GB2183658A - Human manganese superoxide dismintase cDNA, its expression in a host and method of recovery - Google Patents

Description

1 GB 2 183 658 A 1

SPECIFICATION

4 Human manganese superoxide dismutase cDNA, its expression in bacteria and method of recovering enzymatically active human manganese superoxide dismutase Throughout this application, 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 hereby incorporated by reference into 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 (02) was discovered in 1968 by McCord aand Fridovich (1). Superoxide radicals and other highly reactive oxygen species are produced in every respiring 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 + 2W - H202 + 02 and 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 20 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 therapeutic 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 25 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 metal lie-protein composed of identical noncovalently linked subunits, each having a molecular weight of 16,000 daltons 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 eDNA 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-1 2 has recently been cloned and mapped (22). Barra et a]. disclose a 196 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 identical polypeptide subunits (19,23). It is clear, 50 however, 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 half-life 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). 55 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 the human manganese superoxide dismutase polypeptide or an analog or mutant thereof. It is also directed to inserting this cDNA into 60 efficient bacterial expression vectors, to producing human MnSOD polypeptide, analog, mutant and enzyme in bacteria, to recovering the bacterially produced human MnSOD polypeptide, analog, mutant or enzyme. This invention is also directed to the human MnSOd polypeptides, analogs, or mutants thereof so recovered and their uses.

This invention further provides a method for producing enzymatically active human MnSOD in bacteria, as 65 2 GB 2 183 658 A 2 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 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 5 concerns the use of bacterially produced MnSOD or analogs thereof to treat inflammation& Summary of the invention

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 Figure 1 from nucleotide 115 downstream to nucleotide 708 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 15 prepeptide is also shown in Figure 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, 20 using known methods, including but not limited to methods involving cloning vehicles containing either molecule.

Preferably the eDNA or DNA encoding the human manganese superoxide dismutase polypeptide or analog or mutant thereof is incorporated into a plasmid, e.g., pMSE-4 or pMSARB4, and then introduced into a suitable host cell where the DNA can be expressed and the human manganese superoxide dismutase (hMnSOD) polypeptide or analog or mutant thereof produced. Preferred host cells include Escherichia coli, in particular E.

coliA4255 and E. coli All 645. 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 pMSAR134 may be obtained as shown in Figure 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 resulting manganese superoxide dismutase protein may then be recovered.

Veterinary and pharmaceutical compositions containing human MnSOD or analogs or mutants thereof and suitable carriers may also be prepared. This human manganese superoxide dismutase or analogs or mutants may 35 be used to catalyze the following reaction:

20, + 2H -, H201 + 02 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 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' available to the cell in the medium is greater than about 2 ppm.

In a preferred embodiment of the invention the bacterial cell is an Escherichia col! cell containing a plasmid which contains a DNA sequence encoding for the human manganese superoxide dismutase polypeptide e.g.

pMSE-4 or pMS R134 in E. col! strain A4255. The concentration of Mn' in the production medium ranges from aboult 50 to about 1500 ppm, with concentrations of 150 and 750 ppm being preferred.

Tffis 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 55 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 cel 1 wall debris and the soluble proteins are recovered. The soluble proteins are then treated to separate, e.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 disi-nutase or analog or mutant thereof from bacterial cells which contain human manganese superoxide d.Sm.ulase or analog or mutant thereof. The method involves f irst isolating the bacterial cells from the production mediuna and suspending them in suitable solution having a pH of about 7.0 to 8.0. The cells are then disrupted 65 4; 1 3 GB 2 183 658 A and centrifuged and the resulting supernatant is heated for about 30 to 120 minutes at a temperature between 55 and WC, preferably for 45-75 minutes at 58-62'C and more preferably for 1 hour at WC and then cooled to below 1 O'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 5 of about 7.8. Preferably, the dialysis is by ultrafiltration using a filtration membrane smaller than 30K. 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 acetate, pH 5.5 The diaiyzed pooled fractions are10 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 or analogs 15 thereof e.g. met-hMnSOD, or mutants produced by the methods of this invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The sequence of human MnSOD cDNA Figure 1 shows the nucleotide sequence of one strand of a double-stranded DNA molecule encoding the 20 human manganese superoxide dismutase as well as the 198 amino acid sequence of human MnSOD corresponding to the DNA sequence. Figure 1 also shows the nueleotide 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.

