DE3708306A1 - Human manganese superoxide dismutase (hMn-SOD) - Google Patents

Abstract

The present invention relates to a genetic engineering method for the preparation of human Mn superoxide dismutase (hMn-SOD), to the DNA sequences which code for this enzyme, to suitable vectors which contain these DNA sequences, and to host cells which are able to express these DNA sequences, as well as to the enzyme hMn-SOD itself. Proposed uses of this enzyme are also described.

Description

The present invention relates to a genetic engineering process for the production of human Mn superoxide dismutase (hMn-SOD), the DNA sequences that suitable vectors for this enzyme, which these contain DNA sequences and host cells that can express these DNA sequences, as well as the enzyme hMn-SOD itself. Suggestions for using this enzyme are also described.

As a result of various biochemical processes in biological systems (e.g. redox processes in the respiratory chain, oxidations in the cytoplasm), it is known that O ^- ₂ radicals are continuously formed which are highly cytotoxic and can lead to tissue destruction. In pathological situations, e.g. B. in the course of rheumatic diseases, degradations of collagen and synovial fluid by such radicals are discussed (Pasquier, C. et. Al., Inflammation 8, 27-32, 1984). Eukaryotic cells contain two forms of superoxide dismutases, one of which is primarily in cytosol (Cu / Zn-SOD) and the other mainly in mitochondria (Mn-SOD). In liver mitochondria it was found that Mn enzyme is localized in the matrix including the inner membrane, although the Mn-SOD was also detected in the cytosol of the liver cells (Mc Cord JM et. Al., In: Superoxide and Superoxide Dismutases (AM Michelson, JM Mc Cord, I. Fridovich, eds.) Academic Press, NY, 129-138, 1977).

In addition to Mn SOD, there is also Fe SOD in prokaryotes. The latter has also been detected in algae and protozoa and in some plant species (Bridges, SM, Salin, ML, Plant Physiol. 68, 275-278, 1981). These highly active enzymes catalyze the disproportionation O ^- ₂ + O ^- ₂ + 2H⁺₄H₂O₂ + O₂ and prevent the superoxide radicals from accumulating and thus damaging the cells by this dismutation. In addition to the endoplasmic reticulum of the liver, the mitochondrial membranes can be regarded as one of the most important places of O ^- ₂ formation in animal cells, so it is not surprising that mitochondria have their own special SOD (Mn-SOD) available.

The structural gene of a prokaryotic Mn-SOD (E.coli) was cloned and the chromosomal sodA gene localized (Touati, D., J. Bact. 155, 1078-1087, 1983).

The 699 bp nucleotide sequence of a mitochondrial Yeast Mn-SOD was able to elucidate the primary structure both the precursor and the mature protein derived from it - with molecular weights of 26 123 Da for the precursor and 23 059 Da for the mature Protein (Marres, C.A.M. et al., Eur. J. Biochem. 147, 153-161 (1985). The Mn and Cu / Zn-SOD (MW = 14 893, EP-A 1 38 111) in their Molecular weights clearly.  

The complete amino acid sequence of the Mn-SOD Human liver was published by Barra, D., according to the hMn-SOD can be composed of 196 amino acids should (Barra, D. et al., J. Biol Chem. 259, 12 595-12 601, 1984). The Human Cu / Zn-SOD consists of erythrocytes in contrast, from 153 amino acids (Jabusch, J.R., et al., Biochemistry 19, 3210-2316, 1980) and shows none Sequence homologies to hMn-SOD) (Barrac. D. et al., See above).

Generally, superoxide dismutases become one Protective function against certain Inflammatory processes awarded. In particular, should a deficiency in Mn-SOD in developing the rheumatoid arthritis is of importance (Phasquier, C. et al., See above). A protective Effect of SOD against alcohol-induced Liver damage is also assumed (Del Villano B.C. et al., Science 207, 991-993, 1980).  

The cloning and expression of a human SOD is only known for human Cu / Zn-SOD from human liver (EP-A 1 38 111).

Based on the essential properties of Superoxide dismutases, especially hMn SOD, is a Need for therapeutic and / or diagnostic Expected use. It is advantageous to for this purpose, its own, d. H. human Mn-SOD in homogeneous form and in sufficient quantities for To have available. The derived from it The goal is to be expected immunological Reactions e.g. B. after therapeutic use minimize or prevent.

Only the development of recombination technologies of foreign DNA with vector DNA with the possibility, too to establish and stabilize the former in microorganisms express, allows homogeneous proteins of animal or human origin in large quantities. Another goal is to be achieved, namely that the enzyme produced with it - the hMn-SOD - a compared to the authentic genuine hMn-SOD characteristic biological activity spectrum shows.

An object of the present invention was therefore therein, using genetic engineering for the first time to find the DNA sequence coding for this enzyme or to represent and show for the first time, according to what methods this sequence can be obtained.  

According to the invention, this object has been achieved in that one obtained from human cells of placental origin cDNA library with synthetically produced DNA probe molecules was searched and thereby the gene coding for hMn-SOD could be isolated. Around Obtaining the gene for hMn-SOD can be done according to known Methods that isolate mRNA from such cells that produce the desired enzyme. As Starting material are different, e.g. B. metabolically active glandular tissues such as B. leather or placenta suitable. After preparation of the cDNA, the known Methods by primed synthesis with reverser Obtained transcriptase from the isolated mRNA subsequent installation in a suitable Vector and amplification to a complete cDNA library, can this with a defined, radiolabelled DNA probe or a mixture of various such probes can be searched. To the Degeneration of the genetic code to be considered are preferably defined DNA probe mixtures used all possible nucleotide variations for represent the same amino acid or that be selected so that the number of synthesizing DNA probes of a mixture if possible low and the homology to the searched hMn-SOD DNA sequence is as high as possible. Another one Selection criterion for the synthesis of DNA probes can consist in that these are complementary to at least two independent regions, for example near the 3'- and 5'-end of the putative gene sequence.  

This allows you to use at least two separate ones Hybridizations are identified against clones for example, both independent DNA probes are positive Show signals. These clones can then preferably be used for the isolation of the hMn-SOD gene since you can expect them to either essential part or the complete gene for hMn-SOD contain.

The special DNA sequences for the DNA probes according to the invention were used based on those published by Barra, D. et al Amino acid sequence of human Mn-SOD from liver tissues (Barra, D. et al., Oxy Radicals and their scavenger Systems, Vol. 1, 336-339, 1983). In particular, two regions of the putative hMn-SOD DNA sequence used for at least five amino acid residues, preferably for 8 Encode amino acid residues, being a DNA probe length of at least 14, preferably 23 bases advantageous is. It is particularly advantageous if a DNA probe is complementary to that derived hMn-SOD DNA sequence whose genetic information is colinear with amino acid residues 39 to 46 and a second DNA probe is complementary to the corresponding one DNA region responsible for the amino acid residues 200 to 207 of the known amino acid sequence encoded. You can also of course, DNA sequences are also used as probes that are based on other Mn superoxide dismutases can be derived and  different in length. The found differences of this sequence to "Barra sequence" concern the amino acid positions, 42, 88, 109 and 131 (each Glu instead of Gln) and two additional amino acids Gly and Trp between Item 123 and 124, so that the invention DNA sequence of a hMn-SOD of 198 amino acids corresponds.

It was also completely unexpected that, on the other hand, one for cDNA encoding hMn-SOD could be isolated means an amino acid substitution at position 29 (Codon for Gln instead of Lys) and thus in this Point another difference "to the Barra sequence" and to formula Ia, corresponding to formula Ib:

With the help of such a DNA probe, positive clones can are obtained from which a cDNA sequence according to following formula Ia, which can be isolated large portion of a region coding for hMn-SOD contains:

Surprisingly, it was found that the found cDNA encodes an amino acid sequence that differs from the published amino acid sequence (Barra, D. et al., J. Biol. Chem. 259, 12 595-12 601, 1984) in some residues and  different in length. The found differences of this sequence to "Barra sequence" concern the amino acid positions, 42, 88, 109 and 129 (each Glu instead of Gln) and two additional amino acids Gly and Trp between Item 123 and 124, so that the invention DNA sequence of a hMn-SOD of 198 amino acids corresponds. The counting was based on the Amino acid sequence according to Barra, D. et al., (1984) with Lys = 1.

It was also completely unexpected that, on the other hand, one for cDNA encoding hMn-SOD could be isolated means an amino acid substitution at position 29 (Codon for Gln instead of Lys) and thus in this Point another difference "to the Barra sequence" and to formula Ia, corresponding to formula Ib:

Assuming that the Barra sequence is correct has been analyzed using the nucleotide or amino acid sequence according to the invention Possibility to be granted that hereby for the first time and surprising the possible existence of different genes or their allelic forms or isoenzymes for hMn-SOD is shown.

Since cDNA-bearing clones can be obtained which lack the end necessary for the complete hMn-SOD gene, a further object of the present invention was to provide the complete gene for hMn-SOD.
This problem can be solved according to various known strategies. For example, the sequence obtained can itself be used as a DNA probe and the cDNA bank can thus be searched again in order to find a complete gene or a cDNA with the missing end, or the DNA sequence obtained can be used as a hybridization probe against a genomic bank in order to isolate the complete hMn-SOD gene after identification.

Or you use the possibility of oligonucleotides to synthesize the nucleotide sequence of the missing End of the hMn-SOD and with the help of this, after suitable linker ligation, the complete cDNA for to get hMn-SOD. This method has the advantage that, for example, a DNA coding for hMn-SOD can be obtained, the 5'-end directly with the Start codon (ATG) begins.  

It proved to be particularly suitable for solving this task opting for completing the example Amino acid 22 or 26 coding according to the invention cDNAs the DNA sequence of formula II, starting with the 5′-start codon ATG and ending with the codon for Amino acid 31 (His, where AAG [Lys] -1) under Based on the known codon preferences like them are valid for yeast (Sharp, P.M. et al., Nucl. Acids. Res. 14 (13), 5125-5143, 1986)

Likewise, other known synonymous codons can be used for the completion of the hMn-SOD gene or for the in vitro synthesis of the entire gene, e.g. B. those that have an optimal codon-anticodon interaction in bacteria, e.g. BEcoli, and there increase the efficiency of translation (Grosjean, H. Fiers, W., Gene 18, 199-209, 1982; Ikemura, T., J. Mol. Biol. 151, 389-409, 1981) or such Codons that correspond to the genuine ratios in mammalian cells (Grantham, R. et al., Nucleic Acid Research 9, 43-47, 1981).
The latter can preferably be used for transformation and subsequently for expression in mammalian cells.

