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

The present invention relates to a method of producing human Mn-superoxide 
dismutase (hMn-SOD) by genetic engineering, the DNA sequences which code 
for this enzyme, suitable vectors which contain these DNA sequences and 
host cells which are capable of expressing these DNA sequences, and the 
enzyme hMn-SOD itself. Suggestions for the use of this enzyme are also 
described. 
As a consequence of various biochemical processes in biological systems 
(e.g. redox processes in the respiratory chain, oxidation in the 
cytoplasm), O.sub.2.sup.- radicals are continuously formed, as is well 
known, these radicals being highly cytotoxic and capable of resulting in 
tissue damage. The degradation of collagen and synovial fluid by such 
radicals has been discussed with reference to pathological situations, 
e.g. in the course of rheumatically caused diseases (Pasquier, C. et al., 
Inflammation 8, 27-32, 1984). Eukaryotic cells contain two forms of 
superoxide dismutases, one of which occurs predominantly in cttosol 
(Cu/Zn-SOD) whilst the other occurs primarily in the mitochondria 
(Mn-SOD). In liver mitochondria it has been found that Mn enzyme is 
localised in the matrix enclosing the inner membrane, although Mn-SOD has 
also been detected in the cytosol of the liver cells (Mc Cord J. M. et 
al., In: Superoxide and Superoxide Dismutases (A. M. Michelson, J. M. Mc 
Cord, I. Fridovich, eds.) Academic Press, N.Y., 129-138, 1977). 
In prokaryotes there is an Fe-SOD as well as an Mn-SOD. The former has also 
been found in algae and protozoa as well as in some plant species 
(Bridges, S. M., Salin, M. L., Plant Physiol. 68, 275-278, 1981). These 
highly active enzymes catalyse the disproportionation O.sub.2.sup.- 
+O.sub.2.sup.- +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2 and prevent, by 
this dismutation of the superoxide radicals, the concentration thereof and 
hence their damaging effect on cells. Apart from the endoplasmic reticulum 
of the liver, the mitochondrial membranes can be regarded as one of the 
most important sites of O.sub.2.sup.- formation in animal cells, so that 
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 was located (Touati, D., J. Bact. 155, 1078-1087, 
1983). 
The 699 bp long nucleotide sequence of a mitochondrial yeast Mn-SOD was 
determined and the primary structure of both the precursor and also the 
mature protein was derived therefrom--with molecular weight of 26123 Da 
for the precursor and 23059 Da for the mature protein (Marres, C. A. M. et 
al., Eur. J. Biochem. 147, 153-161 (1985). Thus, the Mn- and Cu/Zn-SOD 
(MW=14893, EP-A 138111) differ significantly in their molecular weights. 
The complete amino acid sequence of Mn-SOD from human liver was published 
by D. Barra, and according to this publication the hMn-SOD is supposed to 
consist of 196 amino acids (Barra, D. et al., J. Biol. Chem. 259, 
12595-12601, 1984). Human Cu/Zn-SOD from erythrocytes, on the other hand, 
consists of 153 amino acids (Jabusch, J. R., et al., Biochemistry 19, 
2310-2316, 1980) and shows no sequence homologies with hMn-SOD) (Barra. D. 
et al., see above). 
Generally, the superoxide dismutases are credited with a protective 
function against certain inflammatory processes. In particular, deficiency 
in Mn-SOD is supposed to have some significance in the development of 
rheumatoid arthritis (Pasquier, C. et al., see above). SOD is also assumed 
to have a protective effect against alcohol-induced liver damage (Del 
Villano B. C. et al., Science 207, 991-993, 1980). 
The cloning and expression of a human SOD is known only for human Cu/Zn-SOD 
from human liver (EP-A 138111). 
