CA1341362C - Human manganese superoxide dismutase cdna, its expression in bacteria and method of recovering enzymatically active human manganese superoxide dismutase - Google Patents

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

HUMAN MANGANESE SUPEROXIDE DISMUTASE cDNA, ITS EXPRESSION IN BACTERIA AND
METHOD OF RECOVERING ENZYMATICALLY ACTIVE
HUMAN MANG$NESE SUPEROXIDE DIS~~;fTASE
Throughout this appl ication, various publ ications 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.
Superoxide dismutase (SOD) and the phenomenon of oxygen free radicals (02-) was discovered in 1968 by McCord and Fridovich (1). Superoxide radicals and other high-ly reactive oxygen species are produced in every re-spiring cell as by-products of oxidative 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-reduc-tion reaction 202= + 2H+ ~ H202 + 02 and thus provide a defense mechanism against oxygen toxicity.

~ 3413fi2 There are several known forms of SOD containing differ ent 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 pri marily 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 rang lo ing 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 thera-peutic potential of SOD. The scientific literature s ugge st s that SOD may be usef u1 in a w ide r ange of clinical applications. These include prevention of oncogenesis and of tumor promotion, and reduction of the cytotoxic and cardiotoxic effects of anticancer 2o 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) .
~5 The exploration of the therapeutic potential of human SOD has been 1 invited mainly due to its 1 invited avail-abil ity.
Superoxide dismutase is also of interest because of its :3o anti-inflammatory properties (11) . Bovine-derived su-peroxide 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, suppl ies 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 reac Lions 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.
io Human CuZn SOD is a dimer is metall ic-pr otein composed of identical non-covalently linked subunits, each hav-ing a molecular weight of 16,000 dal tons and containing one atom of copper and one of zinc ( 5) . Each subunit i5 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 2o 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 pro-25 duction 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 summa 3o rized (28) .
Much less is known about manganese superoxide dismutase (MnSOD) . The MnSOD of E. coli R-12 has recently been cloned and mapped (22) . Barra et al. disclose a 196 ~ 34~ 362 amino acid sequence for the MnSOD polypeptide isolated from human liver cells (19) . Prior art disclosures differ, however, concerning the structure of the MnSOD
molecule, particularly whether it has two or four iden-tical polypeptide subunits (19,23) . It is clear, how-ever, that the MnSOD of tide and the CuZn SOD
P YPeP
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 half-1 ife 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 copper SOD and human copper SOD are fully active (20) .
McCord et al. disclose that naturally occurring human manganese superoxide dismutase protects human phagocy-tosing polymorghonuclear (PMN) leukocytes from super-oxide 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 the human manganese super oxide 'S dismutase polypeptide or an analog or mutant thereof.
It is also directed to inserting this cDNA into effi-cient bacterial expression vectors, to producing human MnSOD polypeptide, analog, mutant and enzyme in bacte-ria, to recovering the bacterially produced human MnSOD polypegtide, analog, mutant or enzyme. This in-vention is also directed to the human MnSOD
golypeptides, analogs, or mutants thereof so recovered and their uses.
:~ 5 This invention further provides a method for producing enzymatically active human MnSOD in bacteria, as well as a method for recovering and purifying such enzymatically active human MnSOD.
The present invention also relates to using human manganese superoxide dismutase or analogs or mutants thereof to catalyze the reduction of superoxide radicals to hydrogen peroxide and molecular oxygen. In particular, the present invention concerns using l0 bacterially produced MnSOD or analogs or mutants thereof to reduce reperfusion injury following ischemia and prolong the survival period of excised isolated organs.
It also concerns the use of bacterially produced MnSOD or analogs thereof to treat inflammations.
BRIEF LIST OF DRAWINGS
Figure 1: The nucleotide sequence of DNA encoding human MnSOD analog and corresponding amino acid sequence is shown.
Figure 2: The construction of the ampicillin-resistant plasmid pMSE-4 for expression of MnSOd is shown.
Figure 3: The dependence of SOD activity of MnSOD
produced by plasmid pMSE-4 on the manganese concentration in the growth medium is shown.
Figure 4: The construction of the tetracycline-resistant '~5 plasmid pMSGRB4, which is a high expressor of MnSOD, is shown.

