AU607897B2 – Human manganese superoxide dismutase cDNA and its expression in bacteria and method of recovering enzymatically active human manganese superoxide dismutase
– Google Patents
AU607897B2 – Human manganese superoxide dismutase cDNA and its expression in bacteria and method of recovering enzymatically active human manganese superoxide dismutase
– Google Patents
Human manganese superoxide dismutase cDNA and its expression in bacteria and method of recovering enzymatically active human manganese superoxide dismutase
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AU607897B2
AU607897B2
AU64975/86A
AU6497586A
AU607897B2
AU 607897 B2
AU607897 B2
AU 607897B2
AU 64975/86 A
AU64975/86 A
AU 64975/86A
AU 6497586 A
AU6497586 A
AU 6497586A
AU 607897 B2
AU607897 B2
AU 607897B2
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Prior art keywords
superoxide dismutase
analog
manganese superoxide
human manganese
human
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1985-11-22
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AU6497586A
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Yaffa Beck
Jacob R. Hartman
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Savient Pharmaceuticals Inc
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Savient Pharmaceuticals Inc
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1985-11-22
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1986-11-10
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1991-03-21
1986-11-10
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1987-05-28
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1991-03-21
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C—CHEMISTRY; METALLURGY
C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
C12N9/0004—Oxidoreductases (1.)
C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
A61K38/00—Medicinal preparations containing peptides
A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
A61K38/43—Enzymes; Proenzymes; Derivatives thereof
A61K38/44—Oxidoreductases (1)
A61K38/446—Superoxide dismutase (1.15)
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
A61P35/00—Antineoplastic agents
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
A61P9/00—Drugs for disorders of the cardiovascular system
A61P9/08—Vasodilators for multiple indications
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
A61P9/00—Drugs for disorders of the cardiovascular system
A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Description
COMMONWEALTH OF AUSTRALIA Patent Act 1952, 6J COMPLETE SPECIFICATION
(ORIGINAL)
Class Int. Class Application Number Lodged Complete Specification Lodged Accepted eti~i 111 ai. c L tmei’dCi)IT’ Is secti g.J Published Priority ies Related Art :22 November 1985 12 September 1986 o Name of Applicant BIO-TECHNOLOGY GENERAL CORP.
11 f So Address of Applicant Actual Inventor/s A f SAddress for Service 0 a 375 Park Avenue, New York, N.Y. 10152, United States of America Jacob R. Hartman Yaffa Beck F.B. RICE CO., Patent Attorneys, 28A Montague Street, BALMAIN 2041.
Complete Specification for the invention entitled: HUMAN MANGANESE SUPEROXIDE DISMUTASE cDNA AND ITS EXPRESSION IN BACTERIA AND METHOD OF RECOVERING ENZYMATICALLY ACTIVE HUMAN MANGANESE SUPEROXIDE DISMUTASE The following statement is a full description of this invention including the best method of performing it known to us/fg:- Ia- BACKGROUND OF THE INVENTION r o 000 o i r o o o0 0o 4 oor a a d 000 0o o 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 2 free radicals was discovered in 1968 by McCord and Fridovich 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 A group of metalloproteins known as superoxide dismutases catalyze the oxidation-reduction tion e ion 20 2+ H202 02 and thus provide a defense mechanism against oxygen toxicity.
-2- 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 nave.
containing copper and zinc 68s 4 been found in virtually all eukaryotic organisms Superoxide dismutases containing manganese have been found in organisms ranging from microorganisms to man.
Since every biological macromolecule can serve as a target for the damaging action of the abundant superoxide radical, interest has evolved in the thera- 15 peutic potential of SOD. The scientific literature suggests that SOD may be useful in a wide range of clinical applications. These include prevention of .I oncogenesis and of tumor promotion, and reduction of the cytotoxic and cardiotoxic effects of anticancer drugs (10) protection of ischemic tissues (12) and protection of spermatozoa (13) In addition, there is interest in studying the effect of SOD on the aging process (14).
The exploration of the therapeutic potential of human SOD has been limited mainly due to its limited availability.