In principle it is possible to get the methionine residue through the start codon ATG is encoded and the mature hMn-SOD, beginning with the first amino acid lysine is, according to known methods, for example with CNBr or CNCl. But since in the mature enzyme hMn-SOD further internal methionine residues can occur e.g. B. at positions 23 or 192 - such Procedure cannot be carried out, so in this In case the additional N-terminal methionine residue is obtained remains without influence on the biological activity of the hMn SOD.

However, enzymatic cleavages can also be provided are, known to be suitable synthetic Linkers can be used since it is expected that themselves codons for corresponding, specific amino acids at the desired positions on the vector that the contains hMn-SOD cDNA. For example, Arg- or To name Lys residues for tryptic cleavage or one generally uses codons for Encode protease sensitive amino acids. these can be positioned before or after the start codon or within the coding region.

An additional object of the present invention was it, with the help of genetic engineering methods, the for Sequence encoding hMn-SOD in suitable host cells to express for the first time, the homogeneous enzyme to produce hMn-SOD for the first time using such processes,  to isolate and provide in pure form and the necessary procedure for the first time describe.

According to the invention, this object was achieved by by the DNA sequences coding for hMn-SOD, for example of the formulas IIIa or IIIb

if necessary with appropriate signal or Provide control sequences in suitable vectors inserted and thus transformed suitable host cells were. After cultivating the transformed The polypeptides formed become host cells after known processes isolated and cleaned. The polypeptides obtained correspond to the subsequent ones Formulas IVa and IVb.  

The sequences according to formulas IIIa and IIIb are suitable especially for the production of non-glycosylated hMn-SOD of the formulas IVa and IVb in microorganisms, especially in E.coli or S. cerevisiae. The problem glycosylation in yeast, for example be avoided that mutants are used that The glycosylation of proteins (alg Mutants) (e.g. Huffakter, T.C., Robbins P.W. Proc. Natl. Acad. Sci. USA 80, 7466-7470, 1983).

If it appears necessary or sensible, the complete hMn-SOD gene, for example according to formulas IIIa or IIIb a leader or signal sequence directly the first codon of the first N-terminal amino acid of mature HMn-SOD or in front of the start codon ATG will. This can be achieved that the hMn-SOD transported out of the host cell and easily out of the Culture medium can be isolated.

Such signal sequences have been described; they code for a mostly hydrophobic portion of protein, the through post-translational modification processes in the Host cell is split off (Davis, D.B., Tai. P.-C., Nature 283, 433-438, 1980; Perlman, D., Halvorson, H.O., J. Mol. Biol. 167, 391-409, 1983). If before the first Amino acid of the hMn-SOD has been constructed as an ATG codon is a gene product can be obtained that before Lysine contains an N-terminal methionine. That Signal sequences from prokaryotes are used to To secrete proteins into the periplasm and correct them process is known. (E.g. Davis, B.D., Tai, P.-C., 1980).  

Of course it is after isolation and cloning the hMn-SOD DNA sequence possible through this sequence to specifically modify encoded enzymes. Enzyme modifications can be targeted, for example in vitro mutations with synthetic oligonucleotides take place, whereby the catalytic properties of the hMn-SOD affects and novel enzymatic Activities can be created. The basic methodological steps to carry out such Protein manipulations are known (e.g. Winter, G. et al., Nature 299, 756-758, 1982; Dalbadie - Mc Farland, G. et al., Proc. Natl. Acad. Sci. USA, 79, 6409-6413, 1982).

For cloning, i.e. H. Amplification and preparation, of the hMn-SOD gene, E. coli can be used preferably E.coli C600 (Nelson et al., Virology 108, 338-350, 1981) or JM 101, or E. coli strains at least one of the known sup genotypes. The Cloning can also occur in Gram-positive bacteria such as B. B. substilis. Such systems are described many times.

Both microorganisms and cultures of multicellular organisms come into question as suitable hosts for the expression of the hMn-SOD gene according to the invention.
Microorganisms are to be understood as prokaryotes, ie Gram-negative or Gram-positive bacteria, and eukaryotes such as protozoa, algae, fungi or higher protists. Of the Gram-negative bacteria, the Enterobacteriaceae, z. BEcoli, from the Gram-positive the Bacillaceae and apathogenic Micrococcaceae, e.g. BB subtilis and Staph. carnosus, from the eukaryotes the Ascomycetes in particular the yeasts, e.g. B. Saccharomyces cerevisiae, preferred hosts.

There are a large number of unicellular microorganisms Output vectors are available that are both plasmid as well as being of viral origin. These vectors can be in simple number of copies or as Multicopy vectors are available. Such vectors, the for cloning and expression of the invention hMn-SOD as general for eukaryotic DNA sequences are suitable in many publications and Manuals described (e.g. Maniatis, T. et al. Molecular Cloning, Cold Spring Harbor Laboratory, 1982; Glover, DM. (ed.) DNA Cloning Vol. I, II, 1985) and are commercially available.

In general, plasmid vectors, which are usually an origin of replication and control sequences for the Contain transcription, translation and expression, in Connection with these hosts can be used. These Sequences should come from species that are compatible with the host cells. The vector usually carries in addition to a replication site Recognition sequences that allow in phenotypically selected transformed cell. The selection can be made either by complementation, Suppression or by inactivating a marker respectively. Regarding the two  first-mentioned methods are auxotrophic mutants of Bacteria and yeast that are deficient for one essential metabolite, or Nonsense mutants in which the translation of the affected gene comes to chain termination. Various Suppressor genes, e.g. B. supD. E, F (suppress UAG), supC, G (suppress UAG or UAA) are known. In the third method, the vector is entered Resistance gene against one or more cytotoxic Agents such as B. antibiotics, heavy metals. By Insertion of a foreign DNA into such a marker gene this inactivated so that the newly created phenotype can be distinguished from the original phenotype.

For example, E. coli can be transformed with pBR322, a plasmid derived from E. coli species (Bolivar, et al., Gene 2, 95 (1977). PBR322 contains genes for amplicillin and tetracycline resistance and thus provides simple means to identify transformed cells by converting the phenotype Ap ^r , Tc ^r to Ap ^s , Tc ^r by cloning into, for example, the PstI site in the β- lactamase gene Other methods are equally applicable, for which, for example, the lacZ gene inactivation λ and M 13 vectors as well as with various plasmids (eg pUC, pUR) are significant.These very diverse selection systems are well known and accordingly there is a wealth of literature on them.

In addition to such selection markers, such Vectors, especially expression vectors, Signal sequences that have the correct initiation and ensure termination of the transcription. For the correct transcription of the hMn-SOD gene can therefore these vectors are bacterial or eukaryotic Contained transcription unit consisting of a Promoter, the coding region with the hMn-SOD gene and of the adjoining terminator. Corresponding the nature of the transcription units can affect these conserved prototype sequences such as B. Pribnow box or TTG sequence or CAAT box, TATA box that known termination signals (e.g. AATAAA, TATGT), and contain at least one stop codon, where preferably promoters homologous to the host and terminators can be used. The mRNA formed usually contains a 3'poly (A) sequence and / or a 5'cap structure. For the translation of the hMn-SOD Gene requires a ribosomal binding site (RBS), consisting of Shine / Dalgarno (S / D) sequence and a in addition at a defined distance of generally 3 to 12 Nucleotide standing initiation codon and at least a stop codon. Alternatively, RBSs can be synthetic be produced, which creates homology with the 3 ′ end of the 16S rRNA can be increased (Jay, E. et al. Nucleic Acids Res. 10, 6319-6329, 1982).

Especially in eukaryotic expression systems (e.g. S. cerevisiae) should be preferred Regulation systems for translation host Of origin, because none of the yeasts  Prokaryotic analogous relationships (homology of S / D sequence with the 3'-end of the 16S rRNA) and the signals or the RBS for the initiation of the Translation in a slightly different way than in Prokaryotes are defined (e.g. Kozak, M., Mucleic Acids Res. 9, 5233-5252, 1981; Kozak, M.J. Mol., Biol. 156, 807-820, 1982).

Preferably, the cloning or Expression vector only one Restriction endonuclease recognition site, either is present in the output vector a priori or later can be introduced via appropriate linkers. Left can be easily synthesized either chemically or are commercially available.

Frequently used yeast promoters in the production of corresponding expression plasmids include those promoters which control expression particularly efficiently in the yeast system, such as, for. B. PGK promoter (Tuite, MF et al., The EMBO Journal 1, 603-608, 1982; Hitzeman, RA et al., Science 219, 620-625, 1983), PH05 promoter (Hinnen, A., & Meyhack , B., Current Topics in Microbiology and Immunology 96, 101-117, 1982; Kramer, RA et al., Proc. Natl. Acad. Sci, USA 81, 367-370, 1984), GAPDH promoter (Urdea, MS et al., Proc. Natl. Acad. Sci. USA 80, 7461-7465, 1983), GAL10 promoter (Broach et al. Experimental Manipulation of Gene Expression 83-117, 1983), Enolase (ENO) promoter (Holland, MJ et al., J. Biol. Chem. 256, 1385-1395, 1981), α factor promoter (Bitter, G.-A. et al. Proc. Natl. Acad. Sci. USA 81, 5330-5334; Yakota , T. et al. Miami Winter Symp. 17. Meet. Adv. Gene Technol. 2, 49-52, 1985)) or the ADHI promoter (Ammerer, G., Methods in Enzymology 101, 192-201, 1983; Hitzeman , RA et al. Nature 293, 717-722, 1981).