13413fi2 A DNA molecule which includes cDNA encoding the human manganese superoxide dismutase polypeptide or analog or mutant thereof has been isolated from a human T-cell library. The nucleotide sequence of a double-stranded DNA molecule which encodes human manganese superoxide dismutase polypeptide or analog or mutant thereof has been discovered. The sequence of one strand encoding the polypeptide or analog thereof is shown in Fig. 1 1o fr~n nucleotide 115 downstream to nucleotide 7 08 inclusive. Other sequences encoding the analog or mutant 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 twenty-four (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 human manganese superoxide dismutase polypeptide or analog 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 1 invited to methods involving cloning vehicles containing either molecule.
Preferably the cDNA or DNA encoding the human manganese superoxide dismutase polypeptide or analog or mutant thereof is incorporated into a plasmid, e.g. , pMSE-4 or pMSORB4, and then introduced into a suitable host cell 1 34~ 362 _,_ where the DNA can be expressed and the human manganese superoxide dismutase (hMnSODI polypeptide or analog or mutant thereof produced. Preferred host cells include Escherichia col i, in particular E. col i A4255 and ~oli A1645. The plasmid pMSE-4 in E. coli strain A4255 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250. The plasmid pMS~RB4 may be obtained as shown in FIG. 4 and de-scribed in the Description of the Figures.
1o Cells into which such DNA molecules have been intro-duced may be cultured or grown in accordance with meth-ods 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 re-sulting manganese superoxide dismutase protein may then be recovered.
Veterinary and pharmaceutical compositions containing human MnSOD or analogs or mutants thereof and suitable 2o carriers may also be prepared. This r~uman manganese superoxide dismutase or analogs or mutants may be used to catalyze the following reaction:
202= + 2H+ ~ H202 + 02 and thereby reduce cell injury caused by superoxide radicals.
More particularly, these enzymes or analogs or mutants 3o thereof may be used to reduce injury caused by reperfusion following ischemia, increase the survival time of excised isolated organs, or treat inflammations.
.35 ~3413fi2 _8_ This invention is directed to a method of producing enzymatically active human manganese superoxide dismutase or an analog or mutant thereof in a bacterial cell. The bacterial cell contains and is capable of expressing a DNA sequence encoding the manganese superoxide dismutase or 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++
1o available to the cell in the medium is greater than about 2 ppm.
In a preferred embodiment of the invention the bacteri-al cell is an Escherichia-,coli cell containing a plas-mid 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 about 50 to about 1500 ppm, with concentrations of 150 and 750 ppm being preferred.
This invention also concerns a method of recovering manganese superoxide dismutase or analog 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 or analog or mutant thereof and then the protein fraction is treated to recover human manganese superoxide dismutase or analog or mutant 3o thereof. In a preferred embodiment of the invention, the cell s 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.

1 ~~1~62 _g_ precipitate, a fraction of the soluble proteins containing the hMnSOD or analog or mutant thereof and the fraction containing the hMnSOD or analog or mutant is recovered. The recovered fraction of soluble proteins is then treated to separately recover the human manganese superoxide dismutase or analog thereof.
A more preferred embodiment of the invention concerns a method of recovering human manganese superoxide dismutase or analog or mutant thereof frcxn bacterial cells which contain human manganese superoxide dismutase or analog or mutant thereof. The method involves first isolating the bacterial cells from the production medium and suspending them in suitable solu-tion having a pH of about 7.0 to 8Ø The cells are then disrupted and centrifuged and the resulting super-natant is heated for about 30 to 120 minutes at a tem-perature 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. Pref-erably, the dialysis is by ultrafiltration using a filtration membrane smaller than 30R. Simultaneously with or after dialysis the cooled supernatant optional-ly may be concentrated to an appropriate convenient vol ume e. g. 0 .03 of its or iginal vol ume. The retentate is then eluted on an anion exchange chroma-3o tography 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 ace .~41 X62 -lo-tate, pH 5.5. The dialyzed pooled fractions are then eluted through a ration 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 f ractions 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.
to The invention also concerns purified enzymatically active human manganese superoxide dismutase or analogs thereof e.g. met-hMnSOD, or mutants produced by the methods of this invention.

~ 3~~ 362 DESCRIP'~ O'L N OF THE FIGURES
FIG. 1. Tie Seguence of human ; n~, SOD_ 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 the 5' and 3' untranslated sequences.
FIG. 2. Constr,~ction of pMSE-4: Human MnSO~
ExpressioD Plasmid Plasmid pMSB-4, containing MnSOD on an F~oRI (R~) insert, was digested to completion with N eI and ~rI restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in Fig. 2. The resulting plasmid, pMSB-NN contains the coding region for the mature MnSOD, preceded by an ATG initiation codon. The above plasmid was digested with ,~oRI, ends were filled in with Klenow fragment of Polymerase I and further cleaved with NdeI. The small fragment harboring the MnSOD gene was inserted into pSODal3 which was treated with ddeI and I.
pSODal3 may be obtained as described in pending co-assigned Canadian Patent Application No. 488,832, filed August 15, 1985. This generated plasmid pMSE-4 containing the MnSOD
coding region preceded by the cII ribosomal binding site and under the control of x P~ promoter. Plasmid pMSE-4 has been deposited with the American Type Culture Collection under ATGC Accession No. 53250.