Figure 2. Construction of pMSE-4.. human MnSOD expression plasmid Plasmid pMS8-4, containing MnSOD on an EcoRI (R,) insert, was digested to completion with Noel and Narl restriction enzymes. The large fragment was isotated and ligated with a synthetic oligomer as depicted in Figure 2. The resulting plasmid, pIVIS8-NN contains the coding region for the mature MnSOD, preceded by an ATG 30 initiation codon. The above plasmid was digested with EcoR I, ends were filled in with Klenow fragment of Polymerase 1 and further cleaved with Noel. The small fragment harboring the MnSOD gene was inserted into pSODal 3 which was treated with Noel and Stul. pSODocl 3 may be obtained as described in pending co-assigned U.S. Patent Application Serial No. 644,245, filed August 27, 1984 which is hereby incorporated by reference. This generated plasmid pIVISE-4 containing the MnSOD coding region preceded by the cl] ribosomal binding site and under the control of A PL promoter. Plasmid pIVISE-4 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250.

Figure3. Effect of Mn' concentration on the activity of SOD produced in E. coli The chart in Figure 3 shows the correlation between the specific activity in units/mg of recombinant soluble 40 MnSOD produced by E. coli strain A4255 containing plasmid pIVISE-4 under both non-induction (32'C) and induction (42C) conditions, and the concentration of Mn' (parts per million) in the growth medium.

Figure 4. Construction of pMSAR84.. human MnSOD expression plasmid TetR expression vector, pAQB, was generated from pSODfilTA 1 by complete digestion with EcoRI followed by 45 partial cleavage with BamHI restriction enzymes. pSODfilTA 1 has been deposited with the American Type Culture Collection (ATCC) under Accession No. 53468. The digested plasmid was ligated with synthetic oligomer W- AAMCCGGGMAGATCT - 3' W- G G G CCCAGATCTAGACTAG - 5' 50 resulting in pARB containing theZ PL. promoter.

The EcoRI fragment of MnSOD expression plasmid pIVISE-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 A PL and 0 RBS and confers resistance to tetracycline.

Detailed description of the invention

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 DNA library. The nucleotide sequence of 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 Figure 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 Figure 1. Nucleotides numbers 43 x 4 GB 2183 658 A 4 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 5 DNA molecules containing portions of this sequence.

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. Similarly, 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 Figure 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 15 plasmid, e.g. pIVISE-4 or pMS RB4. These cloning vehicles may be introduced in cells, either procaryotic, e. g., bacterial (Escherichia coli, 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 or analog or mutant thereof if the cDNA was present in the cloning vehicle or will contain DNA which includes a strand, all 20 or a portion of which has the sequence for human MnSOD polypeptide shown in Figure 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 auxottrophic strain of E. coli is A1 645 which has been deposited with the American Type Culture Collection in RockAlle, Maryland, U.S.A. containing plasmid pApoE-Ex2, under ATCC Accession No. 39787. All deposits 25 with the American Type Culture Collection referred to in this application were made pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

AI 645.was obtained from AI 637 by selection for Gall (ability to ferment galactose) as well as loss of tetracycline resistance. it still contains elements of phage A. Its phenotype is C600 r-m' gall thr- leu- lacZ- bl (Acl 857 AHI ABamHI W).

AI 637 was obtained from C600 by inserting transposon containing tetracycline resistance gene into the galactose operon as well as elements of phage A 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 plVISE-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 or 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 or analog or mutant thereof, and the cell expresses the hMnSOD popypeptide or analog or mutant thereof which may then be recovered.

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:

20, + 2 W, 0, + H,O, 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 Figure 1; the N-terminus of the sequence is the lysine encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of the sequence is the lysine encoded by nucleotides 706-708 of Figure 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 natural ly- 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 natural ly- occurring human manganese superoxide dismutase. 60 The term -human manganese superoxide dismutase polypeptide analog" as used herein means a polypeptide which includes a human manganese superoxide 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 polypaptide manganese complex, the polypeptide portion of which includes one or more additional amino acids 65 4 1 GB 2 183 658 A 5 attached to it at either or both ends.