Promoters of other glycolytic enzymes (Kawasaki and Fraenkel, Biochem. Biophys. Res. Comm. 108, 1107-1112, 1982) are also suitable, such as hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase and glucokinase. When constructing suitable expression plasmids, the termination sequences associated with these genes can also be inserted into the expression vector at the 3 'end of the sequence to be expressed, in order to provide for polyadenylation and termination of the mRNA. Other promoters that also have the advantage of growth-controlled transcription are the promoter regions of alcohol dehydrogenase-2, isocytochrome C, the degrading enzymes linked to nitrogen metabolism, the above-mentioned glyceraldehyde-3 -Phosphate dehydrogenase (GAPDH) and the enzymes responsible for the metabolism of maltose and galactose. Promoters that are regulated by the yeast mating type locus, for example promoters of the BARI, MECI, STE2, STE3, STE5 genes, can be used in temperature-regulated systems by using temperature-dependent sir mutations. (Rhine, Ph. D. Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, part I, 181-209 (1981), Cold Spring Habor Laboratory).
These multations influence the expression of the resting mating type cassettes of yeasts and thereby indirectly the mating type dependent promoters. In general, however, any plasmid vector which contains a yeast-compatible promoter, original replication and termination sequences is suitable.

In the event that the expression of hMn-SOD is to take place in bacteria, it is preferable to use promoters which require a high synthesis rate of mRNA and which are also inducible. Known and used promoters include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); Itakura et al., Science 198, 1056 (1977); Goeddel et al. , Nature 281, 544 (1979) including the UV5 promoter (Silverstone, AE et al. Proc. Natl. Acad. Sci. USA 66, 773-779, 1970) and tryptophan (trp) promoter systems (Goeddel et al. , Nucleic Acids Res. 8, 4057 (1980); European application, publication number 00 36 776. Other microbial promoters have also been developed and used. For example, the gene sequence for hMn-SOD can be controlled by the Lamba-P _L promoter, which is known as one of the particularly strong, controllable promoters, can be controlled by a thermolabile repressor cI (eg cI857), of which neighboring restriction sites are known E. coli alkaline phosphate promoter (Ohsuye, K. et al., Nucleic Acids Res. 11, 1283-1294, 1983) and hybrid promoters such as e.g. B. the tac promoter (Amann, E. et al. Gene 25, 167-178, 1983; De Boer, HA et al. Proc. Natl. Acad. Sci. USA 80, 21-25, 1983). The use of such portable promoters (lacuv5, lacZ SD, tac) or vectors for the production of fused and unfused eukaryotic proteins in E. coli can also be found in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982 , in particular p. 412f. The expression and translation of an hMn-SOD sequence in bacteria can also take place under the control of other regulatory systems which can be considered "homologous" to the organism in its untransformed form. For example, promoter-operator systems such as arabinose operator, colicin EI operator, galactose operator, alkaline phosphatase operator, trp operator, xylose A operator, and the like can also be used. Ä. or parts thereof are used.

For cloning or expression of hMn-SOD in Bacteria, for example in E. coli, or in yeast, for example in S. cerevisiae, are well known Vectors are available, of which for the former Host systems distributed the pBR- (Bolivar, F. et al., Gene 2, 95-113, 1977), pUC- (Vieira, I., Messing. I., Gene 19, 259-268, 1982) pOP- (Fuller, F., Gene 19, 43-54, 1982), pAT- (Windass, J.D. et al., Nucleic Acids Res. 10, 6639-6657, 1982) pHV plasmids (Ehrlich, S.D., Proc. Natl. Acad. Sci. USA 75, 1433-1436, 1977), Lambda vectors including phasmids (Brenner, S. et al., Gene 17, 27-44, 1982), Cosmide (Collins, J., Hohn. B.,  Proc. Natl. Acad. Sci. USA 75, 4242-4246, 1979) and the other known vectors (e.g. Maniatis, T. et al., Molecular Cloning, Cold Spring Habor Laboratory, 1982), in particular pBR and pUC derivatives, for example pBR322, pUC18 can be used.

Expression vectors in yeasts are suitable integrating (YIp), replicating (YRp) and episomal (YEp) vectors (Struhl, K. et al. Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1979; Stinchcomb, D.T. et al., Nature 282, 39-43, 1979; Hollenberg, C.P., Current Topics in Microbiology and Immunology 96, 119-144, 1982), preferably YEp13 (Broach, J.R. et al. Gene 8, 121-133, 1979), YIp5 (Struhl, K. et al., 1979 see above, ATCC 37 061) as well as pJDB207 (DSM 3181) or pEAS102. The vector pEAS102 can be obtained by YIp5 with PstI partially and completely digested with BamHI and that isolated 4.3 kb fragment (contains the URA3 gene) with the 4.4 kb BamHI / PstI fragment from pJDB207.

In addition to microorganisms are cultures multicellular organisms also suitable hosts for the expression of hMn-SOD. In principle, each is this Cultures can be used, whether from vertebrate or invertebrate Animal cell cultures. However, there is greatest interest Vertebrate cells, so that the multiplication of Vertebrate cells in culture (tissue culture) in recent Years has become a routine method. (Tissue, Culture, Academic Press, Kruse and Patterson, Editors, 1973). Examples of such useful host cell lines are VERO and HeLa cells, golden hamster ovary  (CHO) cells and W138, BHK, COS-7 and MDCK cell lines. Contain expression vectors for these cells usually an origin of replication, one Promoter that precedes the hMn-SOD to be expressed is localized, a necessary Ribosome binding site, RNA splice site, Polyadenylation site and transcriptional Termination sequences.

When using mammalian cells, the Control functions on the expression vectors often made of viral material. For example, come from the papovavirus promoters commonly used such as polyoma, papilloma viruses, Simian Virus 40 (SV40) and from retroviruses, adenovirus type 2. The early and are SV40 late promoters and their uses described in many ways. It is also possible and often recommended, promoter or control sequences or Use splice signals that match the ones you want Gene sequences are originally linked, provided these control sequences are compatible with the Host cell systems. SV40 vectors are known in which exogenous eukaryotic DNA with their own Promoter sequences and splice signals in addition to the late SV40 promoter deliver a stable transcript.

An origin of replication can be identified either by a corresponding vector construction can be obtained so that this is an exogenous starting point, for example from SV40 or other viral sources (e.g. Polyoma, Adeno, VSV, PBV, etc.) contains or this can by the  chromosomal replication mechanisms of the host cell to be delivered. If the vector is in that Host cell chromosome integrated, that's enough the latter measure mostly.

The transformation of the cells with the vehicles can can be achieved through a variety of processes. For example, it can be done by calcium, whereby either the cells are washed in magnesium and the DNA is added to the cells suspended in calcium or the cells become a co-precipitate of DNA and Exposed to calcium phosphate. In the following Gene expression, the cells are transferred to media, which select for transformed cells.

With intracellular production of hMn-SOD this can Enzyme can be isolated by following the cells Achieve a correspondingly high cell density centrifuged and then enzymatically or mechanically be unlocked. The cleaning of the hMn-SOD according to the invention can be obtained via known biochemical processes for protein or enzyme purification such as B. dialysis, electrophoresis, precipitation, Chromatography or combinations thereof. If the enzyme secreted from the cell will be analog Protein purification procedures performed to hMn-SOD to get from the culture medium in pure form.

The invention cleaned with these methods hMn-SOD shows identical in the genuine enzyme biological activity spectrum in vivo and in vitrol.  

Among these activities are both immunological Properties (e.g. cross reaction with antibodies from genuine hMn-SOD against the hMn-SOD according to the invention) also biochemical and enzymatic activities too understand. For the biological and enzymatic Characterization of the hMn-SOD can e.g. B. the teaching of Marklund, S. (Marklund, S. & Marklund, G., Eur J. Biochem. 47, 469-474, 1974) followed by a strict distinction between the Cu / Zn and Mn containing enzymes, e.g. B. by adding KCN (inhibits Cu / Zn-SOD but not Mn-SOD) or using the different pH dependency of their activities is given (see in particular: Ysebaert-Vanneste, M., Vanneste, W.H., Anal. Biochem. 107, 86-95, 1980).

The polypeptide according to the invention is not only the mature hMn-SOD, which is described in detail, but also any modification of this enzyme. These modifications include e.g. B. shortening of the molecule at the N- or C-terminal end, replacement of amino acids by other residues, whereby the enzyme activity is not significantly changed.
The invention relates not only to gene sequences which code specifically for the described and exemplified hMn-SOD, but also to modifications which can be easily and routinely obtained via mutation, degradation, transposition or addition. Any sequence encoding the hMn-SOD of the invention (ie having the corresponding and known biological activity spectrum) and degenerate compared to that shown is also included; Those skilled in the art are able to degenerate DNA sequences, particularly of the coding regions. Likewise, any sequence which codes for a polypeptide with the activity spectrum of the authentic hMn-SOD and which hybridizes with the sequences shown (or parts thereof) under stringent conditions is included.

It belongs to the state of the art, among which defined hybridization conditions (inclusive Pre-washing, prehybridization, hybridization, washing) is to perform stringent conditions to reach. For the habridization of oligonucleotides against a gene bank ("gene bank screening") preferably under the Wood, I.M. et al. (Proc. Natl. Acad. Sci. USA 82, 1582-1588, 1985) method. To test for a specific DNA sequence with one of the inventive coding for hMn-SOD DNA sequences are habridized - either via in situ Hybridization against plaques or bacterial colonies or via Southern blotting - are those of Maniats, T. et al. methods and conditions described in detail (Maniatis. T. et al., Molecular Cloning, Cold Sping Harber Laboratory, 1982, particularly pp. 326-328 and pp. 387-389. All clear from the background lifting signals accordingly show a positive Hybridization signal on.  

The tasks specified above become more specific solved in that from human tissue, preferably from Human placenta tissue that prepares RNA becomes. During the disruption of tissue culture cells Tissue can be made directly with hot phenol for this type of extraction only in frozen State, advantageously in the presence of powdered or granular dry ice or in liquid nitrogen, can be crushed (e.g. Starmix). Aggregates between mRNA and other RNAs can be generated by formamide or by heating (e.g. 65 ° C) can be dissolved again. A preferred one The RNA isolation method is that of Chirgwin (Chirgwin, J.M. et al., Biochemistry 18, 5294-5299, 1979). The poly (A) ⁺RNA can be derived from that of protein and DNA purified preparation conveniently by Affinity chromatography, e.g. B. Poly (U) -Sepharose or Oligo (dt) cellulose can be isolated as a rule eukaryotic mRNA populations on a poly (A) tail have their 3'-end (Aviv, H., Leder, P., Proc. Natl. Acad. Sci. USA 69, 1409-1412, 1972; Lindberg, U., Persson, T., Europ. J. Biochem. 31, 246-254, 1972). The poly (A) ⁺-RNA can be isolated preferred according to Auffray (Auffray, C., Rougeon, F., Eur. J. Biochem. 107, 303-314, 1980).