~ 3~~ ~s2 FIG. 3 ~f~~~t 9f M~ + C~ntration on the Act ivitv of SOD PrQr,~,iced in E. Coli The chart in FIG. 3 shows the correlation between the specific activity in units/mg of recombinant soluble MnSOD produced by ~. coli strain A4255 containing plasmid pMSE-4 under both non-induction (32oC) and induction (42°C) conditions, and the concentration of Mn++ (parts per million) in the growth medium.
io FIG. 4 ons ~,~~,~on of pMS D RB4: Human MnSOD
Expression. Play TetR expression vector, p~RB, was generated from pSODSIT-11 by complete digestion with F,coRI followed by partial cleavage with CHI restriction enzymes.
i5 pSODs 1T-11 has been deposited with the American Type Culture Collection (ATCC) under Accession No. 5346$.
The digested plasmid was ligated with synthetic oligo-mer 5'- AATTCCCGGGTCTAGATCT - 3' Zo 3'- GGGCCCAGATCTAGACTAG - 5' resulting in poRB containing the a. PL promoter.
The ~I fragment of MnSOD expression plasmid pMSE-4, containing cII ribosomal binding site and the complete ''5 coding sequence for the mature enzyme, was inserted into the unique SRI site of p~RB. The resulting plasmid, pMS 4RB4, contains the MnSOD gene under con-trol of a PL and cII RBS and confers resistance to tetracycl ine.
:30 ~5 13413fi2 A double-stranded DNA molecule which includes cDNA
encoding human manganese super oxide dismutase polypeptide or an analog or mutant thereof has been isolated from a human T-cell DNA library. The nucleo-tide 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 mole-1o cule 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 stranc'l encoding the h~SOD 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.
2o The methods of preparing the cDNA and of determining the sequence of DNA encoding the human manganese super oxide 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 syn-thetic methods can be employed to prepare DNA molecules containing portions of this sequence.
3o Conventional cloning vehicles such as plasmids, e. g. , pBR322, viruses or bacteriophages, e. g. , a, can be modified' or engineered using known methods so as to produce novel cloning vehicles which contain cDNA
encoding human manganese superoxide dismutase polypeptide, or analogs or mutants thereof. Similar 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 mole-cule inserted may be made by various methods including enzymatic or chemical synthesis.
The resulting cloning vehicles are chemical entities to 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 vehi cles may be introduced in cells, either procaryotic, e.g., bacterial tEschericb~ coli, B.subtilis, etc.) or eukaryotic, e. g. , yeast or mammal ian, using tech-niques known to those skilled in the art, such as transformation, transfection and the like. The cells into which the cloning vehicles are introduced will 2o thus contain cDNA encoding human manganese super oxide dismutase polypeptide or 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 '5 shown in Fig. 1 or sequence substantially similar thereto if such DNA was present in the cloning vehicle.
Escherichia ~oli are preferred host cells for the cloning vehicles of this invention. A presently pre-so (erred auxotrophic strain of ,~. r"~~ l is A1645 which has been deposited with the American Type Culture Collec tion in Rockville, Maryland, U. S. A. containing plasmid pApoE-Ex2, under ATCC Accession No. 39787. All de posits with the American Type Culture Collection re 13413fi2 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 tet-racycl ine resistance. It still contains elements of phage ~, . Its phenotype is C600 r m+ gals thr leu lacZ b1 (a cI857 p Hl p BamHl N+) .
to A1637 was obtained from C600 by inserting transposon containing tetracycl ine resistance gene into the galac tose operon as well as elements of phage a including those elements responsible for cI repressor synthesis.
C600 is available from the American Type Culture CoI
i5 -lection, as ATCC Accession No. 23724.
Prototrophic strains of Escherichi~ c~,,i which enable high level polypeptide expression even when grown in a minimal media are even- more preferred as hosts for zo expression of genes encoding manganese super oxide dismutase. One presently preferred prototrophic strain is A4255. Strain A4255 containing the plasmid pMSE-4 has been deposited with the American Type Culture Col-lection under ATCC Accession No. 53250.
The resulting cells into which DNA encoding human man-ganese superoxide dismutase polypeptide or analog or mutant thereof has been introduced may be treated, e. g.
grown or cultured as appropriate under suitable condi--;o tions 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 or analog or mutant thereof, and the cell expresses the hMnSOD polypeptide or analog or mutant thereof which may then be recovered.

13413fi2 As used throughout this specification, the term "superoxide dismutase" (SOD) means an enzyme or a polypeptide acting upon superoxide or oxygen-free rad icals as receptors, or which catalyze the following dismutation reaction 202= + 2H+ ---~ 02 + H202 The term "manganese superoxide dismutase" (MnSOD) as 1o used herein means any superoxide dismutase molecule containing the element manganese, in any of its chemi-cal 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 se quence is the lysine encoded by nucleotides 115-117 of Fig. 1 and the COON terminus of the sequence is the zo 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 S 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 super oxide dismutase.

3341~fi2 The term "human manganese superoxide dismutase polypeptide analog" as used herein means a polypeptide which includes a human manganese super oxide dismutase polypeptide to either or both ends of which one or more additional amino acids are attached.
The term "polypeptide manganese complex analog" as used herein means a molecule which includes a polypeptide manganese complex, the polypeptide por tion 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 manga-nese superoxide dismutase polypeptide analog, in a complex with manganese in any of its chemical forms, and which has the enzymatic activity of naturally-oc-curring human manganese superexide 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 3o dismutase polypeptide mutant in a complex with manga-nese in any of its chemical forms and which has the enzymatic activity of manganese super oxide dismutase.

~ ~~~ ~sz 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 chemi-cal forms and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.
The mutants of hMnSOD polypeptide and hMnSOD which are 1o included as a part of this invention may be prepared by mutating the DNA sequence shown in Fig. 1, the N-ter-minus of which sequence is the lysine encoded by nucleotides 115-117 and the COON terminus of which sequence is encoded by nucleotides 706-708.
The DNA may be mutated by methods known to those of ordinary skill in the art, e. g. Bauer et al. , Gene ?3-81 (1985) . The mutated sequence may be inserted into suitable expression vectors as described herein, 2o 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 ~~5 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 1.15-117 of Fig.
1 and the COON terminus of the sequence being the lysine encoded by nucleotides 706-708 of Fig. 1.