The term---humanmanganese 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 5 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 10 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 Figure 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 706-708.

The DNA may be mutated by methods known to those of ordinary skill in the art, e.g. Bauer et al., Gene 37: 20 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 Figure 1 for human manganese superoxide dismutase, the N-terminus of the sequence being the lysine encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of the sequence being the lysine encoded by nucleotides 706-708 of Figure 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 30 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 peroxide as shown in the following equation:

human MnSOD 202 + 2H+) H,O, + 0, 35 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 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.g., to treat a subject afflicted by inflammations or to reduce injury 40 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 injury to an isolated organ to oxygen-free radicals on perfusion after excision, thus prolonging the survival period of the organ. Additionally, the hMnSOD or analog or mutant thereof may be used to reduce neurological injury on reperfusion following ischemia and to treat borchial pulmonary dysplasia.

A method of producing enzymatically active human manganese superoxide dismutase or an 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 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 50 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 sequence and producing the protein product. The suitable conditions andproduction medium will vary according to the species and strain of bacterium.

The bacterial cell may contain the DNA sequence encoding the superoxide dismutase or 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 auxotrophic strain of E. coli is Al 645. 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 or an 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 Description 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 pIVIS ARB4. A 6 GB 2 183 658 A 6 method of constructing this plasmid is described in the Decsription of the Figures and the plasmid itself is described in Example 5. This plasmid may be constructed from pSODfilTA 1 which has been deposited with the American Type Culture Collection under Accession No. 53468.

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 pIVISE-4 and by E. colf strain A4255 cell 5 containing the plasmid pMSARB4.

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 witha the strain of E. colf and the plasmid it contains, for example E. co/1 A4255 containing plasmid pIVISE-4 is induced at 42'C and maintained at that temperature from about 1 to about 5 hours. The suitable conditions of 10 temperature, time, agitation and aeration for growing the inoculum and from 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 1-B-type growth media Mnll 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 Mnll in the medium is from about 50 to about 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 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 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 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 cells are then disrupted and centrifuged. The resulting supernatant is heated for a period of about 30 to 120 minutes at a temperature between approximately 55 to WC, preferably from 45-75 minutes at 58 to 62C and more preferably one hour at WC, and then cooled to below 1 WC, 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 diaiyzed by ultrafiltration employing a filtration membrane smaller than 30K, most preferably 1 OK. Appropriate buffers include 2 m M 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 40 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 chromatography column having a linear gradient of about 40 to about 200 mM potassium acetate (KOAC) and a 45 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 or analogs or mutants thereof produced by the methods of this invention. In 50 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 Figure 1, the N-terminus of which sequence is the lysine encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 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 55 activity of 3500 units/mg.

1 Examples

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 60 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 veck-or 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 7 GB 2 183 658 A 7 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 1. Fridovich, J. 8161. Chem. 244:6049-55 (1969). 5 C. Beauchamp and 1. Fridovich, Anal. Biochem. 44:276-87 (1971).

0 C EXAMPLE 1

In order to identify MnSOD cDNA clones, mixed ofigomer probes were synthesized according to the published amino acid sequence (18,19).

5'-probe - 30 mer sequence from AA1r, - AA24 (18,19) 5' 3' TTGCATAAMGTGCCTTAATGTGTGGTTC T G T G G G 3.probe - 32 mer sequence from AAM-AA1.9 (18) 5' 31 TCT(3TTACGTMCCCAGTTTATTACGTTCCA 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 25 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 A gt-10 vector. 30 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 &SSC for 16 hrs. The filters were then washed at 50'C with 5xSSC and 0.1 % SDS. Three positive plaques were isolated 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 35 800 bp long, which hybridize to both the 5' and 3' oligonucleotide probes. Phi MS1 J 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 plVIS8-4, pMS1 -4 and pMS1 J, respectively. Restriction analysis and hybridization to the 5' and 3' oligonucleotide probe revealed similar patterns for both pMS8-4 and pMS1 - 4. The following restriction map 40 showing the 5'-----> 3' orientation has been deduced for both plasmids.