Superoxide dismutase is also of interest because of its anti-inflammatory properties Bovine-derived superoxide dismutase (orgotein) has been recognized to possess anti-inflammaitory properti:s and is currently marketed in parts of Europe as a human pharmaceutical.
It is also sold in the United States as a veterinary -3product, particularly for the treatment of inflamed tendons in horses. However, supplies of orgotein are limited. Prior techniques involving recovery from bovine or other animal cells have serious limitations and the orgotein so obtained may produce allergic reactions in humans because of its non-human origin.
Copper zinc superoxide dismutase (CuZn SOD) is the most studied and best characterized of the various forms of superoxide dismutase.
Human CuZn SOD is a dimeric metallo–protein composed of identical non-covalently linked subunits, each having a molecular weight of 16,000 daltons and containing o one atom of copper and one of zinc Each subunit S 1 is composed of 153 amino acids whose sequence has been established The cDNA encoding human CuZn superoxide dismutase has been cloned The complete sequence of the cloned DNA has also been determined 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- S 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 summarized (28).
Much less is known about manganese superoxide dismutase (MnSOD). The MnSOD of j coli K-12 has recently been cloned and mapped Barra et al. disclose a 196
C,
^5 -4amino 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, however, that the MnSOD polypeptide and the CuZn SOD polypeptide are not homologous The amino acid sequence homologies of MnSODs and FeSOD from various sources have also been compared (18).
Baret et al. disclose in a rat model that the half life of human MnSOD is substantially longer than the halflife of human copper SOD; they also disclose that in the rat model, human MnSOD and rat copper SOD are not So°o” effective as anti-inflammatory agents whereas bovine S 1 copper SOD and human copper SOD are fully active McCord et al. disclose that naturally occurring human oomanganese superoxide dismutase protects human phagocyo 0 o tosing 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 25 S dismutase polypeptide or an analog or mutant thereof.
It is also directed to inserting this cDNA into 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 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 concerns the use of bacterially produced MnSOD of or analogs thereof to treat inflammations.
_I -6- SUMMARY OF THE INVENTION A DNA molecule which includes cDNA encoding the human manganese superoxide dismutase polypeptide or analoa cDFNor mutant thereof has been isolated from a human T-cellA 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 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 S” one strand of a double stranded DNA molecule which S 15 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- 2 stranded DNA molecule which contains a nucleotide S«strand having the sequence encoding the human manganese Ssuperoxide dismutase polypeptide or analog or mutant thereof may be incorporated into a cloning vehicle osuch as a plasmid or virus. Either DNA molecule may S2 be introduced into a cell, either procaryotic, e.g., bacterial, or eukaryotic, yeast or mammalian, o0.o using known methods, including but not limited to methods involving cloning vehicles containing either S° molecule.
Preferably the cDNA or DNA encoding the human manganese superoxide dismutase polypeptide or analog or mutant thereof is incorporated into a plasmid, pMSE-4 or pMSARB4, and then introduced into a suitable host cell ~I _IC 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. coli A4255 and E coli 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 pMSARB4 may be obtained as shown in FIG. 4 and described in the Description of the Figures.
Cells into which such DNA molecules have been introduced may be cultured or grown in accordance with methods known to those skilled in the art under suitable conditions permitting transcription of the DNA into 0 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 Ssuperoxide dismutase or analogs or mutants may be used to catalyze the following reaction: 20 2 2H H 2 0- 02 20- +2H H 2 0 2 +0 2 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.
-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 4 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 bacteri- S° o° al cell is an Escherichia coli cell containing a plasmid which contains a DNA sequence encoding for the o human manganese superoxide dismutase polypeptide e.g.
pMSE-4 or pMS RB4 in E. coli 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 2 first treated to recover a protein fraction containing proteins present in the cells including huma 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 Sthereof. In a preferred embodiment of the invention, the cells are first treated to separaLe soluble proteins from insoluble proteins and cell wall deb~ and the soluble proteins are recovered. Th proteins are then treated to separate, ,i ii -9precipitate, 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 from 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- 9 44 tion having a pH of about 7.0 to 8.0. The cells are then disrupted and centrifuged and the resulting supernatant is heated for about 30 to 120 minutes at a tempeiature between 55 and 65°C, preferably for 45-75 minutes at 58-62 0 C and more preferably for 1 hour at C and then cooled to below 10 0 C, preferably to 4 0
C.