In view of the above-mentioned essential properties of the superoxide 
dismutases, particularly hMn-SOD, a demand for its use in therapy and/or 
diagnosis can be expected. For this purpose it is advantageous to have 
access to sufficient quantities of Mn-SOD of the same species, i.e. human, 
in homogeneous form. The projected aim which derives therefrom is to 
minimise or prevent the immunological reactions which can be expected, 
e.g. after therapeutic use. 
Only with the development of technologies for the recombination of foreign 
DNA with vector DNA and the possibility of establishing the former in 
stable form in microorganisms and expressing it therein has made it 
possible to produce homogeneous proteins of animal or human origin in 
large quantities. The objective here is different, namely that the enzyme 
thus prepared, hMn-SOD, should have a biological activity spectrum which 
is characteristic of authentic genuine hMn-SOD. 
An aim of the present invention was therefore to discover or produce the 
DNA sequence coding for this enzyme, for the first time, using genetic 
engineering, and to indicate for the first time the methods by which this 
sequence can be obtained. According to the invention this problem is 
solved by searching through a cDNA gene bank obtained from human cells of 
placental origin with synthetically produced DNA probe molecules, thereby 
isolating the gene which codes for hMn-SOD. In order to obtain the gene 
for hMn-SOD, the mRNA can be isolated, by known methods, from cells which 
produce the desired enzyme. Various starting materials may be used, e.g. 
metabolically active gland tissue such as liver or placenta. After 
production of the cDNA, which can be obtained by known methods by primed 
synthesis with reverse transcriptase using isolated mRNA, subsequent 
incorporation into a suitable vector and amplification to obtain a 
complete cDNA gene bank, the latter can be searched with a defined, 
radioactively labelled DNA probe or a mixture of various probes of this 
kind. In order to take account of the degeneracy of the genetic code, 
defined DNA probe mixtures are preferably used which represent all 
possible nucleotide variations for one and the same amino acid or which 
are selected so that the number of DNA probes of a mixture to be 
synthesised is as small as possible and the homology with the hMn-SOD DNA 
sequence sought is as high as possible. Another criterion for selection in 
the synthesis of DNA probes may require that these probes are 
complementary to at least two independent regions, for example near the 3' 
and 5' ends of the putative gene sequence. In this way, clones which show 
positive signals against, for example, both independent DNA probes can be 
identified by means of at least two separate hybridisations. These clones 
may then preferably be used to isolate the hMn-SOD gene, since they can be 
expected to contain either a substantial part of or the complete gene for 
hMn-SOD. 
The particular DNA sequences used for the DNA probes according to the 
invention were derived from liver tissue using the amino acid sequence of 
human Mn-SOD published by D. Barra et al. (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 which code for at least five 
amino acid groups, preferably for 8 amino acid groups, may preferably be 
used, a DNA probe length of at least 14, preferably 23 bases being 
advantageous. It is particularly advantageous if a DNA probe is 
complementary to the derived hMn-SOD DNA sequence the genetic information 
of which is colinear with the amino acid groups 39 to 46 and a second DNA 
probe is complementary to the corresponding DNA region which codes for 
amino acid groups 200 to 207 of the known amino acid sequence. Similarly, 
of course, DNA sequences which may be derived using other Mn-superoxide 
dismutases may also be used as probes. 
Using a DNA probe of this kind it is possible to obtain positive clones 
from which a cDNA sequence corresponding to the following formula Ia may 
be isolated, containing a large amount of a region coding for hMn-SOD: 
##STR1## 
Surprisingly, it has now been found that the cDNA found codes for an amino 
acid sequence which differs from the published amino acid sequence (Barra, 
D. et al., J. Biol. Chem. 259, 12595-12601, 1984) in some of the groups 
and in their length from one another. The differences discovered in this 
sequence from the "Barra sequence" are concerned with the amino acid 
positions 42, 88, 109 and 131 (in each case Glu instead of Gln) and two 
additional amino acids Gly and Trp between positions 123 and 124, so that 
the DNA sequence according to the invention corresponds to an hMn-SOD of 
198 amino acids. 