51 EvilI,.5 ca i T T,, 1 200 300 400 500 600 700 800 R1 45 The sequence of the cDNA insert of pMS8-4 is shown in Figure 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 AA123-124. Sequence analysis of pMS1-4 and pMSU revealed that the three MnSOD clones were independently derived and confirmed these 55 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.

EXAMPLE 2

Construction of p[v1SE-4.. AMpR Human MnSOD expression Plasmid The starting point for the construction of pMSE-4 is the plasmid pIVIS8-4 which was obtained as described in Example 1. Plasmid pMS8-4, containing human MnSOD cDNA on an EcoRI insert, was digested to completion with. Ndel and Narl restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in Figure 2. The resulting plasmid, pMS8-NN contains the coding region for the mature MnSOD, 65 8 GB 2 183 658 A 8 preceded by an ATG initiation codon. The above plasmid was digested with ECoRI, ends were filled in with Klenow fragment of Polymerase 1 and further cleaved with Noel. The small fragment containing the MnSOD gene was inserted into pSOD 13 which was treated with Noel and Stul. pSOD 13 may be obtained as described in pending, co-assigned U.S. Patent Application Serial No. 644,245, filed August 27, 1984 which is hereby incorporated by reference. This generated plasmid pMSE-4 containing the MnSOD coding region preceded by the cl I ribosomal binding site and under the control of A PL promoter. Plasmid pIVISE-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 pIVISE-4 was introduced into Escherichia coli strain A4255 using known methods. Then the E. col! strain 4255, containing pMSE-4, were grown at WC in Luria Broth (LB) medium containing 100 g/m]) of ampicillin until the Optical Density (OD) at 600 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 15 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 c;eiermined 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 20 contained only slightly more SOD activity than the uninduced counterpart, as assayed by standard methods. See McCord el 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 + + in Growth Media on MnSOD Solubility and Activity The addition of Mn+ 1 in increasing concentrations up to 450 ppm to the growth media of E. colf A4255, containing 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 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 Mnll concentration in the media (Figure 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 35 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. col/A1 645 containing pIVISE-4 were similar to those described above.

TABLE 1

Mn++ Percent Soluble Percent Soluble Specific Activity (PPM) human Mn SOD of human Mn SOD of unitsImg 45 Total human Soluble Bacterial Soluble Proteins MnSOD Induced Proteins 9 0 30.6 7.2 30 50 72.7 15.4 241 50 78.0 16.9 356 82.9 18.8 606 82.0 20.8 338 t51 250 79.2 20.4 380 300 80.8 20.3 381 55 450 89.2 22.4 323 EXAMPLE 5 Cons!ruction of pfvlSAR84.. Tet' Human MnSOD Expression Plasmid Tell expression vector, pARB, was generated from pSODfilTA 1 by complete digestion with Ecoill followed by 60 partial cleavage with BamHI restriction enzymes. pSODfl1TA 1 has been deposited with the American Type Culture Collection under Accession No. 53468. The digested plasmid was ligated with synthetic oligomer W AATT=G G GTWAGATCT - 3' 3'm-- G G G CCCAGATC-1 AGACTAG - 5' 65 9 GB 2 183 658 A 9 resulting in pARB containing the X 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 into the unique EcoRI site of pARB. The resulting plasmid, pMSARB4, contains the MnSOD gene under control of A PL and cII RBS and confers 5 resistance to tetracycline (Figure 4).

EXAMPLE 6 Expression of Human MnSOD from p/14SARB4 Plasmid pMSARB4 was introduced into Escherichia coli strain A4255, using known methods. Cultures were grown at 32% in Luria Broth (LB) containing various concentrations of Mn", until the Optical Density (OD) at 10 600 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" concentration 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).