This can enrich the purified mRNA done that the entire mRNA fraction according to their size is separated - e.g. B. by centrifugation in one Sucrose gradient. The desired mRNA can e.g. B. about known in vitro protein biosynthesis systems (Reticulotytes, oocytes from Xenopus laevis) will.  

The purified mRNA or the enriched fraction serves as a template for the synthesis of the first strand the cDNA, which is generated with the help of the reverse transcriptase and a primer. The primers are either oligo (dT) or synthetic primers can be used, the latter can based on the known amino acid sequence hMn-SOD can be derived and allow the repeated Reverse transcription priming (Uhlen, M. et al. EMBO Journal 1, 249-254, 1982).

In the present invention, the synthesis of the first strand of the cDNA was started with oligo (dT) 12-18 as a primer in the presence of dNTPs.
Various known methods can be used for the synthesis of the second strand of the cDNA, of which the priming with a complementary primer (Rougeon, F., Mach, B., J. Biol. Chem. 252, 2209-2217, 1977) selectively Self priming using a hairpin structure located at the 3'-end of the cDNA (Efstratiadis, A. et al, Cell 7, 279, 1976) or using an Okazaki fragment-like primer formed by RNaseH (Gubler, U., Hoffmann, BJ, Gene 25, 263, 1982). In the present invention, the method as described by Huynh, TV (Huynh, TV et al., In DNA Cloning Vol I, (DM Glover ed.), Chap. 2, pp. 49-78, 1985) was preferred. . The double-stranded cDNA obtained thereafter can be cloned or packaged directly in a suitable vector, for example in a cosmid, insertion or substitution vector, in particular in a lambda vector, preferably in λ gt10 (Huynh, TV et al., 1985). Several known methods are available for cloning in Lambda, of which, for example, the "homopolymer tailing via dA-dT or dC-dG or the linker method with synthetic linkers should be mentioned (Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982; Huynh, TV et al., DNA cloning Vol I (DM, Glover et.) 1985, 1980; Watson, CJ, Jackson, F. dto, 1985, chapter 3). In the case of cloning the The cDNA according to the invention was inserted into the EcoRI site of λ gt 10. The in vitro packaging and cloning of the cDNA according to the invention or the construction of the cDNA gene bank was carried out according to Huynh, TV et al, 1985, pp. 49-78.
With the phage population obtained, the amplification and plaque purification were carried out by infection of a suitable host, in particular E. coli, preferably E. coli C600, or by ensuring the lytic multiplication cycle of lambda.

The cDNA library was searched with radiolabelled synthetic oligonucleotides derived from the published amino acid sequence (Barra, D. et al. Oxy Radicals and their scavenger Systems, Vol. 1, 336-339, 1983) under stringent hybridization conditions. The in situ hybridization method according to Benton and Davis (Benton, WD, Davis, RW, Science 196, 180-182, 1977) was used in the present invention. Two mixtures of eight synthetic 23-mer oligonucleotides each of the formulas Va and Vb, which are colinear with the amino acids 39 to 46 and 200-207, of the type described by Barra, D. et al. (see above) published amino acid sequence and which take into account the degeneration of the genetic code. The last base at the 5 'end of these DNA probes lacks the wobble base for the complete codon for Gln (amino acid 46) or Glu (amino acid 207).
A, G, C and T stand for the corresponding nucleotides, I for inosine.

The oligonucleotide probes can be made according to known methods chemical synthesis processes are produced. For the The present invention became a DNA synthesizer model 381A (Applied Biosystems) is used.  

The synthesis of all possible combinations of these two DNA probes ensure that at least one of the available Oligonucleotides optimal with that for the probe complementary single-stranded DNA region of the sought hMn-SOD genes mate. The use of two independent Pooly of 23-mer oligonucleotides decreases the Possibility that the "wrong" positives are selected will.

After isolation of homogeneous plaques, the based on positive signals after hybridization with the two 23-mer DNA probes were identified seven recombinant phages isolated and from their DNA 500 to 1000 bp EcoRI fragments sequenced will. After sequence analysis of these EcoRI fragments after the Sanger method (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977; Sanger F. et al. FEBS Letters 87, 107-111, 1978) and after subcloning in the EcoRI site of the M13 vector (Bluescribe, Vector Cloning Systems) and transformation in E.coli, for example E.coli JM101, it could be found that the EcoRI fragments contain cDNA inserts which are suitable for hMn-SOD from amino acid 22 (clones BS5, BS8, BS9, BS13, BSXIII) or code from amino acid 26 (clones BS3, BS12).

Surprisingly, however, it was also found that some of the DNA sequences found Deviations from the amino acid sequence of Barra, D. et al. (1984, see above) show:  

The DNA sequence of a 617 bp EcoRI fragment which is obtained from one of the clones obtained, e.g. B. BS8 could be isolated is shown in Fig. 1. The EcoRI fragment contains a 532 bp long sequence coding for hMn-SOD and a 51 bp long non-translated region, including a poly (A) ₃₀ tail. Portions of linker sequences are also shown up to the (complete) EcoRI sites.

Positions 30 to 33 indicate a ThaI interface Position 367 to 372 shows a BamHI site. Surprisingly, codons can be found at position 53 to 61, 155 to 163, 176 to 184 and 500 to 508, the are colinear for potential N-glycosylation sites corresponding amino acid according to the corresponding characteristic general amino acid arrangements Asn-X-Thr or Asn-X-Ser, where X is for valine, Histidine or leucine stands where against the Cu / Zn SOD of the Cytosols has only one such amino acid combination.

On the amino acid differences compared to the Amino acid sequence from Barra, D. et al. (Barra, D. et al., J. Biol. Chem. 259, 12 595-12 601, 1984) by the obtained EcoRI fragment have been derived received earlier.

To the missing bases at the 3 ′ and / or 5 ′ terms of the hMn-SOD DNA partial sequence from the cDNA library To obtain a complete hMn-SOD gene, different strategies can be followed. For example, the cDNA obtained can be used as Hybridization probe against a genomic library used to encode the whole enzyme Obtain sequence, or proceed z. B. according to Kakidani, H. using synthetic complementary to mRNA Oligonucleotides as specific primers for the reverse Transcription (Kakidani, H. et al. Nature 298, 245-249, 1982). However, it is also possible to find the missing end of the cDNA sequence based on the known amino acid sequence (Barra, D. et al., J. Biol. Chem. 259, 12 595-12 601, 1984) chemically synthesize and with the found cDNA connect, whereby a defined end is obtained.  

The latter way was used to manufacture the complete DNA sequence according to the invention for hMn-SOD followed, in which case the 5'-end by two Oligonucleotides of the formulas VIa and VIb has been completed, the advantageously XhoI / CbaI - or XbaI / NcoI - have protruding ends. According to the invention is at the 5'-end of the coding strand the 3'-end of ADHI promoter has been taken into account (Formula VIa)

After combining both synthetic oligonucleotides Formulas VIa and VIb, cloning into a suitable one Vector, for example into a correspondingly modified one pUC18 derivative and adding the ThaI / EcoRI fragment of the cDNA according to the invention from one of the clones obtained, whose 5'-end has at least the ThaI site, can a plasmid can be obtained which is a complete cDNA of the hMn-SOD gene in the correct reading frame accordingly Contains formulas VIIa and VIIb without the THaI positions.

The DNA sequences according to the invention can be found in different expression vectors built in and with the help the control elements described are expressed, preferably in pES103 with the ADHI promoter (DSM 4013). pES103 is obtained in that in the pUC18 derivative pES102, which in the HincII interface Xho linker contains the 1500 bp BamHI / XhoI fragment the ADHI promoter (e.g. Ammerer, G., Methods in Enzymology 101, 192-201, 1983).  

Instead of this ADHI promoter sequence from originally 1500 bp can also be shortened to a length of approx. 400 bp ADHI promoter can be used as a BamHI / XhoI fragment. The shortened ADHI promoter (ADHIk) is obtained by that plasmid pWS323E (DSM 4016) digested with BamHi / XhoI and the ADHIK promoter is isolated.

A suitable one is used for the correct termination Terminator sequence, suitably an ADH terminator, preferably the ADHII terminator behind the hMn SOD ligated. The ADHII terminator (Beier, D.R., Young, E.T., Nature 300, 724-728, 1982) can be digested by SphI pMW5-ADHII (Washington Research Fundation) as 1530 bp long fragment and subsequent HincII digestion as final ADHII terminator (329 bp) can be obtained, or from plasmid pGD2 (DSM 4014) as 336 bp long HindIII / XbaI fragment.

Various yeast vectors are available for expression in yeast into which the expression cassettes with the hMn-SOD gene according to the invention can be incorporated, preferably YEp13 (Broach, J.R. et al., Gene 8, 212-133, 1979; ATCC 37 115), pJDB 207 (DSM 3181, filed on Dec. 28, 1984), YIp5 (Struhl, K. et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1979; ATCC 37 061), pEAS102 (pEAS102 can be obtained by partially YIp5 with PstI and is completely digested with BamHI and that the URA3 gene containing isolated 4.3 kb fragment with the 4.4 kb BamHI / PstI fragment from pJDB207 is ligated).  

With these, an expression cassette with the yeast vectors bearing the hMn-SOD gene according to the invention suitable yeast cells are transformed by known methods will. Can be used as suitable yeast cells for expression preferably all those who are considered are deficient for their own yeast-specific Mn-SOD and which a selectable yeast gene, such as. B. HIS3, URA3, LEU2, SUP, to name a few, included. Such Mutants, for example, by in vitro or in vivo constructed mutated genes and these over a "Transplacement" are about integrative Get transformation (e.g. Winston, F. et al., Methods in Enzymology 101, 211-228, 1983). That too the yeast Mn-SOD gene is for example in Contain plasmid pL41 as a BamHI fragment (from Loon et al., Gene 26, 261-272, 1983). Because the full sequence of this BamHI fragment is known (Marres, C.A.M. et al., Eur. J. Biochem. 147, 153-161, 1985) is the Mn-SOD gene of yeast also accessible without pL41.

The hMn-SOD produced by such transformants can according to known methods of protein isolation and Protein purification can be obtained. Cell disruption can e.g. B. after van Loon et al. (Proc. Natl. Acad. Sci. USA 83, 3820-3824, 1986).