It was also completely unexpected that, on the other hand, a cDNA coding 
for hMn-SOD could be isolated which indicates an amino acid substitution 
at position 29 (codon for Gln instead of Lys) and thus in this point has 
an additional difference from "the Barra sequence" and from formula Ia, 
corresponding to formula Ib: 
##STR2## 
If one assumes that the Barra sequence was correctly analysed, using the 
nucleotide or amino acid sequence according to the invention the 
possibility has to be considered that for the first time, and 
surprisingly, this indicates the possible existence of different genes or 
their allelic manifestations or isoenzymes for hMn-SOD. 
Since it is possible to obtain cDNA-bearing clones which lack the end 
required for the complete hMn-SOD gene, another object of the present 
invention was to prepare the complete gene for hMn-SOD. 
This aim can be achieved by various known strategies. For example, the 
sequence obtained may itself be used as a DNA probe and the cDNA bank can 
be searched once more with it in order to detect a complete gene or a cDNA 
with the missing end or the DNA sequence obtained may be used as a 
hybridisation probe against a genomic bank in order to isolate the 
complete hMn-SOD gene after identifying it. 
Alternatively, there is the possibility of synthesising oligonucleotides in 
which the nucleotide sequence corresponds to the missing end of the 
hMn-SOD and obtaining the complete cDNA for hMn-SOD with the aid of these 
oligonucleotides, after suitable linker ligation. This method has the 
advantage that, for example, a DNA coding for hMn-SOD may be obtained in 
which the 5' end begins directly with the start codon (ATG). 
The DNA sequence of formula II has been found to be particularly suitable 
for solving this problem, completing the cDNAs according to the invention 
which code, for example, from amino acid 22 or 26, this sequence beginning 
with the 5' start codon ATG and ending with the codon for amino acid 31 
(His, whilst AAG [Lys]=1), on the basis of the known codon preferences 
such as those which apply to yeast (Sharp, P. M. et al., Nucl. Acids. Res. 
14 (13), 5125-5143, 1986) 
##STR3## 
Similarly, other known synonymous codons may be used to complete the 
hMn-SOD gene or to synthesise the entire gene in vitro, e.g. those which 
facilitate an optimum codon-anticodon alternation in bacteria, e.g. E. 
coli, and increase the efficiency of translation (Grosjean, H., Fiers, W., 
Gene 18, 199-209, 1982; Ikemura, T., J. Mol. Biol. 151, 389-409, 1981) or 
codons which correspond to the actual conditions in mammalian cells 
(Grantham, R. et al., Nucleic Acid Research 9, 43-47, 1981). The latter 
may preferably be used for transformation and subsequently for expression 
in mammalian cells. 
It is theoretically possible to split off the methionine group which is 
coded by the start codon ATG and which precedes the mature hMn-SOD, 
beginning with the first amino acid lysine, using methods known per se, 
for example using CNBr or CNCl. However, since other internal methionine 
groups may occur, e.g. at positions 23 or 192, in the mature enzyme 
hMn-SOD, such a procedure is impracticable, with the result that in this 
case the additional N-terminal methionine group remains, without affecting 
the biological activity of hMn-SOD. 
However, enzymatic cleaving may also be envisaged, in which suitable 
synthetic linkers may be used in known manner, since codons for 
corresponding specific amino acids can be expected to be located at the 
desired positions on the vector which contains the hMn-SOD cDNA. For 
example, Arg or Lys groups for a tryptic cleavage or codons which code for 
protease-sensitive amino acids will generally be used. These may be 
positioned in front of or behind the start codon or within the coding 
region. 
An additional aim of this invention was to express the sequence coding for 
hMn-SOD in suitable host cells for the first time by genetic engineering, 
to produce the homogeneous enzyme hMn-SOD by such methods for the first 
time, to isolate it and prepare it in pure form and to describe for the 
first time the procedure required for this. 