TABLE2

MnSOD Expression in E. Coli A4255 (pMSAR84) 20 ppmMn++ Percent Soluble hMnSOD of Specific Activity UnitsImg Soluble Bacterial Proteins Soluble Proteins 42' 32' 42' 25 0 10.9 8.0 23 19.8 8.0 227 16.0 8.0 241 17.0 10.0 278 300 16.0 9.3 238 30 EXAMPLE 7

Purification of Enzymatically Active Recombinant Human MnSOD E. coli strain A4255 harboring plasmid pMSAR B4 was fermented in LB supplemented with 750 ppm Mn' 1, at 32% to an A600 of 17.0. Induction of human MnSOID expression was effected by a temperature shift to 42'C for 2 hours at which stage the culture reached A600 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 NaCi. Bacteria were disrupted by a double passage through Dynomill, centrifuged and cell debris were discarded. The supernatant was heated for 1 hour at WC, 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 1 OK 40 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 H20, adjusted to 10 mM potassium phosphate buffer, pH 7.8 and frozen at - WC. 45 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 revealed about 0.77 atoms Mn per anzyme subunit. This is in 50 accordance with published data (23).

TABLE3

Purification of Recombinant Human Mn-SOD 55 stop Total Proteins Yield Specific Activity gm gmSOD % unitsImg Dynomill supernatant 100.0 11.9 100.0 417 60 WC supernatant 24.0 8.2 68.9 1197 Pelicon retentate 20.0 7.5 63.0 1350 DE52 eiuate 7.3 5.7 48.0 2732 CM52 eluate 4.2 4.2 35.3 3500 Values for enzyme purified from 15 L fermentation.

GB 2 183 658 A References 1. McCord, J.M. and Fridovich, L, J. Biol. Chem. 244:6049-55 (1969).

2. Fridovich, 1. in Advances in Inorganic Biochemistry, eds. Eich horn, G. L. and M arzi 11 i, 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.K (CRC Press, Florida), pp. 11 -68 (1982).

5. 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. Liernan-Hurwitz, 1, Dafni, N., Lavie, V. and Groner, Y., Proc. Nad. Acad. Sci. USA 79:2808-11 (1982).

9. Sherman, L., Dafni, N_ Lieman-Hurwitz, J. and Groner, Y., Proc. Nad. Acad. Sci. USA 80:5465-69 (1983).

10. Oberley, L.W and Buettner, G. R., Cancer Research 39:1141-49 (1979). 15 11. Huber, W. and Menander-Huber, K.B., Clinics in Rheum. Dis. 6:46598 (1980). 12. McCord, J.M. and Roy, R.S., Can. J. Physiol. Pharma. 60:1346-52 (1982). Alvarez, J.G. and Storey, B.T., Biol. Reprod. 28:112936 (1983). '4. Talmasoff, J.M., Ono, T. and Cutler, R.G., Proc. Nad. Acad. Sci. USA 77:2777-81 (1980). 15. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J Biol. Chem. 193:265-75 (1951). 20 16. Weser, U. and Hartmann, H.J., FEBS Letters 17:78-80 (1971). 17. Jev,,fett, S.LO., Latrenta, G.S. and Beck, C.M., Arc. Biochem. Biophys. 215:116-128 (1982).

18. Harris, J. 1. and Steinman, H. M., Superoxide and Superoxide Dismutase, M ichelson, A. M., McCord, J. M. and Fridovich, 1. eds., Academic Press, London, pp. 225-230 (1977).

19. Barra, D., Schinina, M.E., Simmaco, W Bannister, J.V., Bannister, W.H. , Rotilio, G. and Bossa, F.,J.

Biol. Chem. 259:12595-601 (October 25,1984).

13.

20. Baret, A., Jadot, G., and Michelson, A.M., Biochemical Pharmacology 33:2755-60 (September 1, 1984).

21. MicCord, J. M. and Sal in, M. L., Movement, Metabolism and Bactericidal Mechanisms of Phagocytes, Ross, A., Patriarea, P.L., Romeo, D. (eds) pp. 257-264 (1977).

22. Touati, D_ Journal of Bacteriology 155:1078-87 (1983).

23. PAcCord, J.M., Boyle, J.A., Day, Jr., E.D., Rizzolo, L.J. and Salin, M.L., Superoxide and Superoxide 30 Dismutase, Michaelson, A.M., McCord, J.M., and Fridovich, 1. (eds) Academic Press, London pp. 129-138 (1977).

24. European -Patent Publication No. 0131843 A], published January 23, 1985, corresponding 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. 111allewell, et a[., Nucleic Acids Res. 5, (1985).

26. European Patent Publication 0138111 AI, published April 24,1985, corresponding to European 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.