The expression systems already mentioned and established are available for the expression of hMn-SOD in bacteria, preferably E. coli, for example E. coli HB101, C600, JM101. For this purpose, the DNA sequences according to the invention have to be brought under the control of a strong E. coli promoter (see above), not under a eukaryotic promoter. Examples of such known promoters are lac, lacuv5, trp, tac, trp-lacuv5, λ P _L , ompF, bla.
The obligatory use of a ribosomal binding site to ensure efficient translation in E. coli has already been described in detail earlier.

To detect expression of hMn-SOD activity by E.coli are the bacteria after incubation in one suitable, conventional culture medium and the supernatant of the hMn-SOD activity as described (e.g. Marklund, S., Marklund, G., 1974; Ch. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276-287, 1971; H.P. Misra and I. Fridovich, Arch. Biochem. Biophys. 183, 511-515, 1977; B.J. Davis, Annals of the NY Academy of Sciences 121, 404-427, 1964; M. Ysebaert-Vanneste and W.H. Vanneste, Anal. Biochem. 107, 86-95, 1980).

The detection of the expressed hMn-SOD gene can also be done by Labeling of the proteins in maxi cells are performed. Plasmid-encoded proteins can be obtained using the Maxi cell technique (Sancar, A. et al., J. Bacteriol, 137, 692-693, 1979) can be selectively labeled in vivo. The E.coli Strain CSR603 (CGSC 5830) has none DNA repair mechanisms. By a suitable one UV radiation dose will destroy the bacterial chromosome, some of the much smaller ones and in multiple copies per In contrast, cell-present plasmid DNAs remain functional  receive. After killing everyone not harmed themselves multiplying cells by the antibiotic D-cycloserine and The endogenous mRNA will be used up in the remaining Cells only transcribed and plasmid-encoded genes translated. The proteins formed can be built in radioactively labeled and detected by ³⁵S-methionine will. E.coli CSR603 is made using the usual methods Expression plasmids transformed and on agar plates containing amplicillin on transformed bacteria selected. The preparation of the maxi cells and that The proteins are labeled according to the instructions of A. Sancar (1979, see above). A is used as the molecular weight standard ¹⁴C-methylated protein mixture (Amersham) used. As The plasmid that controls only the promoter without the contains hMn-SOD gene.

After transformation of the host, expression of the Gene and fermentation or cell cultivation under Conditions under which the proteins of the invention can be expressed, the product can usually by known chromatographic separation methods extracted in order to obtain a material that contains the proteins with or without leader and tailing sequences. The hMn-SOD according to the invention can with a leader sequence on N-terminus are expressed which, as already described, can be removed from some host cells. Unless, as already described, this is a split off of the leader polypeptide (if any) required to to obtain mature hMn SOD. Alternatively, the sequence can be like this be modified that the mature enzyme in the microorganism is produced directly. In this case, the  Precursor sequence of the yeast mating pheromone MF-alpha-1 used to correct "maturation" of the fused protein and the excretion of the products in the growth medium or the periplasmic space guarantee. The "secretion" of the hMn-SOD in Yeast mitochondria can be done by just before the hMn-SOD gene is the leader sequence for the yeast Mn.SOD gene is provided.

The purification of hMn-SOD from cells can be done according to known Procedure.

The genetically obtained hMn-SOD according to the invention are due to the biological-enzymatic Spectrum of action on the one hand and because of now Available amount of high purity enzyme with the highest possible immunological identity to the genuine hMn-SOD on the other hand for every kind of prevention, treatment and / or diagnostics in the area of inflammatory, degenerative, neoplastic or rheumatic Diseases for wound healing, autoimmune diseases and suitable for transplants - or for prevention and  Therapy of diseases with a deficiency hMn-SOD go hand in hand or are causally connected with it. For example, the clinical Possible uses are those from Bannister W.H. and Bannister J.V. (Biological ans Clinical Aspects of Superoxide and Superoxide Dismutase, Vol. 11B, Elsevier / North-Holland, 1980) and from Michelson, A.M., McCord, J.M., Fridovich (Superoxide and Superoxide dismutases, Acedemic Press, 1977) are. Furthermore, the following are clinical Consider possible uses: at Perfusion wounds, stroke, alcohol-damaged liver, Premature babies, possibly pancreatitis, acute Respiratory diseases (ARDs), emphysema, dialysis-impaired Kidneys, osteoarthritis, rheumatoid arthritis, radiation-induced damage, sickle cell anemia.

The hMn SODs according to the invention are also suitable for increasing the shelf life of solid or liquid foods.
The hMn-SODs according to the invention can be administered either systemically or topically, in the first case using conventional parenteral application routes (e.g. iv, imsc, ia) and in the second case the correspondingly known administration forms (e.g. pastes, ointments, Gels, lozenges or chewable tablets, powders, and other galenic formulations and pharmaceutically acceptable excipients which enable the local absorption of the hMn-SOD preparation) are suitable. Approximately 4 mg daily, for example, can be used as a therapeutically effective dose range according to individual criteria (e.g. patients, disease severity, etc.).

Legends for the pictures:

Fig. 1: EcoRI fragment from clone BS8 with the 532 bp long coding region from amino acid 22 of the mature hMn-SOD, the 51 bp 3 ′ ut region and the sequence parts of the linker. The potential N-glycosylation sites (underlined), the only ThaI site and the BamHI site (underlined) are indicated.

Fig. 2: schematic strategy for the construction of plasmid HSOD4, which contains the synthetic 5'-end of the hMn-SOD gene as an XhoI / NcoI fragment.

Fig. 3: Restriction maps of the plasmids HSOD2 and HSOD3, and of the plasmid HSOD4 constructed from them.

Fig. 4: Construction of a plasmid (HSOD6) with the complete cDNA for hMn-SOD, as XhoI / EcoRI fragment.

Fig. 5: Preparation of the ThaI / EcoRI fragment of the hMn-SOD cDNA from clone BS8.

Fig. 6: Construction of plasmid p154 / 2, which contains the ADHI promoter as a 1500 bp BamHI / XhoI fragment.

Fig. 7: Construction of plasmid p150 / 2 with the ADHI promoter and ADHII terminator (336 bp XbaI / HindIII fragment) necessary for the expression of hMn-SOD.

Fig. 8: Completion of the final plasmids (pKH1 and pKH2) with the ADHI promoter or the ADHIk promoter and the ADHII terminator, for further insertion of the hMn-SOD cDNA via the XhoI / EcoRI site. The plasmid pKH2 corresponds to pKH1, except that pKH2 contains the ADHIk promoter instead of the ADHI promoter.

Fig. 9: Construction of the HSOD7 expression cassette with the shortened approx. 400 bp long ADHI promoter (ADHIk). The construction with the ADHI promoter of the original length is carried out analogously starting from pKH1.

Fig. 10: Construction of plasmids with the URA3 gene lying within the yeast Mn-SOD gene as a marker in different orientations to the Mn-SOD gene (SODY7, SODY8), for the production of a yeast Mn-SOD mutant suitable for expression. The gene transplacement in the corresponding yeast strain (DBY747) was carried out with SODY7 and SODY8.

The following examples, which do not limit the invention are intended to illustrate the invention in detail.

used material

Unless specifically stated in the following examples, were the following materials, solutions, plasmids, vectors and microorganisms used:  

ADHI promoter: DSM 4013 (pES103), deposited on (1500 bp BamHI / XhoI) 27. 2. 87 ADHI promoter, abbreviated: DSM 4016 (pWS323E), deposited on (400 bp BamHI / XhoI) 27. 2. 87 ADHII terminator: DSM 4014 (pGD2), deposited on (336 bp XbaI / HindIII) 27. 2. 87 BamHI buffer: 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl₂, 100 µg / ml BSA core buffer: 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 50 mM NaCl denaturing solution: 0.5 M NaOH, 1.5 M NaCl Denhardt solution (50 ×): 1 g polyvinylpyrrolidone MG 360 000, 1 g Ficoll, 1 g bovine serum albumin (BSA) ad 100 ml H₂O E.coli C600: F ^- , supE44, thil, thr1, leuB6, lacY1, tonA21, g ^- (ATCC 23 724) E.coli JM101: supE, thi, Δ (lac-pro AB), [F ′, traD36, proAB, lacI ^q Z, Δ M15] High buffer: 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol (DTT) HincII buffer: 10 mM Tris-HCl pH 7.5, 60 mM NaCl, 10 mM MgCl₂, 1 mM 2-mercaptoethanol, 100 µg / ml BSA hybridization solution: as prehybridization solution, without salmon sperm DNA Klenow reaction solution: 22 µl DNA / H₂O, 2.5 µl 10 × NTR buffer (0.5 M Tris-HCl pH 7.2, 0.1 M MgSO₄, 1 mM DTT, 500 µg / ml BSA) 1 µl each 2 mM dATP, dGTP, dCTP, dTTP, 2.5 U Klenow fragment (0.5 µl) Lambda buffer: 100 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM EDTA LB agar: LB liquid medium, 15 g / l Bacto agar (Difco ) LB liquid medium: 10 g / l Bacto-Trypton (Difco), 5 g / l yeast extract (Difco), 5 g / l NaCl, 10 M NaOH ad pH 7.4 Ligation solution: 66 mM Tris-HCl pH 7 , 6, 10 mM MgCl₂, 5 mM DTT, 1 mM ATP, 1 U T4-DNA ligase neutralization solution: 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl nitrocellulose filter: Schleicher & Schuell, membrane filter BA 85 NruI Buffer: 50 mM KCl, 50 mM NaCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂ prehybridization solution: 5 × SSC, 5 × Denhardt's solution, 50 mM Na phosphate buffer pH 6.8, 1 mM Na₂P₄O₇, 100 µM ATP, 0.1% SDS, 30-100 (50) µg / ml denatured, ultrasonically treated salmon sperm DNA puC18: Pharmica pURA3: DSM 4015, deposited on February 27, 87 S. cerevisiae DBY747: a. leu2, his3, trpl, ura3 (Yeast Genetic Stock Center, Berkeley) SC-URA medium: 0.67% BYNB (Difco), 2% glucose, 2% 50 × AS mix (per liter: 1 g histidine, 6 g leucine, 2.5 g tryptophan, 4 g lysine, 1.2 g adenine, 2 g arginine, 1 g methionone, 6 g phenylalanine, 5 g threonine, 6 g isoleucine) SmaI buffer: 10 mM Tris-HCl pH 8 , 0, 20 mM KCl, 10 mM MgCl₂, 10 mM 2-mercaptoethanol, 100 µg / ml BSA SphI buffer: 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 10 mM 2-mercaptoethanol, 100 µg / ml BSA SSC (20 ×): 3.0 M NaCl, 0.3 M Na₃ citrate, pH 7.0 SSPE (20 ×): 3.6 M NaCl, 0.2 M Na₂HPO₄, 20 mM EDTA , with NaOH (10 N) ad pH 7.4 TE buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA ThaI buffer: 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂ top agarose: LB liquid medium, 0.7% agarose (Seaken FM agarose) wash solution: 1 M NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS

1. Construction of a cDNA library

A fresh human placenta became cube-sized Pieces of tissue in liquid nitrogen snap frozen and the tissue is pulverized at below -80 ° C.  