Human MnSOD or analogs or mutants thereof may be prepared from cells into which DNA or cDNA encoding human manganese superoxide dismutase, or its analogs or mutants have been introduced. This human MnSOD or analogs or mutants may be used to catalyze the dismutation or univalent reduction of the superoxide anion in the presence of protons to form hydrogen per-oxide as shown in the following equation"
human MnSOD
io 2 p 2 ~ + 2 H+ ~ H2 02 + 02 Veterinary and pharmaceutical compositions may also be prepared which contain effective amounts of hMnSOD or one or more hMnSOD analogs or mutant and a suitable i5 carrier. Such carriers are well-known to those skilled in the art. The hMnSOD or analog or mutant thereof may be administered directly or in the form of a composition to the animal or human subject, e.9., to treat a subject afflicted by inflammations or to 2o reduce injury to the subject by oxygen-free radicals on reperfusion following ischemia or organ transplantation. The hMnSOD or 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 25 injury to an isolated organ by oxygen-free radicals on perfusion after excision, thus prolonging the survival period of the organ. Additionally. the hMnSOD or ana log or mutant thereof may be used to reduce neurological injury on reperfusion following ischemia 3o and to treat bronchial pulmonary dysplasia.
A method of producing enzymatically active human manga-nese superoxide dismutase or an analog or mutant there-of in a bacterial cell has also been discovered. The bacterial cell contains and is capable of expressing a DNA sequence encoding the human manganese super oxide dismutase or 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 of 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 has been introduced by recombinant DNA techniques. The bacterium must be capable of expressing the DNA se-quence 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 encod-ing the super oxide dismutase or analog in the body of a vector DNA molecule such as a plasmid. The vector or 'o 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 bacteri-''S al cell is an Escherichia coli cell. A preferred auxo-trophic strain of ~,. coli is A1645. A preferred proto trophic strain of E. col i is A4255 The E, col i cell of this invention contains a plasmid which encodes for human manganese superoxide dismutase or an analog or 3~ 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 De-scription of the Figures and the plasmid itself is described in Example 2. This plasmid has been deposit-ed with the ATCC under Accession No. 432 50.
In another preferred embodiment of this invention, the bacterial cell contains the plasmid pMS 4RB4. A method of constructing this plasmid is described in the De scription of the Figures and the plasmid itself is described in Example 5. This plasmid may be construct ed from pSOD ~T-11 which has been deposited with the American Type Culture Collection under Accession No.
53468.
In specific embodiments of the invention, an enzymati-cally active human manganese superoxide dismutase ana-log is produced by E. coli strain A4255 cell contain-ing the plasmid pMSE-4 and by E,, coli strain A4255 cell containing the plasmid pMS pRB4.
The suitable production medium for the bacterial cell 2o 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 ~.~ cc,~j, A4255 containing plasmid p~E-4 is induced at 42oC and maintained at that tem-perature from about 1 to about 5 hours. The suitable conditions of temperature, time, agitation and aeration f or gr ow ing the inocul um and f or gr ow ing the cul tur a to a desired density before the production phase as well 3o as for maintaining the culture in the production period may vary and are knaan to those of ordinary skill in the art.

The concentration of Mn++ ion in the medium that is nece ssary to pr oduce enzymatical 1y act ive MnSOD w i1 l 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 grawth mediums the concen-tration of Mn++ in the medium is from about 50 to about 1500 ppm.
The specific ingredients of the suitable stock, cul-ture, 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 h~an manganese superoxide dismutase or 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 or 2o analog or mutant thereof and then the protein fraction is treated to recover human manganese superoxide dismutase or 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 :3o containing the human manganese superoxide dismutase or analog or mutant thereof and the fraction is recovered.
The fraction is then treated to separately recover the human manganese superoxide dismutase or 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 about 7 .0 or 8 .0 . The cell s ar a then di sr upted and centrifuged. The resulting supernatant is heated for a period of about 30 to 120 minutes at a temperature between approximately 55 to 65°C, preferably for 45-75 minutes at 58 to 62°C and more preferably one hour at 60°C, and then cooled to below 10°C, preferably to about 4°C. Any precipitate which may form during cool-ing 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 30R, most preferably 10R. 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 2o 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 f rac-tions are then eluted through a ration exchange chrana-3o tography column having a 1 inear gradient of about 40 to about 200 mM potassium acetate (BOAC) 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 ~s ~ X41362 solution, e.g. water or a buffer solution of about 10 mM potassium phosphate hav ing a pH of about 7.8 .
The invention also concerns purified, i.e.
substantial 1y free of other substance s of human or igin, human manganese superoxide dismutase or analogs or mutants 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 1o has the amino acid sequence shown in Fig. 1, the N-terminus 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 pur if ied Met-hMnSOD hav ing a specific activity of 3500 units/mg.

