its Nterminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig. 1A and continuing to the lysine encoded by nucleotides 706-708 of Fig. IB which is the COOH terminus of the polypeptide.

2. A plasmid according to claim 1 designated pMS£-4 and deposited in Escherichia coli strain A4255 under ATCC Accession No. 53250.

3. A plasmid according to claim l designated 15 pMSaRB4.

4. A host cell into which has been introduced a plasmid according to any one of claims 1 to 3.

5. A eucaryotic cell according to claim 4.

6. A procaryotic cell according to claim 4.

7. A bacterial cell according to claim 6.

8. An Escherichia coli cell according to claim 7.

9. An Escherichia coli cell according to claim 8 containing the plasmid designated pMSE-4 and deposited under ATCC Accession No. 53250.

10. An Escherichia coli cell according to claim β containing the plasmid designated pMSaRB4.

11. An enzymatically active polypeptide analog of human manganese superoxide dismutase comprising 199 amino acids, the sequence of which has methionine at its Nterminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig. 1A and continuing to the lysine encoded by nucleotides 706-708 of Fig. IB which is the COOH terminus of the polypeptide.

12. An enzymatically active analog of human manganese superoxide dismutase comprising at least two polypeptides according to claim ll.

13. A composition comprising a carrier and the human manganese superoxide dismutase analog according to claim 12 free of other proteins of human origin.

14. A complex comprising the human manganese superoxide dismutase analog according to claim 12 and manganese in any of its chemical forms complexed therewith, the complex having the enzymatic activity of naturallyoccurring, human manganese superoxide dismutase.

15. A complex according to claim 14 having a specific activity greater than 3500 units/mg.

16. A veterinary composition comprising an effective amount of a human manganese superoxide dismutase analog according to claim 12, and a suitable carrier.

17. A pharmaceutical composition comprising an effective amount of a human manganese superoxide dismutase analog according to claim 12, and a suitable carrier. 5 18. A method of catalyzing the reaction

18. 20^- + 2H*------------------- H 2 O 2 + o 2 which comprises contacting the reactants under suitable conditions with human manganese superoxide dismutase or an analog thereof according to claim ll. 10 19. A method of reducing injury caused by superoxide radicals to cells in vitro which comprises catalyzing the reduction of the superoxide radicals according to claim 18. 20. A method of prolonging the survival period of 15 excised isolated organs which comprises incorporating an effective amount of human manganese superoxide dismutase according to claim ll in the perfusion medium.

19. 21. A method of producing a human manganese superoxide dismutase analog which comprises growing a 20 host plasmid system according to claim 4 under conditions permitting production of the human manganese superoxide dismutase analog, and recovering the analog so produced.

20. 22. A method of producing an enzymatically active analog of human manganese superoxide dismutase having 25 substantially the same amino acid sequence as, and the 47 · biological activity of, naturally-occurring human manganese superoxide dismutase which comprises: growing a culture of bacterial cells in a production medium supplemented with a non-growth inhibitory amount of Mn** such that the concentration of Kn“ in the medium while the bacterial cells are growing is greater than 2 ppm, wherein the bacterial cells contain a plasmid which contains DNA encoding the analog of human manganese superoxide dismutase and the cells are capable of expressing the DNA encoding the analog of human manganese superoxide dismutase and wherein the culture is grown under suitable conditions such that the DNA is expressed and the analog of human manganese superoxide dismutase is produced in the bacterial cells; and recovering the enzymatically active analog of human manganese superoxide dismutase so produced.

21. 23. A method according to claim 22, wherein the bacterial cells comprise Escherichia coli cells.

22. 24. A method according to claim 22 or claim 23. wherein the plasmid contains DNA encoding a human manganese superoxide dismutase analog, the analog consisting essentially of at least two polypeptides, each comprising 199 amino acids, the sequence of each polypeptide having methionine at its N-terminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig. 1A and continuing to the lysine encoded by nucleotides 706-708 of Fig. IB which is the COOH terminus of the polypeptide.

23. 25. A method according to claim 24, wherein the plasmid is designated pMSE-4 and is deposited in Escherichia coli strain A4255 under ATCC Accession Nc. 53250.

24. 26. A method according to claim 24, wherein the 5 plasmid is designated pMSARB4.

25. 27. A method according to any one of claims 22 to 26, wherein the production medium is a casein hydrolysate medium.

26. 28. A method according to any one of claims 22 to 10 26, wherein the production medium is LB medium.

27. 29. A method according to any one of claims 22 to 28, wherein the Mn** concentration is from 50 to 1500 ppm.

28. 30. A method according to claim 29, wherein the 15 Mn** concentration is 150 ppm.

29. 31. λ method according to claim 29, wherein the Mn** concentration is 750 ppm.

30. 32. A method of recovering human manganese superoxide dismutase or an analog thereof from bacterial cells 20 which contain the human manganese superoxide dismutase or analog thereof which comprises: (a) treating the bacterial cells so as to recover a protein fraction containing proteins present in the cells including the human manganese superoxide dismutase or analog thereof; and (b) treating the protein fraction so as to recover the human manganese superoxide dismutase or analog thereof.