The pulverized tissue material became then the RNA according to Chirgwin, J.M. et al. described procedure extracted and prepared (Chirgwin, J.M. et al., Biochemistry 18, 5294-5299, 1979).

The poly (A) -RNA according to Aviv, H. and Leder, P. (Proc. Natl. Acad. Sci, USA 69, 1409-1412, 1972) was prepared from the RNA obtained afterwards.
The synthesis of the cDNA was carried out using the "cDNA synthesis system" (Amersham RPN 1256).

The packaging was done with Gigapack (Vector cloning systems). All further methodical steps for cloning into the EcoRI site of λ gt10 were carried out according to the instructions from Huynh TV et al. (DNA Cloning Vol 1, DM Glover ed., IRL Press, Chapter 2, 1985), except that E.coli C600 was used as the "plating bacteria". The titer of the λ gt10 phage representing the cDNA library was 1.2 × 10¹⁰ pfu / ml, the number of independent clones was 1 × 10⁶.

2. Amplification of the λ gt10 library

A suitable E. coli host strain (C600, genotype F-, supE44, thil, thrl leuB6 lacY1 tonA21 lambda - (M.A. Hoyt et al., 1982, Cell 31, 5656) was overnight in LB medium supplemented with 0.2% maltose at 37 ° pre-bred.

This overnight culture was 5 min at 3000 rpm centrifuged and in ice-cold 10 mM MgSO₄ solution suspended so that the OD600 nm was 4.0. The so prepared Mg cells were stored at 4 ° C and could be used for a week.  

12 × 200 μl of Mg cells were mixed in a sterile tube with a phage suspension (50,000 pfu each of the cDNA library / plate) and incubated at 37 ° C. for 20 min.
6-7 ml of melted top agarose (containing 10 mM MgSO₄, final concentration), heated to 42 ° C., were then pipetted into each tube, mixed and mixed with 12 LB agar plates (13.5 cm in diameter) preheated at 37 ° C. with 10 mM MgSO₄ poured out and incubated at 37 ° C for 6-12 hours.

3. Primary screening to identify recombinant λ phages a) Preparation of the nitrocellulose filter

The plates prepared in this way were cooled to 4 ° C. after the incubation. Nitrocellulose filters numbered in pencil were placed on the plate surface and the positions on the plates were marked with pinpricks. About 1 min after they were completely moist, the filters were carefully removed again, placed in denaturing solution and incubated for 1 min at room temperature (RT).
This was followed by neutralization in neutralization solution for 5 min at RT and incubation for 30 sec in 2 × SSPE, also at RT.

Up to 3 additional prints were made from each plate made, the filters on each 30 sec longer left on the plate. The positions of the Pinpricks were accurate on the following filters transfer.

The filters were lying on filter paper in the open air dried and the DNA by baking for two hours Fixed at 80 ° C. The plates were kept until the result the subsequent hybridization.

b) Preparation of the 32 P-labeled DNA probes

The synthetic oligonucleotide mixtures were prepared on a 381A DNA synthesizer (Applied Biosystems), purified by polyacrylamide gel electrophoresis (20% in 8 M urea, T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982, p173ff) and via Sephadex G50 ( Pharmacia) desalted.
The DNA probes synthesized in this way are complementary to RNA base sequences which code a) for amino acids 39-46 and b) 200-207 (D. Barra et al., Oxy Radicals and their scanvenger Systems, Vol. 1, 336 -339, 1983) and have the following base sequences:

where A, G, C and T for the corresponding nucleotides, I stands for inosine.

The chemically synthesized DNA sample mixtures were each dissolved in water at a concentration of 20 pM / ul.

Reaction set

20-100 pM gamma³²-PATP (<3000 Ci / mmol, Amersham), lyophilized from ethanolic solution, 20-100 pmol Oligonucleotide, 1 ul 10 x kinase buffer (0.7 M Tris-HCl pH 7.6, 0.1 m MgCl₂, 50 mM dithiothreitol, 10 units T4 Polynucleotide Kinase (BRL), water ad 10 ul.

The reaction took place at 37 ° C for 60 min and was followed by Addition of 25 mM EDTA stopped. Not built in Radioactivity was removed by Exclusion chromatography on a 1 ml Biogel P6-DG (Biorad) column, made in a 1 ml Disposable syringe. TE buffer was used as eluent used.

c) In situ hybridization

To remove remains of agarose and bacteria from the Remove nitrocellulose during hybridization lead to strong background radiation Filters in a not too small volume pre-wash solution a few hours up to overnight with panning Incubated at 65 ° C. To nonspecific binding sites for Saturate DNA on the nitrocellulose filters this 1-12 hours at 37 ° C in the previously in a vacuum degassed prehybridization solution incubated.  

The radioactive used for hybridization labeled DNAs (approx. 1 × 10⁹ cpm / µg) were used required amount of preheated to 37 ° C and degassed hybridization solution added. To the Concentration of the respective DNA sample in the It was only to keep the hybridization solution as high as possible used so much hybridizing liquid that the Filters were just covered with liquid. The Hybridization took place at 37 ° C for 12-18 hours.

The nitrocellulose filters were then according to the method of Wood et al. (Proc. Natl. Acad. Sci. Vol 82, 1585-1588, 1985) 3 times in 6 × SSC and 0.05% SDS rinsed (4 ° C) and 2 × 30 min at 4 ° C washed as well. Then the filters were in a freshly prepared solution, the 3 M Tetramethylammonium chloride (Me4NCl), 50 mM Tris-HCl pH 8, 2 nM EDTA and 0.05% SDS contains, 3 times Rinsed room temperature (RT), washed 2 × 30 min (RT) and finally 3 × 30 min at 49 ° C (Oligonucleotide mixture a)) or at 52 ° C. (Oligonucleotide mixture b)) washed in air dried (oligonucleotide mixture b) and on paper glued. X-ray films were kept at -70 ° C for 2-8 days Exposed using an "intensifiyin screen".  

4. Plaque cleaning

Since the high density of the plaques used in the no single plaques isolated could be, the recombinant lambda phages through multiple successive searches at the same time reduced plaque density. After the autoradiograms were developed, regions became isolated from the agar plate, (from three performed Primary screenings were 2 out of 28 regions, out of 35 Regions 1 and out of 15 regions 5 positive) on two nitrocellulose filters hybridized in duplicate gave a positive hybridization signal. This was the desired place with the narrow end of a sterile Pasteur pipette pricked out of the agar and in 0.3-0.6 ml Lambda buffer (100 mM Tris HCl pH 7.5, 10 mM MgCl₂ and 1 mM EDTA). But it can also use SM buffers (Maniatis T., Molecular-Cloning, 1982, p. 70) will. After adding a drop of chloroform, let the phages from the agar overnight at 4 ° C diffuse out and plated each one Phage suspension again in several dilutions. From plates that had 300-1000 plaques again a nitrocellulose filter deduction and hybridized against each of the two DNA probes. This The process was repeated, with single plaques were followed up until all plaques on a plate gave a positive hybridization signal.  

5. Analysis of the phage clones obtained a) Titration of the λ phages

The phage suspensions were diluted 1:10 with lambda buffer, mixed by repeated swirling and plated out. After incubation at 37 ° C., the plaques formed on the bacterial lawn could be counted and the titer (plaque forming units (pfu)) determined.
The titer was 2.2-8.6 × 10¹⁰ pfu / ml for the purified phage suspensions.

b) Preparation of the Lambdaphagen DNA

After isolation and titration of the homogeneous Phage clones were these in a density of 2 × 10⁶ pfu / 13.5 cm petri dish (with the culture media of the Composition: 1.5% agarose, 10 g / l trypton, 5 g / l Yeast extract, 5 g / l NaCl, 10 mM MgSo₄ and 0.2% Glucose) with 200 ul C600 Mg cells (4 OD₆₀₀) plated, incubated for 5 hours at 37 ° C and then cooled to 4 ° C. Elution of the phages was done by overlaying the plates with 8 ml each Lambda buffer and a few drops of chloroform and slight swiveling at 4 ° C overnight. The through Centrifugation (15,000 rpm, 15 min, 4 ° C) cleaned The supernatant was finally removed and the phages by centrifugation at 50,000 rpm (Beckman Ti50 rotor) Pelleted at RT for 30 min. After adding 500 µl each Lambda buffer and incubation with ribonuclease A (RNase A, 10 µg / ml and deoxiribonuclease (DNase, 1 µg / ml, 30 min at 37 ° C, the salt concentration  by adding 25 ul of 0.5 M DETA, 12 ul of 1 M each Tris-HCl pH 8.0 and 6.5 µl 20% SDS increased and the existing enzymes by incubation at 70 ° C for 15 min deactivated. After extraction with phenol and extraction twice with chloroform / isoamyl alcohol (24: 1) the DNA was run at equal volumes Add 0.1 vol. 3 M sodium acetate pH 5.2 and 2 vol. Alcohol precipitated, centrifuged, with 70% alcohol washed, dried and in 50 µl TE buffer added.

c) Restriction analysis

2 ul of DNA solution were mixed with 5 units of EcoRI HIGH buffer incubated for 2 hours at 37 ° C, the fragments obtained on a 1% agarose gel (T. Maniatis et al., 1982, p149ff) under a voltage of 1-5 volts / cm separated, the fragments with lengths between 500 and 1000 base pairs eluted from the gel (G.M. Dretzen et al., Anal. Biochem. 112, 295-298, 1981) and finally subjected to a sequence analysis.