Any precipitate which forms is to be removed e.g. by centrifugation, and the cooled supernatant is dialyzed against an appropriate buffer e.g. 2 mM potassium phosphate buffer having a pH of about 7,8. Preferably, the dialysis is by ultrafiltration using a 25 ffiltration membrane smaller than 30K. Simultaneously 0 with or after dialysis the cooled supernatant optional- ’06.o ly 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 acehL.- M tate, pH 5.5. The dialyzed pooled fractions are then eluted through a cation exchange chromatography column having a linear gradient of about 40 to about 200 mM potassium acetate and a pH of 5.5. The peak fractions containing the superoxide dismutase are collected and pooled. Optionally the pooled peak fractions may then be dialyzed against an appropriate solution e.g. water or a buffer solution of about 10 mM potassium phosphate buffer having a pH of about 7.8.
The invention also concerns purified enzymatically active human manganese superoxide dismutase or analogs thereof e.g. met-hMnSOD, or mutants produced by the methods of this invention.
9 90 o o o o 0 o, 0 ia 0 0 o 0 0 c, 1 -11- BRIEF DESCRIPTION OF THE FIGURES FIG. 1. The Sequence of human MnSOD cDNA FIG. 1 shows the nucleotide sequence of one strand of a double-stranded DNA molecule encoding the human manganese superoxide dismutase as well as the 198 amino acid sequence of human MnSOD corresponding to the DNA sequence. FIG. 1 also shows the nucleotide sequence of one strand of a double stranded DNA molecule encoding a prepeptide to the mature human MnSOD consisting of i0 twenty-four amino acids and the amino acid sequence corresponding to that DNA sequence. Also shown are the 5′ and 3′ untranslated sequences.
the 5′ and 31 untranslated sequences.
o oo 0O 0 tia0 0 FIG. 2. Construction of pMSE-4: Human MnSOD Exr ession Pl amir 0 0 0 00 00 ‘0 Qs 000 a 0 a 0 0 0 0 a g I
NT
Plasmid pMS8-4, containing MnSOD on an EcoRI (R insert, was digested to completion with NdeI and NarI restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in FIG. 2. The resulting plasmid, pMS8-NN contains the coding region for the mature MnSOD, preceded by an ATG initiation codon. The above plasmid was digested with E coRI, 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 pSOD a 13 which was treated with NdeI and SI. pSODa L.S. P-‘EeV t ,74 tao 13 may be obtained as described in4jp ig assdq is-Sea Hct 3 195g gt 27, 1984- which is hereby incorporated by reference. This generated plasmid pMSE-4 containing the MnSOD coding region preceded by the cll ribosomal binding site and under the control of X PL promoter.
Plasmid pMSE-4 has been deposited with the American Type Culture Collection under ATCC Accession No. 53250.
01
LU
-12- FIG. 3 Effect of Mn Concentration on the Activity of SOD Produced in E. Coli The chart in FIG. 3 shows the correlation between the specific activity in units/mg of recombinant soluble MnSOD produced by E. coli strain A4255 containing plasmid pMSE-4 under both non-induction (32°C) and induction (42 C) conditions, and the concentration of Mn (parts per million) in the growth medium.
FIG. 4 Construction of pMS ARB4: Human MnSOD Expression Plasmid TetR expression vector, pARB, was generated from pSODb1T-11 by complete digestion with .v.RI followed by partial cleavage with BamHI restriction enzymes.
15 So pSODIT-ll has been deposited with the American Type °Co Culture Collection (ATCC) under Accession No. 53468.
o The digested plasmid was ligated with synthetic oligomer a5′- AATTCCCGGGTCTAGATCT 3′ GGGCCCAGATCTAGACTAG ,o .resulting in pARB containing the X PL promoter.