1 ~41~fi2 The Examples which follow are set forth to aid in un-derstanding 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 to 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 focus-ing (IEF) gels. Such methods are well-known to those .or ordinary skill in the art and are described in nu-merous publications including by way of example the following:
T. Maniatis, E. F. Fritsch and J. Sombrook, Molecular Cloning,: ~ysbo~at~~~y Manual, Cold Spring Harbor Labo-ratory, New York (1982) .
J. M. McCord and I . Fr idov ich, J. BiQ~ 'hem. 2 44: 6049-55 (1969) .
C. Beauchamp and I . Fr idov ich, ~,al . Biochem. 44 : 276-87 (1971) .
:30 :35 ~ X41 X62 In order to identify MnSOD cDNA clones, mixed of igomer probes were synthesized according to the published amino acid sequence (18,19) .
5'-,robe - 30 mer sequence from AA15 AA24 (18,19) 5' 3' TTG CATAATTTG TG CCTT AATG TG TGGTT C
T G T G
G G
3'-probe - 32 mer sequence from AA179-AA189 (18) 5, 3' TCTGTTACGTTTTCCCAGTTTATTACGTTCCA
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 pr obe ) .
The 5'-probe was employed to screen 300,000 plaques of a T-cell cDNA library cloned into the ~ gt-10 vector.
Hybridization to phage plaque replicas immobilized on nitrocellulose filters was performed according to 341 ~fi2 standard procedures (Maniatis et al. ) 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 iso-lated and named Phi MS8, Phi MS1 and Phi. MS1J.
EcoRI digests of DNA from Phi MS8 and Phi MS1 showed that they both have cDNA inserts approximately 800 by long, which hybridize to both the 5' and 3' ofigonu cleotide probes. Phi MS1J carried only 450 bg cDNA
1o insert which hybridized only to the 5' end probe.
The SRI inserts of the three phage clones were sub-cloned into the ,SRI site of pBR322 thus yielding pMSB-4, pMSl-4 and pMSlJ, respectively. Restriction analysis and hybridization to the 5' and 3' oligonu-cleotide probes revealed similar patterns for both pMSB-4 and pMSl-4. The following restriction map showing the 5' ---~ 3' orientation has been deduced for both plasmids.

~~3~~~~2 S~

The sequence of the cDNA insert pMSB-4 is shown of in Fig. 1. The predicted amino differs from acid sequence io the publ fished amino acid sequence (19) in that Glu appears instead of Gln in three (3) locations (AA
42, 88, 108) and an additional two amino acids. Gly and Trp appear between AA123-124 Sequence analys is of pMSl-4 and pMSlJ revealed that the three MnSOD clones were independently derived and confirmed these differences fran the published amino acid sequence.

The sequence upstream of the N-terminal Lysine of ma ture MnSOD predicts a pre-peptide sequence of 24 amino 2o acids.

:35 C s ruc R ss The starting point for the construction of pMSE-4 is the plasmid pMSB-4 which was obtained as described in Example 1.
Plasmid pMSB-4, containing human MnSOD cDNA on an ~gRI
insert, was digested to completion with ~I and T~,~I
restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in Fig. 2.
The resulting plasmid, pMSB-NN contains the coding region for the mature MnSOD, preceded by an ATG initiation codon.
The above plasmid was digested with SRI, ends were filled in with Klenow fragment of Polymerise I and further cleaved with 1 eI. The small fragment containing the MnSOD gene was inserted into pSODal3 which was treated with T~gI and S,~I.
pSODal3 may be obtained as described in pending, co-assigned Canadian Patent Application No. 488,832, filed August 15, 1985. This generated plasmid pMSE-4 containing the MnSOD
coding region preceded by the cII ribosomal binding site and under the control of ~1 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.

1;41362 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 Lucia Broth (LB) medium containing 100 g/ml) of ampi-cill in unt i1 the Opti cal Densi ty (OD) at 6 00 nm was 0.7. Induction was performed at 42°C. Samples taken 1o at various time intervals were electrophoresed sepa-rated on sodium dodecyl sulfate - polyacrylamide gels electrophoresis (SDS-PAGE) . The gels showed increases in human MnSOD levels up to 120 minutes post-induc-tion, at which stage the recombinant MnSOD protein c~prised 27% of total cellular groteins as determined by scanning of Coomassie-blue stained gel. Sonication of samples for 90 sec. in a W-375* sonicator and parti-tioning of pr oteins to sol uble (s ) and non-sol uble (p) fractions by centrifugation at 10,000 g for 5 min.
2o revealed that most of the recombinant MnSOD produced was non-soluble. The induced soluble protein fraction contained only slightly more SOD activity than the uninduoed counterpart, as assayed by standard methods.
See McCord et al., . Apparently a portion of the ~gOD 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 ef-fect, inactive.
* trade mark.

~ 341 362 ++
Activity The addition of Mn++ in increasing concentrations up to 450 ppm to the growth media of E. coli A4255, contain-ing pMSE-4, prior to a 2 hr. induction at 42°C had no adverse effect on the overall yield of human MnSOD.
Analysis of sonicated protein fractions soluble (s) and to non-soluble (p) on sodium dodecyl sulfate - polyacryl-amide gel electrophoresis (SDS-PAGE) , showed increased solubil ization of the recombinant protein with in-creased Mn++ concentrations (Table 1) . An assay of SOD
activity (see McCord et al. , ) suggests a corre-i5 lation 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++ concen-trations activated previously inactive soluble enzyme.
2o 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 stain-ing of isoelectric focusing (IEF) gels (see Beauchamp 25 et al , . ) r eveal ed that mul ti f orms of the r ecom-binant MnSOD were identical to those of native human liver MnSOD.
Results for human MnSOD production by E. coli A1645 :3o Containing pMSE-4 were similar to those described above.
,3 S