C Ull! N1 S 11 1 1. Purified DNA encoding human manganese superoxide dismutase polypeptide or an analog or mutant thereof.

2. A DNA molecule which comprises cDNA encoding human manganese superoxide dismutase polypeptide or an analog or mutant thereof.

3. A double-stranded DNA molecule which encodes a human manganese superoxide dismutase pre- 50 polypeptide or an analog or mutant thereof, one strand of which comprises substantially the nucleotide sequence shown in Figure 1 from nucleotide number 43 downstream to nucleotide number 708.

4. Adouble-stranded DNA molecule which encodes human manganese superoxide dismutase polypeptide or an analog or mutant thereof, one strand of which comprises substantially the nucleotide sequence shown in Figurel from nucleotide number 115 downstream to nucleotide number 708 inclusive.

5. A cloning vehicle which comprises the DNA molecule of claim 1.

6. A plasmid which comprises the DNA molecule of claim 1.

7. A plasmid according to claim 6 designated pMSE-4, having the restriction map shown in Figure 2 and deposited in E. colf strain A4255 under ATCC Accession No. 53250.

8. A plasmid according to claim 6, designated pMSARB4, and having the restriction map shown in Figure 4. 60 9. A cell into which the DNA of claim 1 has been introduced. iG. A procaryotic cell according to claim 9. 1 1. A bacterial cell according to claim 9. 12. A cell according to claim 11 containing the plasmid pMSE-4 and deposited under ATCC Accession No.

53250.

1 4 11 GB 2 183 658 A 11 13. A cell according to claim 11 containing the plasmid pIVISAR B4.

14. A eukaryotic cell according to claim 9.

15. A method of producing a human manganese superoxide dismutase polypeptide oran analog or mutant thereof which comprises treating a cell according to claim 9 so that the DNA directs expression of the human manganese superoxide dismutase polypeptide or analog or mutant thereof and the cell expresses the human manganese superoxide dismutase polypeptide or analog or mutant thereof and recovering from the cell the human manganese superoxide dismutase polypeptide or analog or mutant thereof so expressed.

16. A purified polypeptide comprising 222 amino acids having the sequence shown in Figure 1, the N-terminus of which sequence is the methionine encoded by nucleotides 43- 45 of Figure 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 1.

17. A purified polypeptide consisting essentially of 222 amino acids having the sequence shown in Figure 1, the N-terminus of which sequence is the methionine encoded by nucleotides 43-45 of Figure 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 1.

18. A bacterially-produced polypeptide comprising 198 amino acids having the portion of the amino acid sequence shown in Figure 1, the N-terminus of which sequence is the lysine encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 1.

19. A bacterial ly- produced polypeptide free of substances of human origin which polypeptide comprises 198 amino acids having the portion of the amino acid sequence shown in Figure 1, the N-terminus of which sequence is the lysine encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 1.

20. A bacterial ly- produced polypeptide consisting essentially of 198 amino acids having the portion of the amino acid sequence shown in Figure 1, the N-terminus of which polypeptide is encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of which polypeptide is the lysine encoded by nucleotides 706-708 of Figure 1.

21. A bacterially- produced polypeptide free of substances of human origin consisting essentially of 198 25 amino acids having the portion of the amino acid sequence shown in Figure 1, the N-terminus of which polypeptide is the lysine encoded by nucleotides 115-117 of Figure 1 and the COOH terminus of which polypeptide is the lysine encoded by nucleotides 706708 of Figure 1.

22. Human manganese superoxide dismutase comprising at least two polypeptides each in accordance with claim 19.

23. A method of producing a polypeptide in accordance with claim 18 which comprises treating a cell containing DNA encoding and capable of directing expression of the polypeptide so that the cell expresses the polypeptide, and recovering from the cell the polypeptide so expressed.

24. Human manganese superoxide dismutase or an analog thereof resulting from the expression in bacteria of the DNA sequence shown in Figure 1 from nucleotide number 115 downstream to nucleotide number 708. 35 25. A polypeptide (a) encoded by human manganese superoxide dismutase DNA; (b) capable of being produced in bacteria; and (c) capable of catalyzing the following reaction:

20,- + 2H±--.H202 + 02 26. A veterinary composition comprising an effective amount of a human manganese superoxide dismutase in accordance with claim 22 and a suitable carrier.