The EZSRI 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, pMS6RB4, contains the MnSOD gene under control of X PL and cII RBS and confers resistance to tetracycline.
-13- 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 a double-stranded cDNA molecule which encodes human manganese superoxide dismutase polypeptide or an analog or mutant thereof has been discovered. The sequence of one strand of DNA molecule encoding the human manganese superoxide dismutase polypeptide or analog thereof is shown in Fig. 1 and includes nucleotides numbers 115 to 708 inclusive. The sequence of one strand encoding hMnSOD analog or mutant is substantially similar to the strand encoding the o0 o hMnSOD polypeptide. The nucleotide sequence of the 0co 0 o prepeptide of human manganese superoxide dismutase is o also shown in Fig. 1. Nucleotides numbers 43 through 00 0 0oo0 2 114 inclusive code for this prepeptide.
o ooo 20 So 0 0 The methods of preparing the cDNA and of determining the sequence of DNA encoding the human manganese superoxide dismutase polypeptide, analog or mutant o a 0 0 “ooo. thereof are known to those skilled in the art and are o°o described more fully hereinafter. Moreover, now that the DNA sequence which encodes the human manganese superoxide dismutase has been discovered, known synoqo thetic methods can be employed to prepare DNA molecules containing portions of this sequence.
0 3 30 Conventional cloning vehicles such as plasmids, e.g., pBR322, viruses or bacteriophages, X, can be modified or engineered using known methods so as to produce novel cloning vehicles which contain cDNA encoding human manganese superoxide dismutase -14polypeptide, 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 Fig. 1 for human manganese superoxide dismutase polypeptide or segments substantially similar thereto. The DNA molecule inserted may be made by various methods including enzymatic or chemical synthesis.
The resulting cloning vehicles are chemical entities which do not occur in nature and may only be created by the modern technology commonly described as recombinant DNA technology. Preferably the cloning vehicle is a plasmid, e.g. pMSE-4 or pMS RB4. These cloning vehicles may be introduced in cells, either procaryotic, .o bacterial (Escherichia coli, B.subtilis, etc.) °o or eukaryotic, yeast or mammalian, using techo u niques known to those skilled in the art, such as .oo transformation, transfection and the like. The cells into which the cloning vehicles are introduced will ooo,* 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 or a portion i 0of which has the sequence for human MnSOD polypeptide S shown in Fig. 1 or sequence substantially similar thereto if such DNA was present in the cloning vehicle.
6 4 Escherichia coli are preferred host cells for the cloning vehicles of this invention. A presently pre- 0 ferred auxotrophic strain of E. coli is A1645 which has been deposited with the American Type Culture Collection in Rockville, Maryland, U.S.A. containing plasmid pApoE-Ex2, under ATCC Accession No. 39787. All deposits with the American Type Culture Collection re- SA (3 I\ 2, V. -r^ ferred to in this application were made pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.
A1645 was obtained from A1637 by selection for Gal (ability to ferment galactose) as well as loss of tetracycline resistance. It still contains elements of phage X Its phenotype is C600 rm+ gal thr- leulacZ- bl (\cl857 A H ABamHl 0 A1637 was obtained from C600 by inserting transposon containing tetracycline resistance gene into the galactose operon as well as elements of phage X including those elements responsible for cI repressor synthesis.
C600 is available from the American Type Culture Col- 15 S 5 lection, as ATCC Accession No. 23724.
00 0 Prototrophic strains of Escherichia coli which enable d0 0 0 a high level polypeptide expression even when grown in a o 2 minimal media are even- more preferred as hosts for a0 o 2 expression of genes encoding manganese superoxide dismutase. One presently preferred prototrophic strain is A4255. Strain A4255 containing the plasmid pMSE-4 has been deposited with the American Type Culture Cold Y lection 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.
o 000 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 polypeptide or analog or mutant thereof which may then be recovered.