~ ~413fi2 Mn++ Percent Percent Specific (ppm) Soluble Soluble Activity human Mn human Mn units/mg SOD of SOD of Sol ub1 a Total human Soluble Bac- Proteins MnSOD terial Proteins Induced io 0 30 .6 7 .2 30 50 72.7 15.4 241 100 78.0 16.9 356 150 82 .9 18 . 8 6 06 200 82.0 20.8 338 250 79.2 20.4 380 300 80.8 20.3 381 450 89.2 22.4 323 :z 0 :30 ~5 ~ 341362 R
TetR expression vector, ppRB, was generated from pSODs 1T-11 by complete digestion with SRI followed by partial cleavage with CHI restriction enzymes.
pSOD ~T-11 has been deposited with the American Type Culture Collection under Accession No. 53468. The 1o digested plasmid was ligated with synthetic oligomer 5'- AATTCCOGGGTCTAGATCT - 3' 3'- GGGCCCAGATCTAGACTAG - 5' 1s resulting in p~R.B containing the a PL promoter.
The EcoRI fragment of MnSOD expression plasmid pMSE-4, containing cII ribosomal binding site and the complete coding sequence for the mature enzyme, was inserted 2o into the unique SRI site of poRB. The resulting plasmid, pMSdRB4, contains the MnSOD gene under con-trol of ~ PL and cII RBS and confers resistance to tetracycline (Fig. 4) .
2s ;; S

~ 341362 Plasmid pMS~RB4 was introduced into Escherichia coli strain A4255, using known methods. Cultures were grown at 32°C in Luria Broth (LB) containing various concen-trations 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 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++ con-centration in growth media. This is in contrast with observations for the AmpR plasmid pMSE-4. ( See Example 4.) However, maximum SOD activity and expression level were dependent on Mn++ supplementation (Table 2) .

~jaSOD Egpression in E. Col i A4255 Ij~MS aRB4) ppm Mn+~ Percent Soluble Specific Activity hMnSOD Units/mg Sol ubl a of Soluble Proteins Bacter ial Pr oteins to 420 320 420 0 10.9 8.0 23 50 19 .8 8 .0 227 100 16.0 8.0 241 200 17.0 10.0 278 300 16.0 9.3 238 s E. coli strain A4255 harboring plasmid pMSQR84 was fermented in LB supplemented with 750 ppm Mn~+, at 32°C
to an A600 of 17Ø Induction of human MnSOD expres-sion was effected by a temperature shift to 42°C for 2 hour s at which stage the cul tur a r eached A600 of 43 .0 .
1o 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. Bact* ria were disrupted by a double passage through Dynomill, cen-trifuged and cell debris were discarded. The superna-is Cant was heated for 1 hour at 60°C, cooled to 4°C and the cleared supernatant was concentrated to 0.03 origi-nal volume and dialyzed against 2 mM potassium phos-phate buffer, pH 7.8, on a Pelicon ultra filtration unit equipped with a lOR membrane: The crude enzyme preparation was loaded onto a DE52*column, washed thor-oughly 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 con-taining human MnSOD were pooled, dialyzed against H20, 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) .
:35 *Trade Marks v 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 re-vealed about 0.77 atoms Mn per enzyme subunit. This is in accordance with publ fished data (23) .

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~ ~~~ X62 1. McCord, J.M. and Fridovich, I., J-Biol. (:hem.
244:6049-55 (1969) .
2. Fridovich, I. in A~yances ~n Inorganic Biochem-istry, eds. Eichhorn, G.L. and Marzilli, L.G.
(Elsevier/North Holland, New York) , pp. 67-90 (1979) .
l0 3 . Fr eeman, B. A. and Cr apo, J. D. , I~arQr atory In-ve~tion 47:412-26 (1982) .
4. Steinman, H. M. in S~,ineroxic~ Dismutase, ed.
Oberley, L.W. (CRC Press, Florida) , pp. 11-68 (1982) .
5. Hartz, J.W. and Deutsch, H. F. , J. yiol. Ch em.
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. , Schin ina, M.W., Rotilio, W.H., Bannister, W.H. and Bossa, F. , FEB,~Letters 120: 53-56 (I980) .

8 . Lieman-Hurwitz, J. , Dafni, N. , Lavie, V. and Groner, Y. , g,Loc. Natl. Acad. ~ci. USA 79:2808 11 (1982) .

9. Sherman, L. , Dafni, N. , Lieman-Hurwitz, J. and Groner, Y., Proc. Natl. Acad. yR~i. USA 80:5465-69 (1983).

10. Oberley, L.W. and Buettner, G. R., cancer Re-se~rch 39:1141-49 (1979).

11. Huber, W. and Menander-Huber, R. B. , C1 inics i,~
Rheum. Dis. 6:465-98 (1980) .

12. McCord, J. M. and Roy, R. S. , Can. J. Physiol .
Pharma. 60:1346-52 (1982).
l0 13. Alvarez, J.G. and Storey, B.T., Biol. Re rp od.
28:1129-36 (1983).
14. Tal masof f, J. M. , Ono, T. and Cutl er , R. G. , roc. Natl. Acad. Sci. U iA 77:2777-81 (1980) .
15. Lowry, 0. H. , Rosebrough, N. J. , Farr, A. L. and Randall, R.J., J. Biol,~ Chem. 193:265-75 (1951) .
2o 16. Weser, U, and Hartmann, H.J., FEBS Letters 17:78-80 (1971) .
17 . Jewett, S. L0. , Latrenta, G. S. and Beck, C. M. , Arc. Biochem. Bioghys. 215:116-128 (1982) .
18. Harris, J.I. an d Steinman, H.M., Superoxide an d yur_zeroxide Dismutase, Michelson, A. M., McCord, J. M. and Fridovich, I. eds. , Academic Press, London, pp. 225-230 (1977).
19. Barra, D., Schinina, M.E., Simmaco, M., Bannis-ter, J.V., Bannister, W.H., Rotilio, G. and Bossa, F. , J. B~.ol. Chem. 259:12595-601 (Octo-ber 25, 1984) .