27. A pharmaceutical composition comprising an effective amount of a human manganese superoxide 45 dismutase in accordance with claim 22 and a suitable carrier.

28. A method of catalyzing the reaction 20z + 2H + --- H,O, + 02 which comprises contacting the reactants under suitable conditions with human manganese superoxide dismutase or analog thereof in accordance with claim 24.

29. A method of reducing injury to cells caused by superoxide radicals which comprises catalyzing the reduction of the superoxide radicals in accordance with claim 28.

30. A method of reducing injury to a subject occurring upon reperfusion following ischemia which comprises administering to the subject an effective amount of human manganese superoxide dismutase or analog thereof in accordance with claim 24.

31. A method of prolonging the survival period of excised isolated organswhich comprises adding an effective amount of human manganese superoxide dismutase in accordance with claim 24 to the perfusion medium.

32. A method of treating a subject afflicted with inflammations which comprises administering to the subject an effective amount of human manganese superoxide dismutase or an analog thereof in accordance with claim 24.

33. A method of producing enzymatically 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 65 12 GB 2 183 658 A 12 dismutase or analog which comprises maintaining the bacterial cell under suitable conditions and in a suitable production medium, the production medium being supplemented with an amount of Mn' so that the concentration of Mn' 1 in the medium is greater than about 2 ppm.

34. A method according to claim 33, wherein the bacterial cell is an Escherichia coli cell.

35. A method according to claim 33, wherein the bacterial cell contains a plasmid, the plasmid containing the DNA sequence encoding the manganese superoxide dismutase or analog incorporated therein.

36. A method according to claim 33, wherein the suitable production medium is a casein hydroiysate medium.

37. A method according to claim 33, wherein the suitable production medium is LB medium.

38. A method according to claim 33, wherein the Mn' concentration is from about 50 to about 1500 ppm. 10 39. A method according to claim 38, wherein the Mn' concentration is about 150 ppm.

40. A method according to claim 38, wherein the Mn' concentration is about 750 ppm.

41. A method according to claim 34, wherein the bacterial cell is Escherichia coli strain A4255 containing plasmid pIVISE-4 and deposited under ATCC Accession No. 53250.

42. A method according to claim 35, wherein the plasmid is pIVISE-4 having the restriction map shown in 15 Figure 2 and deposited under ATCC Accession No. 53250.

A3. A method according to claim 34, wherein the bacterial cell is Escherichia coli strain A4255 containing p!asmid pIVISARB4 having the restriction map shown in Figure 4.

44. A method according to claim 35, wherein the plasmid is pIVISARB4 having the restriction map shown in Figure 4.

45. Bacterially-produced human manganese superoxide dismutase or analog or mutant thereof.

46. A polypeptide having substantially the same amino acid sequence as human superoxide dismutase polypeptide, said poiypeptide having been prepared in a unicellular microorganism.

47. A method of recovering human manganese superoxide dismutase or analog thereof from bacterial cells which contain human manganese superoxide dismutase which comprises:

(a) treating the bacterial cells so as to recover a protein fraction containing proteins present in the cells including human manganese superoxide dismutase; and (b) treating the protein fraction so as to recover the human manganese superoxide dismutase.

48. A method in accordance with claim 47 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; (e) treating the fraction of soluble proteins containing the human manganese superoxide dismutase or analog thereof so as to separately recover the human manganese superoxide dismutase or analog thereof.

49. A method of claim 47 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 about 7.0 to about 8.0; 40 (c) disrupting the suspended bacterial cells; (d) centrifuging the disrupted bacterial cells; (e) heating the resulting supernatant for a period ranging from about 30 to about 120 minutes at a temperature ranging from about 55 to about WC; (f) cooling the heated supernatantto below about 'ITC; (9) 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; 50 dialyzing the pooled fractions against about 40 mM potassium acetate, pH 5.5; (1) eluting the dialyzed pooled fractions through a cation exchange chromatography column with a linear gradient of about 40 to about 200 mM potassium acetate, pH 5.5; (m) collecting and pooling peak fractions of the eluent containing superoxide dismutase.