-16- As used throughout this specification, the term “superoxide dismutase” (SOD) means an enzyme or a polypeptide acting upon superoxide or oxygen-free radicals as receptors, or which catalyze the following dismutation reaction: 202- 2H 02 H 2 0 2 The term “manganese superoxide dismutase” (MnSOD) as 0used herein means any superoxide dismutase molecule containing the element manganese, in any of its chemical forms.
The term “human manganese superoxide dismutase 15 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- S quence is the lysine encoded by nucleotides 115-117 of Fig. 1 and the COOH terminus of the sequence is the 0 0n 20 0 lysine encoded by nucleotides 706-708 of Fig. 1.
n The term “polypeptide manganese complex” as used herein 0 04 means a molecule which includes a human manganese superoxide dismutase polypeptide in a complex with Smanganese in any of its chemical forms and which has O the enzymatic activity of naturally-occurring human manganese superoxide dismutase.
The term “human manganese superoxide dismutase” as used 30 herein means a molecule which includes at least two human manganese superoxide dismutase polypeptides in a complex with manganese in any of its chemical forms and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.
-P~L
-17- 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 polypeptide manganese complex, the polypeptide portion of which includes one or more additional amino acids attached to it at either or both ends.
The term “human manganese superoxide dismutase analog” as used herein means a molecule that includes at least o0°o two polypeptides at least one of which is human mangao nese superoxide dismutase polypeptide analog, in a o.o complex with manganese in any of its chemical forms, and which has the enzymatic activity of naturally-occurring human manganese superoxide dismutase.
The term “human manganese superoxide dismutase polypeptide mutant” as used herein means a polypeptide having an amino acid sequence substantially identical to that of the human manganese superoxide dismutase polypeptide but differing from it by one or more amino acids.
The term “polypeptide manganese complex mutant” means a molecule which includes a human manganese superoxide S 30 dismutase polypeptide mutant in a complex with manganese in any of its chemical forms and which has the enzymatic activity of manganese superoxide dismutase.
-18- 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 hMnSQD which are 0 included as a part of this invention may be prepared by mutating the DNA sequence shown in Fig. 1, the N-terminus of which sequence is the lysine encoded by nucleotides 115-117 and the COOH terminus of which sequence is encoded by nucleotides 706-708.
S o o 0 3 The DNA may be mutated by methods known to those of ordinary skill in the art, e.g. Bauer et al., Gene 31: 0° 73-81 (1985). The mutated sequence may be inserted into suitable expression vectors as described herein, 20 20 ,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 enzymnatically active form of human manganese 2 superoxide dismutase is believed to be a protein having at least two, and possibly four, identical subunits, each of which has approximately 198 amino acids in the sequence shown in Fig. 1 for human manganese superoxide dismutase, the N-terminus of the sequence being the lysine encoded by nucleotides 115-117 of Fig.
1 and the COOH terminus of the sequence being the lysine encoded by nucleotides 706-708 of Fig. 1.
-19- 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 peroxide as shown in the following equation: human MnSOD 202 2H
H
2 0 2 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 So 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 Scomposition to the animal or human subject, to Streat a subject afflicted by inflammations or to o 20 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 injury to an isolated organ by oxygen-free radicals on o perfusion after excision, thus prolonging the survival o0″ period of the organ. Additionally, the hMnSOD or analog or mutant thereof may be used to reduce a a0 neurological injury on reperfusion following ischemia 30 and to treat bronchial 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 is greater than about 2 ppm.
The bacterial cell can be any bacterium in which a DNA 0 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 .o conditions and production medium will vary according to o 15 the species and strain of bacterium.
o a The bacterial cell may contain the DNA sequence encodo o ing the superoxide dismutase or analog in the body of a vector DNA molecule such as a plasmid. The vector or 2 plasmid is constructed by recombinant DNA techniques to have the sequence encoding the SOD incorporated at a Ssuitable position in the molecule.