d) sequence analysis

The subcloning of the restriction fragments into one for the sequence determination according to Sanger (F. Sanger et al., Proc. Natl. Acad. Sci. 74, 5463-5467, 1977; F. Sanger et al. FEBS-Letters 87, 107-111, 1978) suitable vector (Bluescribe M13 + or M13-, vector cloning systems (C. Yanisch-Perron et al., Gene 33, 103-119, 1985)) was carried out according to the usual methods for carrying out restriction and ligation of DNA fragments and transformation of E. coli host cells (T. Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Press, p104, 146ff, 396; DNA cloning, IRL-Press 1985, Vol. 1, chapter 6). 100 ng isolated EcoRI cDNA fragments were inserted via EcoRI sites into the correspondingly prepared dsDNA form (replicative form, 50 ng) of the vector (by incubation for 2-12 hours at 14 ° C. in 10 μl ligation solution) and with This recombinant construction (with the designations BS3, BS5, BS8, BS9, BS12, BS 13, BSXIII) transformed competent E. coli (strain JM 101) cells.
The single-stranded DNA of the recombinant phages was isolated and sequenced according to Sanger. The sequences read were processed using suitable computer programs (R. Staden, Nucl. Acid. Res. 10, 4731-4751, 1982).
The isolated clone 8 (BS8) contains the coding sequence from amino acid 22 of the mature enzyme (Fig.1)

6. Construction of an expression cassette

To express the hMn-SOD in yeast Completion of the isolated cDNA, as well as the Construction of an expression cassette necessary, whereby the ADHI promoter in its original length (approx. 1500 bp, Methods in Enzymology, Vol. 101, Part C, 192-201, 1983), the same in abbreviated form (ADHIK approx. 40 bp) and the ADHII terminator (Dr. R. Beier and E.T. Young, Nature 300, 724-728, 1982) is used.  

a) Completion of the gene

Since the isolated cDNA clone 8 at the N-terminus lacks the number of bases corresponding to the 21 amino acid (AS), the gene was completed according to the reported amino acid sequence (D. Barra et al., J. Biol. Chem. 259, 12 595-12 601, 1984) taking into account the yeast codon selection (PM Sharp et al., Nucl. Acids. Res. 14, 5125-5143, 1986) 2 pairs of oligonucleotides as XhoI-XbaI fragment (OP1, according to formula VIa ) or XbaI-NcoI fragment (OP2, corresponding to formula VIb) constructed and synthesized (381A) DNA synthesizer, applied biosystems). OP1 was converted into plasmid V 17 (originated from pUC18 (J. Vieira and J. Messing, Gene 19, 259, 1982) via XhoI / XbaI after HincII restriction and insertion of XhoI linkers (New England Biolabs, d (CCTCGAGG) and SmaI restriction of the corresponding plasmid pES102 with subsequent insertion of NcoI linkers (New England Biolabs, d (CCCATGGG)), inserted (Fig. 2), OP2 via XbaI / NcoI: 4 μg of V 17 DNA with 10 each were used for this Units XbaI and NcoI or XhoI and XbaI in 40 μl CORE buffer digested for 2 hours at 37 ° C. and purified by gel electrophoresis (0.7% agarose, see above). 5 μl each of the synthesized single strands of OP1 and of OP2 (each 10 pM / ul) were mixed, incubated for 10 min at 65 ° C. and slowly cooled to RT, in each case 1/10 of them were each described with 50 ng double-cut vector (XhoI / XbaI for OP1 and XbaI / NcoI for OP2) as described above Conditions ligated (plasmids HSOD2 and HSOD3, Fig. 2). Finally, HSOD2 and HSOD3 were scaI / XbaI (ie after double digestion g combined with ScaI and XbaI in CORE buffer for 2 hours at 37 ° C after purification and isolation of the cut vectors by gel electrophoresis and ligation under the conditions described above HSOD4 (Fig. 2, 3), this was prepared for the uptake of the ThaI / EcoRI cDNA fragment by NcoI restriction, followed by Klenow fill-in and EcoRI restriction: 5 μg of DNA were in 50 μl of high buffer with 18 units of NcoI at 37 ° C incubated for several hours, the cut DNA purified by gel electrophoresis, isolated and half of it incubated in 30 ul Klenow reaction solution for 1 hour at RT.
After the reaction had ended by adding 2 μl of 0.5 M EDTA and incubating the reaction solution at 70 ° C. (10 min), the DNA was purified by gel electrophoresis, isolated and cut again with 7.5 units of EcoRI in 20 μl of HIGH buffer, again cleaned and insulated (Fig.5).

The ThaI / EcoRI cDNA fragment was prepared as follows:
Competent E. coli (strain JM 101) host cells were transformed with the plasmid BS8, which contains the isolated cDNA clone 8 (see above), and the plasmid was prepared under suitable conditions (T. Maniatis et al., 1982, p368).
After restriction with ThaI (10 μg plasmid were digested in 40 μl ThaI buffer with 25 units of ThaI for 8 hours at 60 ° C.), the 759 bp ThaI fragment was trimmed with EcoRI (see above), with subsequent gel electrophoretic purification and isolation of the corresponding Fragments, the ThaI / EcoRI fragment (Fig. 4) thus obtained was prepared with the correspondingly prepared plasmid HSOD4 (approx. 100 ng fragment was ligated with 50 ng cut vector in 10 μl ligation solution. (See above)) to HSOD6 (Fig. 5 ) combined. Plasmid HSOD6 thus contains the complete c-DNA for hMn-SOD including Met. The reading frame is retained.

b) Construction of the expression cassette

Plasmid HSOD6 was mixed with Xho and EcoRI (5 Units / µg DNA) was double digested in CORE buffer XhoI fragment (gene) isolated and in the plasmid PKH1 or PKH2 inserted via XhoI / EcoRI. The plasmids PKH1 or PKH2 were prepared as follows (Fig. 6, 7, 8): In plasmid PES 103, which identified the ADHI promoter as 1500 bp BamHI-XhoI fragment in PES 102 (PES 102 is a pUC18 Derivative, which in the HincII interface XhoI-Linker contains the construction of the BamHI-XhoI fragments is described in Methods in Enzymology 101, 192-201) contains, were after SmaI restriction (1 µg plasmid was mixed with 5 units SmaI digested in SmaI buffer for 2 hours at 37 ° C), Purification and isolation BgIII linker inserted (T. Maniatis et al., 1982, p396). The resulting Plasmid (P154 / 1, Fig. 6) was removed by EcoRI restriction (see above), Klenow fill-in (see above) and religation (1 µg DNA was in 40 ul ligation solution (see above) overnight Incubated at 14 ° C) changed to plasmid 154/2 (Fig. 6).

Also based on plasmid pES103 was used double digestion with XhoI and HindIII in CORE buffer the linker - XhoI.EcoRI.XbaI.HindIII - (Fig. 7, synthesized on the 381a DNA synthesizer). This linker contains the sequence

In the resulting plasmid 150/1 was over XbaI / HindIII (double digestion in CORE buffer) ADHII terminator inserted (plasmid 150/2 (Fig. 7)). The ADHII terminator was obtained as follows: plasmid pMW5 ADHII (Washington Research Fundation) was with HindIII (core buffer), then with SphI (in SphI buffer) digested and since isolated 605 bp fragment in the vector V18 cloned and into the HincII interface XbaI linker (Biolabs, CTCTAGAG) built in (ligation so.). A 335 bp XbaI / SphI fragment was found in pUC18 (XbaI / SphI) ligated in (pGD2).

The vector V18 was obtained in that in pUC18 in the Hindi linker was inserted in the SmaI site and the HindIII position at the original location is missing, so that the multicloning station in V18 EcoRI. SstI. KpnI.HindIII.BamHI.XbaI.SalI.PstI.SphI reads.

The ADHII terminator eventually became duplicate Digestion with XbaI / HindIII in CORE buffer according to the usual methods (see above) isolated. Plasmid 150/2 contains up to the usual XhoI / EcoRI inserting gene for gene expression necessary units of approx. 1500 bp (promoter), 7 bp (XhoI linker), 6 bp (EcoRI linker), 7 bp (XbaI linker), 329 bp (terminator). These units were now over BamHI / HindIII (double digestion in CORE buffer) in inserted the vector 154/2 (Fig. 8). In that way Plasmid PKH1 obtained (Fig. 8) was obtained in an analogous manner the ADHI promoter through the shortened promoter ADHIk replaced as BamHI / XhoI fragment (412 bp) (pKH2, Fig. 9).  

In both plasmids, XhoI / EcoRI was eventually used (see above) the complete cut out of HSOD6 cDNA gene (see above) inserted. The plasmids thus obtained HSOD7 / 1 and HSOD7 / 2 (Fig. 9) differ from each other only through the different ADHI promoters and ADHIk (see above). The so finished Expression cassettes were placed on BglII / HindIII (after double digestion of the plasmids in CORE buffer and Isolation of the expression cassettes cut out) in the appropriately prepared Yeast transformation vectors YEp13 (J.R. Broach et al., Gene 8, 121-133, 1979), pJDB207 (DSM 3181, deposited on Dec. 28, 84), pEAS102, YIp5 (K. Struhl et al., Proc. Natl. Acad. Sci. USA 76, 1035-1039, 1979, ATCC 37 061) inserted via the interfaces BamHI and HindIII.

7. Preparation of a yeast Mn-SOD mutant suitable for expression

The gene for yeast Mn SOD (A.P.G.M. from Loon et al., Gene 26, 261-272, 1983) is a BamHI fragment in the vector PL 41 (Fig. 10) included and the sequence complete published (C.A.M. Marres et al. Eur. J. Biochem. 147, 153-161, 1985). After restriction with BamHI (2 µg plasmid were with 5 units in 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl₂, 100 ug / ul bovine serum albumin Digested at 36 ° C for 2 hours) the 2045 bp was long BamHI fragment containing the gene as usual by Gel electrophoresis purified and isolated and over BamHI into the vector VO (pUC18, but without HindIII interface) was cloned.  