50. A method according to claim 49, wherein in step (e), the resulting supernatant is heated for about 45 to 55 about 75 minutes at about 58 to about 62T.

51. A method according to claim 49, wherein the supernatant is heated for about 60 minutes at about 60T.

52. A method according to claim 49, wherein instep (f), the heated supernatant is cooled to about 4T.

53. A methrd according to claim 49, wherein in step (g), the precipitate is removed by centrifugation.

54. A method according to claim 49, wherein in step (h), the cooled supernatant is dialyzed by ultra-filtration 60 employing a filtration membrane smaller than 30K.

55. A method according to claim 49, wherein in step (h), the appropriate buffer is a 2 mM potassium p,iespha'út-. buffer having a pH of about 7.8.

56. A method according to claim 49, wherein in step (i), the buffered solution is at least 20 mM potassium phosphate and has a pH of about 7.8.

13 GB 2 183 658 A 13 57. A method according to claim 49, wherein after dialyzing the cooled supernatant, the dialyzed supernatant is concentrated to an appropriate volume; 58. A method according to claim 57, wherein the appropriate volume is about 0.03 of the supernatant's original volume.

59. A method according to claim 49 further comprising dialyzing the pooled peak fractions against an 5 appropriate solution.

60. A method according to claim 59, wherein the appropriate solution is H20.

61. A method according to claim 59, wherein the appropriate solution is a buffer solution of about 10 mM potassium phosphate buffer having and a pH of about 7.8.

62. A method in accordance with claim 49, wherein the superoxide dismutase is human superoxide 10 dismutase.

63. A method in accordance with claim 49, wherein the superoxide dismutase is human manganese superoxide dismutase.

64. Human manganese superoxide dismutase or an analog thereof purified by the method of claim 47.

65. Human manganese superoxide dismutase substantially free of other substances of human origin. 15 66. A non -natural ly-occurri ng molecule having human manganese superoxide dismutase activity comprising at least two human manganese superoxide dismutase polypeptides.

67. Human manganese superoxide dismutase polypeptide comprising a polypeptide of 198 amino acids, a portion of the amino acid sequence of which is shown in Figure 1, the N- terminus of which sequence is the lysine encoded by nucieotides 115-117 of Figure 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 1.

68. A polypeptide manganese complex comprising a human manganese superoxide dismutase polypeptide in a complex with manganese in any of its chemical forms which complex has the enzymatic activity of natural lyoccurring human manganese superoxide dismutase.

69. Human manganese superoxide dismutase comprising at least two human manganese superoxide dismutase polypeptides in a complex with manganese in any of its chemical forms and having the enzymatic activity of natural ly-occu rri ng human manganese superoxide dismutase.

70. A human manganese superoxide dismutase polypeptide analog comprising a human manganese superoxide dismutase polypeptide to either or both ends of which one or more additional amino acids are attached.

71. A polypeptide manganese complex analog comprising a polypeptide manganese complex, the polypeptide portion of which includes one or more additional amino acids attached to it at either or both ends.

72. A human manganese superoxide dismutase analog comprising 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 dismutase analog has the enzymatic activity of natural ly- occurring human 35 manganese superoxide dismutase.

73. A human manganese superoxide dismutase polypeptide mutant comprising 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.

74. A polypeptide manganese complex mutant comprising human manganese superoxide dismutase polypeptide mutant in a complex with manganese in any of its chemical forms and which mutant has the enzymatic activity of manganese superoxide dismutase.

75. A human manganese superoxide dismutase mutant comprising 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 45 manganese superoxide dismutase.

76. A human manganese superoxide dismutase analog comprising at least two polypeptides, at least one of which has the amino acid sequence shown in Figure 1, the N-terminus of which sequence is the lysine encoded by nucleotides 115-117 of Figure 1, and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Figure 1, plus an additional methionine residue at the N-terminus.

77. The purified human manganese superoxide dismutase analog of claim 76 having a specific activity greater than about 3500 units/mg Printed for Her Majesty's Stationery Office by Croydon Printing Company (U K) Ltd, 4187, D8817356. Published by The Patent Office, 25 Southampton Buildings, London, WC2A 'I AY, from which copies may be obtained.