In a preferred embodiment of the invention the bacteri- 25 25, al cell is an Escherichia coli cell. A preferred auxotrophic strain of E. coli is A1645. A preferred prototrophic strain of E. coli is A4255 The E. coli cell of this invention contains a plasmid which encodes for human manganese superoxide dismutase 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 De- I- ~L i z L -21scription 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 pMSRB4. A method of constructing this plasmid is described in the Description of the Figures and the plasmid itself is described in Example 5. This plasmid may be constructed from pSOD T-11 which has been deposited with the American Type Culture Collection under Accession No.
53468.
In specific embodiments of the invention, an enzymatically active human manganese superoxide dismutase anao 15 log is produced by E. coli strain A4255 cell contain- °o o ing the plasmid pMSE-4 and by E. coli strain A4255 cell containing the plasmid pMSARB4.
The suitable production medium for the bacterial cell can be any type of acceptable growth medium such as 0, casein hydrolysate or LB (Luria Broth) medium, the .o latter being preferred. Suitable growth conditions will vary with the strain of E. coli and the plasmid it contains, for example E. coli A4255 containing plasmid pMSE-4 is induced at 42 C and maintained at that temperature from about 1 to about 5 hours. The suitable 8,8 8conditions of temperature, time, agitation and aeration for growing the inoculum and for growing the culture to a desired density before the production phase as well as for maintaining the culture in the production period may vary and are known to those of ordinary skill in the art.
-22o o0 a O eo o S0 0 00′ I The concentration of Mn ion in the medium that is necessary to produce enzymatically active MnSOD will vary with the type of medium used.
In LB-type growth media Mn concentrations of 150 ppm to 750 ppm have been found effective. It is preferred that in all complex types of growth mediums the concentration of Mn++ in the medium is from 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 15 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 3 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.
0 C! g oll -z i ~e I I- r r; -23- 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 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 65°C, preferably for 45-75 minutes at 58 to 6°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 cooling is removed, e.g. by centrifugation and then the cooled supernatant is dialyzed against an appropriate buffer. Preferably the cooled supernatant is dialyzed 15 S 15 by ultrafiltration employing a filtration membrane smaller than 30K, most preferably 10K. Appropriate a buffers include 2 mM potassium phosphate buffer having n a pH of about 7.8. After or simultaneously with this dialysis the cooled supernatant may optionally be concentrated to an appropriate volume, e.g. 0.03 of the supernatant’s original volume has been found to be convenient. The retentate is then eluted on an anion exchange chr cmatography column with an appropriate buffered solution, a solution at least 20 mM 5 potassium phosphate buffer having a pH of about 7.8.
The fractions of eluent containing superoxide dismutase o 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 pH of The peak fractions containing the superoxide dismutase are collected and pooled. Optionally the pooled peak fractions may then be dialyzed against an appropriate -24solution, e.g. water or a buffer solution of about 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 particular, it concerns a human manganese superoxide dismutase analog having at least two polypeptides, at least one of which polypeptides has the amino acid sequence shown in Fig. 1, the Nterminus of which sequence is the lysine encoded by nucleotides 115-117 of Fig. 1 and the COOH terminus of which sequence is the lysine encoded by nucleotides 706-708 of Fig. 1 plus an additional methnione residue 15 5 at the N-terminus (Met-hMnSOD). A preferred embodiment o of this invention concerns purified Met-hMnSOD having a specific activity of 3500 units/mg.
0 L, -L IL .L j
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 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 0 detailed description for conventional methods employed for assaying the polypeptides produced by such host vector systems or determining the identity of such 0 0° polypeptides by activity staining of isoelectric focus- Sing (IEF) gels. Such methods are well-known to those ao 15 or ordinary skill in the art and are described in nuo merous publications including by way of example the following: T. Maniatis, E.F. Fritsch and J. Sombrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboo oo ratory, New York (1982).
J.M. McCord and I. Fridovich, J. Biol. Chem. 244:6049- 55 (1969) C. Beauchamp and I. Fridovich, Anal. Biochem. 44:276-87 o° C 0 (1971) C rr~-r~ir_; -26- EXAMPLE 1 In order to identify MnSOD cDNA clones, mixed oligomer probes were synthesized according to the published amino acid sequence (18,19).