According to the invention, this aim was achieved by inserting the DNA 
sequences coding for hMn-SOD, for example of formula IIIa or IIIb 
##STR4## 
optionally provided with corresponding signal or control sequences, into 
suitable vectors and transforming suitable host cells therewith. After 
cultivation of the transformed host cells the polypeptides formed are 
isolated and purified by methods known per se. The polypeptides obtained 
correspond to the following formulae IVa and IVb. 
##STR5## 
The sequences shown in formulae IIIa and IIIb are particularly suitable for 
the preparation of non-glycosylated hMn-SOD of formulae IVa and IVb in 
microorganisms, particularly in E. coli or S. cerevisiae. The problem of 
glycosylation in yeast, for example, can be avoided by using mutants which 
are deficient in the glycosylation of proteins (alg mutants) (e.g. 
Huffaker, T. C., Robbins P. W., Proc. Natl. Acad. Sci. USA 80, 7466-7470, 
1983). 
If necessary or advisable, the complete hMn-SOD gene, for example according 
to formula IIIa or IIIb, may be preceded by a leader or signal sequence 
directly before the first codon of the first N-terminal amino acid of the 
mature hMn-SOD or before the start codon ATG. This ensures that the 
hMn-SOD can be transported from the host cell and readily isolated from 
the culture medium. 
Signal sequences of this kind have been described; they code for a 
generally hydrophobic protein content, which is split off by 
post-translational modification processes in the host cell (Davis, D. B., 
Tai. P. -C., Nature 283, 433-438, 1980; Perlman, D., Halvorson, H. O., J. 
Mol. Biol. 167, 391-409, 1983). If an ATG codon has been constructed in 
front of the first amino acid of the hMn-SOD, a gene product may be 
obtained which contains an N-terminal methionine in front of the lysine. 
The use of signal sequences of prokaryotes in order to secrete proteins 
into the periplasma and process them correctly is known (see Davis, B. D., 
Tai, P. -C., 1980). 
Obviously, after isolating and cloning the hMn-SOD DNA sequence, it is 
possible specifically to modify the enzyme coded by this sequence. Enzyme 
modifications may be effected, for example, by controlled in vitro 
mutations with synthetic oligonucleotides, thereby influencing the 
catalytic properties of hMn-SOD and obtaining new enzymatic activities. 
The basic procedural steps for performing these 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 the cloning, i.e. amplification and preparation, of the hMn-SOD gene it 
is possible to use E. coli, preferably E. coli C600 (Nelson et al. 
Virology 108, 338-350, 1981) or JM 101, or E. coli strains with at least 
one of the known sup-genotypes. However, the cloning may also be carried 
out in the gram-positive bacteria such as B. subtilis. Systems of this 
kind have been described many times. 
Suitable hosts for the expression of the hMn-SOD gene according to the 
invention include both microorganisms and also cultures of multicellular 
organisms. The term microorganisms includes prokaryotes, i.e. 
gram-negative or gram-positive bacteria and eukaryotes such as protozoa, 
algae, fungi or higher Protista. Of the gram-negative bacteria, the 
Enterobacteriaceae, for example E. coli are preferred hosts, whilst of the 
gram-positive bacteria the Bacillaceae and apathogenic Micrococcaceae, 
e.g. B. subtilis and Staph. carnosus are preferred hosts, and of the 
eukaryotes the Ascomycetes, particularly the yeasts, e.g. Saccharomyces 
cerevisiae are preferred hosts. 
For single-cell microorganisms there are a plurality of starting vectors 
available which may be of both plasmidic and viral origin. These vectors 
may occur in a single copy or as multicopy vectors. Vectors of this kind 
which are suitable for the cloning and expression of the hMn-SOD according 
to the invention and for eukaryotic DNA sequences in general have been 
described in a number of publications and manuals (e.g. Maniatis, T. et 
al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982; Glover, D. M. 
(ed.) DNA Cloning Vol. I, II, 1985) and are commercially obtainable. 