The vector VO was obtained by adding 1 µg pUC18 was cut with HindIII (CORE buffer) linearized fragment from the gel according to known Methods isolated, the protruding ends with 2 U Klenow polymerase (ligase buffer + 0.2 mM dNTP) filled and after 30 min at RT by adding 2 U T4-DA ligase was regulated overnight at 14 ° C.

The resulting plasmid SODY1 (Fig. 10) was purified by NruI restriction (1 µg of plasmid was digested with 5 units of NruI in NruI buffer for 2 hours at 36 ° C) by gel electrophoresis and by inserting a HindIII linker (CAAGCTTG) to SODY3 (Fig. 10) changed. Finally, the URA3 gene (obtained from pURA3) was inserted into the HindIII site: 4 μg of SODY3 were digested with 20 units of HindIII in CORE buffer at 37 ° C. for 2 hours and dephosphorylated:
40 ul H₂O, 10 ul 1 mM EDTA, 5 ul 1 M Tris-HCl pH 9.5, 1 ul 100 mM spermidine 1 ul calf intestinal alkaline phosphatase (CIAP, 1 mg / ml H₂O) were added to 40 ul digestion and added at Incubated at 36 ° C. After 15 min, another 1 ul of CIAP was added and incubated for a further 15 min. The dephosphorylated vector was also purified by agarose gel electrophoresis. 2 µg of plasmid pURA3 were cut with HindIII (see above) and a 1.2 kb fragment which contains the yeast gene URA3 was also isolated and inserted into the prepared vector (see above).

The resulting plasmids SODY7 and SODY8 contain the URA3 gene within the yeast Mn gene and differ in the orientation of the URA gene relative to the Mn-SOD gene (Fig. 10).

The orientation of the URA3 gene relative to the Mn SOD gene is detectable because the URA3 gene is asymmetrical PstI position contains.

The plasmid SODY7 and SODY8 were used in strain DBY 747 (Genotype a, leu2, his3, trp1, ura3, Yeast Genetic Stock Center, Berkeley) carried out a "gene transplacement" (Methods in Enzymology 101, 202-211 and 211-228). The Strain DBY 747 was made with the BamHI fragment from SODY7 and SODY8 transformed (J.D. Beggs, Nature 275, 104, 1978). For this, 20 ug SODY7 or SODY8 with 50 U BamHI in 200 ul BamHI buffer (150mM NaCl, 6mM Tris-HCl pH 7.9, 6 mM MgCl₂, 1 mM DTT) cut and the total digestive mix (without the pUC portion to separate) extracted with phenol (Maniatis, T. et al., Molecular Cloning, 1982, pp. 458f) and by Ethanol precipitation concentrated (addition of 20 ul 3 M Sodium acetate pH 5.5, 500 ul ethanol). The DNA was in 10 µl of water taken up and ready for transformation used by yeast.

The transformants were based on uracil prototrophy selected.

Individual transformants were placed in 5 ml overnight SC-URA medium grown at 28 ° C. The cells were harvested by centrifugation, according to van Loon et al. open-minded (Proc. Natil. Acad. Sci. USA 83, 3820-3824, 1986) and examined for their content of Mn-SOD.  

For gel electrophoretic detection of Mn-SOD on the one hand and Cu / Zn-SOD on the other hand have already been existing work regulations (Ch. Beauchamp and I. Fridovich, Anal. Biochem, 44, 276-287, 1971; H.P. Misra and I. Fridovich, Arch. Biochem. Biophys. 183, 511-515, 1977; B.J. David, Annals of the NY Academy of Sciences Vol. 121, 404-427, 1964) applied.

The separation of the proteins with subsequent negative staining with nitroblue tetrazolium has proven to be very effective (BJ Davis, 1964; Ch. Beauchamp and I. Fridovich, 1971). Staining with dianisidine can increase sensitivity (HP Misra and I. Fridovich, 1977). For further characterization, a spectrophotometric assay (Hyland, K. et al. Anal. Biochem. 135, 280-287, 1983) with alkaline dimethyl sulfoxide as O ^- ₂-generating system and with cytochrome c as "scavenger" was used.

The distinction between Mn-SOD on the one hand and Cu / Zn-SOD, on the other hand, is made by adding KCN (see above and M. Ysebaert-Vanneste and W.H. Vanneste, Anal. Biochem. 107, 86-95, 1980). The SODY7 / 2 tribes, SODY7 / 6, SODY7 / 8 and SODY7 / 10 did not contain Mn-SOD Activity.

Claims (36)

1. DNA sequences that are all or partial carry genetic information for an enzyme that the enzymatic, biochemical and immunological Properties of human Mn superoxide dimutase (hMn-SOD) having. 2. DNA sequences according to claim 1, characterized in that that they have the genetic information for an hMn SOD an amino terminal leader or signal sequence contain. 3. DNA sequences according to claim 1, characterized in that that they code for the mature enzyme hMn-SOD. 4. DNA sequences according to claim 1 to 3, characterized characterized that this is the first codon for the first amino acid of the mature HMn-SOD Translation start codon is prepended. 5. DNA sequences according to claim 4, characterized in that the last codon for the last amino acid of the hMn-SOD is followed by a stop codon. 6. DNA sequences according to one of claims 1 to 5, characterized characterized in that they correspond to the nucleotide sequences Formulas Ia, Ib, II, IIIa, IIIb, Va, Vb, VIa, VIb, VIIa, VIIb correspond. 7. DNA sequences with a DNA sequence according to one of the Claims 1 to 6 under stringent conditions hybridize, these DNA sequences being synthetic, be semi-synthetic or natural in origin can and with the DNA sequences according to one of the  Claims 1 to 6 and their mutations of any kind can be related and for an enzyme with the enzymatic, biochemical and immunological Coding properties of the hMn-SOD. 8. Replicating vectors with at least one Selection marker, preferably with a Detection site for at least one restriction enzyme outside the origin of replication and others essential gene areas, but within one Selection marker, characterized in that this one of the DNA sequences according to one of claims 1 to 7 contain. 9. Replicating vectors according to claim 8, characterized in that they are of viral origin, preferably lambda phages, in particular λ gt10 or M13 phages. 10. Replicating vectors according to claim 9, characterized characterized as being of plasmid origin, preferably pUC18. 11. Plasmids, one of the DNA sequences according to one of the Containing claims 1 to 7, characterized in that that these plasmids are one of said DNA sequences carry the expression cassette containing, prokaryotic and stably transform eukaryotic host cells can be replicable in one of these host cells and the genetic information it contains for hMn-SOD is correctly transcribed and translated. 12. Plasmids according to claim 11, characterized in that they are YEp13, pJDB207, pEAS102, YIp5.   13. Plasmids according to claim 11, characterized in that they are pUC plasmids, preferably pUC18. 14. Plasmids according to one of claims 11 to 13, characterized characterized that the one bearing them Expression cassette the elements promoter, hMn-SOD Structural gene with initiation and stop codon, terminator or promoter, all in the correct reading direction. 15. Plasmids according to claim 14, characterized in that the promoter of the fully approx. 1500 bp long ADHI Promoter or the shortened approx. 400bp long ADHIk promoter and the terminator of the ADHII terminator are. 16. Transformed host cells coding for hMn-SOD containing genetic information. 17. Transformed host cells according to claim 16, characterized characterized as having replicating vectors one of claims 8 to 10 or plasmids according to one of claims 11 to 15 contain replicate them and express and synthesize the hMn SOD accumulate intracellularly, into the periplasm or transport extracellularly. 18. Transformed host cells according to claim 17, characterized characterized that these are prokaryotes, especially Enterobacteriaceae, Bacillaceae, apathogenic Micrococcaceae, preferably E. coli, in particular E.coli C600, E.coli JM101. 19. Transformed host cells according to claim 17, characterized characterized that these are eukaryotes, preferably S. cerevisiae.   20. Transformed host cells according to claim 17, characterized characterized as being mammalian cells. 21. A process for the preparation of hMn-SOD, characterized in that

• a) the mRNA is isolated from human tissue, preferably placental tissue, and the poly (A) ⁺RNA is prepared,
• b) the double-stranded cDNA is synthesized from this poly (A) RNA and a cDNA library is constructed therefrom,
• c) a DNA coding for hMn-SOD, whole or partial sequence from said cDNA bank is isolated by means of DNA probes, preferably those corresponding to formulas Va and Vb,
• d) this DNA sequence is optionally completed up to the start or stop codon and optionally provided with a leader or signal sequence in front of the start codon and cloned into a vector or plasmid,
• e) with this complete DNA sequence coding for hMn-SOD, an expression cassette suitable for the host cells is constructed.
• f) transforming a suitable host cell with a vector or plasmid carrying this expression cassette,
• g) the hMn-SOD synthesized by this transformed host is isolated and purified.

22. The method according to claim 21, characterized in that the DNA sequences according to claims 1 to 7 are defined. 23. The method according to claim 21, characterized in that the vectors and plasmids according to claims 8 to 10 and 11 to 15 are defined. 24. The method according to claim 21, characterized in that the host cells according to claims 16 to 20 have defined properties. 25. The method according to claims 21 to 24, characterized characterized in that an HMn SOD can then be produced which is the enzymatic, biochemical and immunological properties of the authentic hMn-SOD having. 26. The method according to claim 21 to 25, characterized characterized in that an HMn SOD can then be produced is, preferably with the amino acid sequences according to Formulas IVa or IVb. 27. Polypeptide with the enzymatic, biochemical and immunological properties of hMn-SOD in im essentially pure form.   28. Polypeptide according to claim 27, characterized in that that it is free from native glycolization. 29. Polypeptide according to claims 27 and 28, characterized characterized in that it contains the amino acid methionine before 1. has amino acid of the N-terminus. 30. Polypeptide according to one of claims 27 to 29, characterized characterized in that it contains a leader peptide. 31. Polypeptide, characterized in that it is the Has amino acid sequences according to formulas (IVa) or (IVb). 32. Polypeptide according to claim 31, characterized in that that it's the amino acid methionine before the 1st amino acid of the N-terminus of the mature HMn-SOD. 33. Polypeptide according to claim 31 and 32, characterized characterized in that it contains a leader peptide. 34. Polypeptide, producible according to one of claims 21 to 26. 35. Use of a polypeptide according to one of the claims 27 to 34 for therapeutic treatment. 36. agents for therapeutic treatment of humans, characterized in that it is pharmaceutical inert carriers an effective amount of Polypeptides according to any one of claims 27 to 34 contains.