30 mer sequence from AA 1 5
AA
2 4 (18,19) •0 5′ 3′ Io TTGCATAATTTGTG CCTTAATGTG TGGTT C S 1 T G T G o G G 3′-probe 32 mer sequence from AA 7 9
-AA
1 8 9 (19) 3′
TCTGTTACGTTTTCCCAGTTTATTACGTTCCA
S0, G G G G S The 5′-probe consisting of 30 nucleotides corresponds 0 00, 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 i above. The 3′-probe is a mixed probe consisting of 16 j 25 Sdifferent sequences as shown above. (When more than Sone 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 X gt-10 vector.
Hybridization to phage plaque replicas immobilized on nitrocellulose filters was performed according to Ls -rY -L -27standard procedures (Maniatis et al. supra) except that the hybridization was performed at 500C in 8xSSC for 16 hrs. The filters were then washed at 50 C with 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 800 bp long, which hybridize to both the 5′ and 3′ oligonucleotide probes. Phi MS1J carried only 450 bp cDNA insert which hybridized only to the 5′ end probe.
The E__RI inserts of the three phage clones were subcloned into the EcoRI site of pBR322 thus yielding pMS8-4, pMS1-4 and pMS1J, respectively. Restriction °o .15 analysis and hybridization to the 5′ and 3′ oligonucleotide probes revealed similar patterns for both pMS8-4 and pMSl-4. The following restriction map showing the 5′ 3′ orientation has been deduced for both plasmids.
s e -28- S PuII PUII S aI S u Rl 100 200 300 400 500 600 700 800 bp The sequence of the cDNA insert of pMS8-4 is shown in Fig. 1. The predicted amino acid sequence differs from the published amino acid sequence (19) in that Glu appears instead of Gin in three locations (AA 42, S88, 108) and an additional two amino acids, Gly and Trp appear between AA 2 3 1 2 4 Sequence analysis of pMS1-4 and pMSlJ revealed that the three MnSOD clones were independently derived and confirmed these differences from the published amino acid sequence.
The sequence upstream of the N-terminal Lysine of mature MnSOD predicts a pre-peptide sequence of 24 amino acids.
I LI j_ -29- EXAMPLE 2 Construction of pMSE-4: Amp
R
Human MnSOD Expression Plasmid The starting point for the construction of pMSE-4 is the plasmid pMS8-4 which was obtained as described in Example 1. Plasmid pMS8-4, containing human MnSOD cDNA on an E.coRI insert, was digested to completion with NdeI and NaLI restriction enzymes. The large fragment was isolated and ligated with a synthetic oligomer as depicted in Fig. 2. The resulting plasmid, pMS8-NN contains the coding region for the mature MnSOD, pre- 0 0 ceded by an ATG initiation codon. The above plasmid 400 0 was digested with EcoRI, ends were filled in with 15 SKlenow fragment of Polymerase I and further cleaved with NdeI. The small fragment containing the MnSOD ee- w a r-te–i-o-pc l3which was treated wi’ o NdeI and S.tI. pSOD 13 may be obtained as described in pending, co-assigned U.S. Patent Applicta-ion Serial No. 644,245, filed August 27, 1984 w ich is hereby incorporated by reference. Th generated plasmid pMSE-4 containing the MnSOD ing region preceded by the cII ribosomal bindin site and under the control of X PL promoter. -Plasmid pMSE-4 has been deposited with the Amer can Type Culture Collection under ATCC Accession o. 53250. All methods utilized in the abovy. processes are essentially the same as those dec/4.