In general, plasmid vectors which as a rule contain a replication origin 
and control sequences for transcription, translation and expression may be 
used in conjunction with these hosts. These sequences must originate from 
species which are compatible with the host cells. The vector usually 
carries, in addition to a replication site, recognition sequences which 
make it possible to phenotypically select the transformed cells. The 
selection may be carried out either by complementation, suppression or by 
deactivation of a marker. With regard to the first two methods, there are 
auxotrophic mutants of bacteria and yeast which are deficient in an 
essential product of metabolism, or nonsense mutants in which chain 
breakage occurs on translation of the gene in question. Various suppressor 
genes, e.g. supD, E, F (which suppress UAG), supC, G (which suppress UAG 
or UAA), are already known. In the third process, the vector carries a 
resistance gene against one or more cytotoxic agents, such as antibiotics, 
heavy metals. The insertion of a foreign DNA into a marker gene of this 
kind deactivates the latter so that the newly formed phenotype can be 
distinguished from the original phenotype. 
For example, E. coli can be transformed with pBR322, a plasmid which 
originates from E. coli species (Bolivar, et al., Gene 2, 95 (1977). 
pBR322 contains genes for ampicillin and tetracycline resistance and thus 
provides simple means of identifying transformed cells, by converting the 
phenotype Ap.sup.r, Tc.sup.r into Ap.sup.s, Tc.sup.r by cloning in, for 
example, the PstI site in the .beta.-lactamase gene. Other methods may 
equally be used, for which, for example, the lacZ-gene deactivation in 
.lambda. and M 13 vectors and in various plasmids (e.g. PUC, pUR) is 
important. These very versatile selection systems have long been known and 
accordingly there is a wide range of literature on this subject. 
In addition to selection markers of this kind, these vectors, particularly 
expression vectors, must contain signal sequences which ensure correct 
initiation and termination of the transcription. For the correct 
transcription of the hMn-SOD gene, therefore, these vectors may contain a 
bacterial or eukaryotic transcription unit consisting of a promoter, the 
coding region with the hMn-SOD gene and the adjoining terminator. 
Depending on the nature of the transcription units, these may contain 
conserved prototype sequences such as, for example, Pribnow-box or TTG 
sequence or CAAT-box, TATA-box, the known termination signals (for example 
AATAAA, TATGT), and at least one stop codon, whilst preferably promoters 
and terminators which are homologous with respect to the host are used. 
The mRNA formed usually contains a 3' poly(A) sequence and/or a 5' cap 
structure. Translation of the hMn-SOD gene requires a ribosomal binding 
site (RBS) consisting of a Shine/Dalgarno (S/D) sequence and an initiation 
codon at a defined spacing therefrom, generally of 3 to 12 nucleotides, 
and at least one stop codon. Alternatively, RBSs may be prepared 
synthetically, thereby increasing the homology with the 3' end of the 16S 
rRNA (Jay, E. et al. Nucleic Acids Res. 10, 6319-6329, 1982). 
In eukaryotic expression systems, in particular, (for example S. 
cerevisiae), it is preferable to use regulatory systems for the 
translation which originate from the host, since in yeasts the conditions 
are analogous to those which apply to prokaryotes (homology of the S/D 
sequence with the 3' end of the 16S rRNA) and the signals or the RBS for 
initiating the translation are defined in a different way than in 
prokaryotes (e.g. Kozak, M., Nucleic Acids Res. 9, 5233-5252, 1981; Kozak, 
M., J. Mol. Biol. 156, 807-820, 1982). 
Preferably, the cloning or expression vector has only one restriction 
endonuclease recognition site which either is present in the starting 
vector from the outset or can be inserted subsequently by means of 
suitable linkers. Linkers may either be obtained by a simple chemical 
synthesis or they are commercially available. 