20. Baret, A. , Jadot, G. , and Michelson, A. M. , R~ochem~ca~ Phar acoloav 33:2755-60 (September 1, 1984) .
21. McCord, J. M, and Sal in, M. L. , Movement, Metabo-~ ism aHd Bactericidal Mechanisms of Pha9~yjtes, Ross, A. , Patriar ca, P. L. , Rome a, D. (eds ) pp.
257-264 (1977) .
22. Touati, D. , Journal of acte;,yoloav 155: 1078-87 (1983) .
23 . McCord, J. M. , Boyle, J. A. , Day, Jr. , E. D. , Rizzolo, L.J. and Salin, M.L., Superoxi a and ~u~roxide Dismutase, Michaelson, A. M. , Mc-Cord, J. M., and Fridovich, I. (eds) Academic Press, London pp. 129-138 (1977).
24. European Patent Publication No. 0131843 Ai, 2o publ fished January 23, 1985, carresponding to European Patent' Application No. 84107717.5, filed July 3, 1984, which claims priority of U. S. Serial No: 514,188, filed July 15, 1983.
25. Hallewell, et al . , N'~l_.P,~ic Aci~,~ Res. 5, (1985) .
26. European Patent Publication 0138111 Al, pub-lished Agril 24, 1985, corresponding to Europe-3o 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.

~ 341362 27. E1~0 Journal, Vol. 4, No. 1, pp. 77-84 (January 1985 .
28. Abstracts of the Fourth International Confer-ence on Superoxide and Superoxide Dismutase, Rome, Italy, September 1-6, 1985.
1o

Claims (47)

1. A plasmid for expression in a suitable bacterial host cell of an enzymatically active human manganese superoxide dismutase analog wherein the analog consists 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. 1 and continuing to the lysine encoded by nucleotides 706-708 of Fig. 1 which is the COOH terminus of the polypeptide, the plasmid comprising DNA
encoding such polypeptide and suitable regulatory elements arranged within the plasmid so as to permit expression of the polypeptide and formation of the human manganese superoxide dismutase analog in the host cell.

2. A plasmid according to claim 1, designated pMSE-4, having the restriction map shown in Fig. 2 and deposited in Escherichia coli strain A4255 under ATCC Accession No. 52350.

3. A plasmid according to claim 1, designated pMS.DELTA.RB4, and having the restriction map shown in Fig. 4.

4. A bacterial cell into which the plasmid of claim 1 has been introduced.

5. A bacterial cell according to claim 4 containing the plasmid pMSE-4 and deposited under ATCC Accession No. 53250.

6. A bacterial cell according to claim 4 containing the plasmid pMS~RB4.

7. A method of producing a human manganese superoxide dismutase polypeptide which comprises treating a bacterial cell of claim 4 so that the DNA directs expression of the human manganese superoxide dismutase polypeptide in the bacterial cell and recovering from the bacterial cell the human manganese superoxide dismutase polypeptide so expressed.

8. A bacterially-produced polypeptide analog of human manganese superoxide dismutase of 199 amino acids comprising methionine attached to the N-terminus of a portion of the amino acid sequence of Fig. 1 the sequence of which is lysine encoded by nucleotides 115-117 of Fig. 1 and continuing to the lysine encoded by nucleotides 706-708 of Fig. 1 which is the COOH terminus of the polypeptide.

9. An analog of human manganese superoxide dismutase comprising two polypeptides, each in accordance with claim 8.

10. A method of producing a human manganese superoxide dismutase in accordance with claim 9 which comprises treating a bacterial cell containing DNA encoding and capable of directing expression of human manganese superoxide dismutase polypeptide so that the bacterial cell expresses the polypeptide and forms human manganese superoxide dismutase therefrom, and recovering from the bacterial cell the superoxide dismutase so expressed and formed.

11. Human manganese superoxide dismutase analog resulting from the expression in bacteria of the DNA sequence shown in Fig. 1 from nucleotide number 115 to nucleotide number 708.

12. A veterinary composition comprising human manganese superoxide dismutase in accordance with claim 9 and a carrier.

13. A pharmaceutical composition comprising human manganese superoxide dismutase in accordance with claim 9 and a carrier.

14. A method of catalyzing the reaction which comprises contacting the reactants with human manganese superoxide dismutase in accordance with claim 9.

15. A method of reducing injury to cells caused by superoxide radicals in vitro, which comprises catalyzing the reduction of the superoxide radicals in accordance with claim 14.

16. A method of prolonging the survival period of excised isolated organs which comprises adding human manganese superoxide dismutase in accordance with claim 9 to the perfusion medium.

17. A method of producing an enzymatically active human manganese superoxide dismutase in a bacterial cell which contains and is capable of expressing the superoxide dismutase having methionine at its N-terminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig. 1 and continuing to the lysine encoded by nucleotides 706-708 of Fig. 1 which comprises maintaining the bacterial cell in a production medium supplemented with an amount of Mn++ so that the concentration of Mn++ in the medium is greater than about 2 ppm.