-29a gene was inserted into pSOD c 13 which was treated with NdeI and Stul. pSOD 13 may be obtained as described in US patent No. 4,742,004 issued May 3 1988, which is hereby incorporated by reference. This generated plasmid pMSE-4 containing the MnSOD coding region preceded by the cII ribosomal binding site and under the control of X PL 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.
t e l C_ EXAMPLE 3 I Expression of the Recombinant Human MnSOD Plasmid pMSE-4 was introduced into Escherichia coli strain A4255 using known methods. Then the E. coli strain 4255, containing pMSE-4, were grown at 32°C in Luria Broth (LB) medium containing 100 g/ml of ampicillin until the Optical Density (OD) at 600 nm was 0.7. Induction was performed at 42 0 C. Samples taken i0 Sat various time intervals were electrophoresed separated on sodium dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE). The gels showed increases S°in human MnSOD levels up to 120 minutes post-induc- Stion, at which stage the recombinant MnSOD protein 15 comprised 27% of total cellular proteins as determined S” by scanning of Coomassie-blue stained gel. Sonication I of samples for 90 sec. in a W-375 sonicator and parti- Stioning of proteins to soluble and non-soluble (p) fractions by centrifugation at 10,000 g for 5 min.
revealed that most of the recombinant MnSOD produced was non-soluble. The induced soluble protein fraction contained only slightly more SOD activity than the uninduced counterpart, as assayed by standard methods.
fi TSee McCord et al., supra. Apparently a portion of the 25 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.
l I-
I
-31- EXAMPLE 4 Effect of Mn in Growth Media on MnSOD Solubility and Activity The addition of Mn in increasing concentrations up to 450 ppm to the growth media of E. coli A4255, containt o lfao 60 o,7 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 and non-soluble on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed increased solubilization of the recombinant protein with in- So creased Mn++ concentrations (Table 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 2 an apparent optimum at 150 ppm Mn concentration in the media (Fig. Furthermore increased Mn concentrations activated previously inactive soluble enzyme.
Soluble protein fractions of induced cultures grown at 0 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 stains ing of isoelectric focusing (IEF) gels (see Beauchamp 25 et al, suira.) revealed that multi forms of the recombinant MnSOD were identical to those of native human a0 o liver MnSOD.
0 0 Results for human MnSOD production by E. coli A1645 containing pMSE-4 were similar to those described above.
i_ 1 i II- -32- TABLE I Mn (ppm) Per cent Sol ubl e human Mn SOD of Total human MnSOD Induced Per cent Sol uble human Mn SOD of Soluble Bacterial Proteins Specif ic Activity units/mg Sol ubl e Pr otein s 00 0 0o~0 0 o 0<0 0 100 15150 200 250 300 450 30.6 72.7 78.0 82.9 82 .0 79.2 80.8 89.2 7.2 15.4 16.9 18.8 20.8 20.4 20.3 22.4 241 356 606 338 380 381 323 -33- Construction of pMSARB4: Tet R Human MnSOD Expression Plasmid expression vector, pARB, was generated from pSO 31T-11 by complete digestion with EcoRI followed partial cleavage BamHI restriction enzymes.
pSODB1T-ll has been deposited the American Type Culture Collection under Accession No. 53468. The digested plasmid ligated synthetic oligomer AATTCCCGGGTCTAGATCT 3' GGGCCCAGATCTAGACTAG resulting in pARB containing X PL promoter.
0 LcRI fragment pMSE-4, cll ribosomal binding site and coding sequence for mature enzyme, inserted Sinto unique pARB. oi, plasmid, pMSARB4, contains gene control cII RBS confers resistance to tetracycline (Fig. 4).
Q 3 -34- EXAMPLE 6 pMSARB4 introduced into Escherichia coli strain A4255, using known methods. Cultures were grown at 32°C Luria Broth (LB) various concentrations until Optical Density (OD) 600 nm reached 0.7. Induction performed 42 0
C.
Samples taken time intervals electrophoresed on SDS-PAGE. hMnSOD level increased induction up 120 minutes, which stage it comprised about 15% total cellular proteins as determined scanning Coomassie Blue stained gel.
The induced soluble, regardless Mn++ concentration growth media. This is contrast observations Amp pMSE-4. (See Example However, maximum SOD activity dependent Mn supplementation (Table 2).
0 00 0o 000 TLE 2& Expressip~jn in- E.Coli A4255 ft)MSARB4) Percent Soluble h Bacterial Proteins Specific Activity Units>Download PDF in English