Frequently used yeasts promoters in the production of corresponding 
expression plasmids contain promoters which control the expression 
particularly efficiently in the yeast system, such as PGK promoter (Tuite, 
M. F. et al., The EMBO Journal 1, 603-608, 1982; Hitzeman, R. A. et al., 
Science 219, 620-625, 1983), PH05 promoter (Hinnen, A., & Meyhack, B., 
Current Topics in Microbiology and Immunology 96, 101-117, 1982; Kramer, 
R. A. et al., Proc. Natl. Acad. Sci. USA 81, 367-370, 1984), GAPDH 
promoter (Urdea, M. S. 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, M. J. et al., 
J. Biol. Chem. 256, 1385-1395, 1981), .alpha.-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 ADHI 
promoter (Ammerer, G., Methods in Enzymology 101, 192-201, 1983; Hitzeman, 
R. A. et al., Nature 293, 717-722, 1981). 
It is also possible to use promoters of other glycolytic enzymes (Kawasaki 
and Fraenkel, Biochem. Biophys. Res. Comm. 108, 1107-1112, 1982), 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 may also be included in the expression vector 
at the 3' end of the sequence which is to be expressed, in order to 
provide polyadenylation and termination of the mRNA. Other promoters which 
also have the advantage of transcription controlled by growth conditions 
are the promoter regions of alcohol dehydrogenase-2, isocytochrome C, the 
degradation enzymes coupled to nitrogen metabolism, the above-mentioned 
glycerine aldehyde-3-phosphate dehydrogenase (GAPDH) and the enzymes which 
are responsible for metabolising maltose and galactose. Promoters which 
are regulated by the yeast mating type locus, for example promoters of the 
genes BAR1, MECI, STE2, STE3 and STE5, may be used in 
temperature-regulated systems by the use of temperature-dependent sir 
mutations (Rhine, Ph.D. Thesis, University of Oregon, Eugene, Oreg., 
(1979), Herskowitz and Oshima, The Molecular Biology of the Yeast 
Saccharomyces, Part I, 181-209 (1981), Cold Spring Harbour Laboratory)). 
These mutations affect the expression of the resting mating type cassettes 
of yeast and thus indirectly the mating type dependent promoters. 
Generally, however, any plasmid vector which contains a yeast-compatible 
promoter, origin of replication and termination sequences, is suitable. 
If the expression of hMn-SOD is to take place in bacteria, it is preferable 
to use promoters which result in a high rate of synthesis of mRNA and 
which are also inducible. Known promoters which are used contain 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, 
A.E. 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 patent application, publication No. 0036 776). Moreover, other 
microbial promoters have also been developed and used. The gene sequence 
for hMn-SOD may be transcribed, for example, under the control of the 
lambda-P.sub.L promoter. This promoter is known as one of the particularly 
powerful, controllable promoters. Control is possible by means of a 
thermolabile repressor cI (e.g. cI857), to which adjacent restriction 
cutting sites are known. Furthermore, it is also possible to use the 
promoter of alkaline phosphatase from E. coli (Ohsuye, K. et al., Nucleic 
Acids Res. 11, 1283-1294, 1983) and hybrid promoters such as, for example, 
the tac-promoter (Amann, E. et al., Gene 25, 167-178, 1983; De Boer, H.A. 
et al., Proc. Natl. Acad. Sci. USA 80, 21-25, 1983). The use of promoters 
of this kind (lacuv5, lacZ SD, tac) which can be carried and vectors for 
preparing fused and non-fused eukaryotic proteins in E. coli is described 
in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 
1982, especially page 412ff. The expression and translation of an hMn-SOD 
sequence in bacteria may also be carried out under the control of other 
regulatory systems which may be regarded as "homologous" to the organism 
in its untransformed state. For example, it is also possible to use 
promoter-operator systems such as arabinose operator, colicin El operator, 
galactose operator, alkaline phosphatase operator, trp operator, xylose A 
operator and the like or parts thereof. 