31. 33. A method according to claim 32 which comprises: (a) treating the cells to separate soluble proteins from insoluble proteins and cell wall debris; (b) recovering the soluble proteins; (c) treating the soluble proteins so recovered to separate a fraction of the soluble proteins containing the human manganese superoxide dismutase or analog thereof; (d) recovering the fraction of soluble proteins containing the human manganese superoxide dismutase or analog thereof; and (e) treating the fraction of soluble proteins containing the human manganese superoxide dismutase or analog thereof so as separately to recover the human manganese superoxide dismutase or analog thereof.

32. 34. A method according to claim 32 which comprises: (a) isolating the bacterial cells from the production medium; (b) suspending the isolated bacterial cells in a suitable solution having a pH of 7.0 to 8.0; (c) disrupting the suspended bacterial cells; (d) centrifuging the disrupted bacterial cells; (e) heating the resulting supernatant for a period ranging from 30 to 120 minutes at a temperature ranging from 55 to 65 ®C; (f) cooling the heated supernatant to below lo°C; (g) removing any precipitate from the cooled supernatant; (h) dialyzing the cooled supernatant against an appropriate buffer; (i) eluting the retentate on an anion exchange chromatography column with an appropriate buffered solution; (j) collecting and pooling fractions of the eluent containing human manganese superoxide dismutase; (k) dialyzing the pooled fractions against 40 mM potassium acetate, pH 5.5; (l) eluting the dialyzed pooled fractions through a cation exchange chromatography column with a linear gradient of 40 to 200 mM potassium acetate, pH 5.5; and (m) collecting and pooling peak fractions of the eluent containing superoxide dismutase or an analog thereof.

33. 35. A method according to claim 34, wherein in step 5 (e), the resulting supernatant is heated for 45 to 75 minutes at 58 to 62 °C.

34. 36. A method according to claim 34, wherein the supernatant is heated for 60 minutes at 60°C.

35. 37. A method according to any one of claims 34 to 10 36, wherein in step (f), the heated supernatant is cooled to 4 e C.

36. 38. A method according to any one of claims 34 to 37, wherein in step (g), the precipitate is removed by centrifugation. 15

37. 39. A method according to any one of claims 34 to 38, wherein in step (h), the cooled supernatant is dialyzed by ultra-filtration employing a filtration membrane smaller than 3OK.

38. 40. A method according to any one of claims 34 to 20 39, wherein in step (h), the appropriate buffer is a 2 mM potassium phosphate buffer having a pH of 7.8.

39. 41. A method according to any one of claims 34 to 40, wherein in step (i), the buffered solution is at least 20 mM potassium phosphate and has a pH of 7.8.

40. 42. A method according to any one of claims 34 to 41, wherein after dialyzing the cooled supernatant, the dialyzed supernatant is concentrated to an. appropriate volume.

41. 43. A method according to claim 42, wherein the appropriate volume is 0.03 of the supernatant’s original volume.

42. 44. A method according to any one of claims 34 to 43, further comprising dialyzing the pooled peak fractions against an appropriate solution.

43. 45. A method according to claim 44, wherein the appropriate solution is H z 0.

44. 46. A method according to claim 44, wherein the appropriate solution is 10 mM potassium phosphate buffer having a pH of 7.8.

45. 47. Human manganese superoxide dismutase or an analog thereof purified by a method according to any one of claims 34 to 46.

46. 48. Use of a human manganese superoxide dismutase analog according to claim 11 for reducing injury to a subject occurring upon reperfusion following ischemia.

47. 49. Use of a human manganese superoxide dismutase analog according to claim 11 for treating a subject afflicted with inflammations.

48. 50. A plasmid according to claim 1, substantially as hereinbefore described and exemplified.

49. 51. A plasmid according to claim 1, substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.

50. 52. A host cell according to claim 4, substantially as hereinbefore described and exemplified.

51. 53. A human manganese superoxide dismutase analog according to claim 11, substantially as hereinbefore described and exemplified.

52. 54. A human manganese superoxide dismutase analog according to claim 11, substantially as hereinbefore described with reference to the accompanying drawings.

53. 55. A method of producing a human manganese superoxide dismutase analog according to claim 11, substantially as hereinbefore described and exemplified.

54. 56. A human manganese superoxide dismutase analog according to claim 11, whenever produced by a method claimed in any one of claims 21-33 or 55.

55. 57. A composition according to claim 13, substantially as hereinbefore described.

56. 58. A complex according to claim 14, substantially as hereinbefore described.

57. 59. A veterinary composition according to claim 16, substantially as hereinbefore described.

58. 60. A pharmaceutical composition according to claim 17, substantially as hereinbefore described.

59. 61. A method according to claim 18, substantially as hereinbefore described and exemplified.

60. 62. A method according to claim 19, substantially as hereinbefore described. 5

61. 63. A method according to claim 20, substantially as hereinbefore described.

62. 64. Use according to claim 48 or 49, substantially as hereinbefore described.