18. A method according to claim 17, wherein the bacterial cell is an Escherichia coli cell.

19. A method according to claim 17, wherein the bacterial cell contains a plasmid, the plasmid containing the DNA
sequence encoding the human manganese superoxide dismutase incorporated therein.

20. A method according to claim 17, wherein the suitable production medium is a casein hydrolysate medium.

21. A method according to claim 17, wherein the suitable production medium is LB medium.

22. A method according to claim 17, wherein the Mn++
concentration is from 50 to 1500 ppm.

23. A method according to claim 22, wherein the Mn++
concentration is 150 ppm.

24. A method according to claim 22, wherein the Mn++
concentration is 750 ppm.

25. A method according to claim 18, wherein the bacterial cell is Escherichia coli strain A4255 containing plasmid pMSE-4 and deposited under ATCC Accession No. 53250.

26. A method according to claim 19, wherein the plasmid is pMSE-4 having the restriction map shown in Fig. 2 and deposited under ATCC Accession No. 53250.

27. A method according to claim 18, wherein the bacterial cell is Escherichia coli strain A4255 containing plasmid pMS.DELTA.RB4 having the restriction map shown in Fig. 4.

28. A method according to claim 19, wherein the plasmid is pMS.DELTA.RB4 having the restriction map shown in Fig. 4.

29. Bacterially-produced human manganese superoxide dismutase produced by the method of claim 17.

30. A method of recovering human manganese superoxide dismutase from bacterial cells harboring cloning vehicles which express human manganese superoxide dismutase having methionine at its N-terminus immediately adjacent to the lysine encoded by nucleotides 115-117 of Fig.1 and continuing to the lysine encoded by nucleotides 706-708 of Fig. 1 which comprises:

(a) isolating the bacterial cells from the production medium;
(b) suspending the isolated bacterial cells in a buffered solution;
(c) disrupting the suspended bacterial cells;
(d) separating soluble proteins from insoluble proteins and cell wall debris;
(e) recovering the soluble proteins;
(f) separating a fraction of the soluble proteins containing the human manganese superoxide dismutase;
(g) recovering the fraction of soluble proteins containing the human manganese superoxide dismutase;
and (h) treating the fraction of soluble proteins containing the human manganese superoxide dismutase so as to separately recover the human manganese superoxide dismutase.

31. A method of claim 30 wherein the buffered solution in step (b) has a pH of 7.0 t.o 3.0;
and wherein step (d) comprises centrifuging the disrupted bacterial cells obtained in (c) to obtain a supernatant containing said soluble proteins;
and wherein step (e) comprises recovering the supernatant;
and wherein step (f) comprises:

(i) heating the supernatant obtained in (e) for a period ranging from 30 to 120 minutes at a temperature ranging from 55 to 65°C;
(ii) cooling the heated supernatant to below 10°C;
and wherein step (g) comprises:
(i) removing any precipitate from the cooled supernatant obtained in (f); and (ii) dialyzing the cooled supernatant;
and wherein step (h) comprises:
(i) subjecting the supernatant obtained in step (g) to anion exchange chromatography wherein the retentate is eluted with a buffered solution;
(ii) collecting and pooling fractions of the eluent containing human manganese superoxide dismutase;
(iii) dialyzing the pooled fractions against 40 mM potassium acetate, pH 5.5;
(iv) 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 (v) collecting and pooling peak fractions of the eluent containing superoxide dismutase.

32 . A method according to Claim 31, wherein step (f) (i) comprises heating for 45 to 75 minutes at 58 to 62°C.

33. A method according to claim 31, wherein step (f)(i) comprises heating for 60 minutes at 60°C.

34. A method according to claim 31, wherein step (f)(ii) comprises cooling to 4°C.

35. A method according to claim 31, wherein in step (g)(i) the precipitate is removed by centrifugation.

36. A method according to claim 31, wherein the dialyzing in step (g)(ii) comprises ultra-filtration employing a filtration membrane smaller than 30K.

37. A method according to claim 31, wherein the dialyzing in step (g)(ii) is carried out against a 2 mM potassium phosphate buffer with a pH of about 7.8.

38. A method according to claim 31, wherein in step (h)(i) the buffered solution is a 2 mM potassium phosphate buffer with a pH of about 7.8.

39. A method according to claim 31, wherein the dialyzed supernatant obtained in step (g)(ii) is concentrated to a smaller volume.

40. A method according to claim 39, wherein the smaller volume is 0.03 of the supernatant's original volume.

41. A method according to claim 31 wherein step (h)(v) further comprises dialyzing the pooled peak fractions.

42. A method according to claim 41 wherein the dialyzing is carried out against a solution of 10mm potassium phosphate buffer having a pH of 7.8.

43. Human manganese superoxide dismutase purified by the method of claim 30.

44. A non-naturally-occurring molecule having human superoxide dismutase activity comprising a human manganese superoxide dismutase polypeptide of claim 8.

45. A polypeptide manganese complex comprising a human manganese superoxide dismutase polypeptide of claim 8 in a complex with manganese in any of its chemmical forms, which complex has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.

46. A purified human manganese superoxide dismutase analog according to claim 9, having a specific activity greater than about 3500 units/mg.

47. Use of the human manganese superoxide dismutase analog according to claim 9, for reducing injury to cells caused by superoxide radicals, or for preparing a medicament therefor.