For the cloning or expression of hMn-SOD in bacteria, for example in E. 
coli, or in yeasts, for example in S. cerevisiae, there are well known 
vectors available, of which, for the former host systems, it is 
advantageous to use the pBR plasmids (Bolivar, F. et al., Gene 2, 95-113, 
1977), pUC plasmids (Vieira, I., Messing I., Gene 19, 259-268, 1982) pOP 
plasmids (Fuller, F., Gene 19, 43-54, 1982), pAT plasmids (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), cosmids 
(Collins, J., Hohn, B., Proc. Natl. Acad. Sci. USA 75, 4242-4246, 1979) 
and the other vectors known from the literature (e.g. Maniatis, T. et al., 
Molecular Cloning, Cold Spring Harbor Laboratory, 1982), particularly pBR 
and pUC derivatives, for example pBR322 and pUC18. 
Suitable expression vectors in yeasts are 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 37061) and pJDB207 
(DSM 3181) or pEAS102. The vector pEAS102 may be obtained by digesting 
YIp5 partially with PstI and totally with BamHI and ligating the isolated 
4.3 kb fragment (which contains the URA3 gene) with the 4.4 kb BamHI/PstI 
fragment of pJDB207. 
In addition to microorganisms, cultures of multicellular organisms are also 
suitable host organisms for the expression of hMn-SOD. In theory, any of 
these cultures may be used, whether obtained from vertebrate or 
invertebrate animal cultures. However, the greatest interest has been in 
vertebrate cells, with the result that the multiplication of vertebrate 
cells in culture (tissue culture) has become a routine method in recent 
years (Tissue Culture, Academic Press, Editors Kruse and Patterson, 
(1973)). Examples of useful host cell lines of this kind include VERO and 
HeLa cells, Golden Hamster Ovary (CHO) cells and W138, BHK, COS-7 and MDCK 
cell lines. Expression vectors for these cells generally contain a 
replication site, a promoter which is located in front of the hMn-SOD to 
be expressed, together with any necessary ribosome binding site, RNA 
splicing site, polyadenylation site and transcriptional termination 
sequences. 
When used in mammalian cells, the control functions in the expression 
vector are often obtained from viral material. For example, the promoters 
normally used originate from papova viruses such as polyoma viruses, 
papilloma viruses, Simian Virus 40 (SV 40) and from retroviruses and 
adenovirus Type 2. The early and late promoters of SV 40 and their 
applications have frequently been described. Furthermore it is also 
possible and often desirable to use promoter or control sequences or 
splicing signals which are originally linked to the desired genetic 
sequences, provided that these control sequences are compatible with the 
host cell systems. Thus, SV40 vectors are known in which an exogenic 
eukaryotic DNA with its own promoter sequences and splicing signals, as 
well as the late SV40 promoter, will yield a stable transcript. 
A replication starting point may either be provided by corresponding vector 
construction in order to incorporate an exogenic site, for example from SV 
40 or other viral sources (e.g. polyoma, adeno, VSV, PBV, etc.) or it may 
be provided by the chromosomal replication mechanisms of the host cell. If 
the vector is integrated into the host cell chromosome, the latter measure 
is usually sufficient. 
Transformation of the cells with the vehicles can be achieved by a number 
of processes. For example, it may be effected using calcium, either by 
washing the cells in magnesium and adding the DNA to the cells suspended 
in calcium or by subjecting the cells to a coprecipitate of DNA and 
calcium phosphate. During the subsequent gene expression, the cells are 
transferred to media which select for transformed cells. 
In the intracellular production of hMn-SOD the enzyme may be isolated by 
centrifuging the cells off after a suitably high cell density has been 
reached and then enzymatically or mechanically opening them up. 
Purification of the hMn-SOD according to the invention may be carried out 
by known biochemical methods for purifying proteins or enzymes, such as 
dialysis, electrophoresis, precipitation, chromatography or combinations 
of these methods. If the enzyme is secreted from the cell, analogous 
methods of protein purification are carried out in order to obtain hMn-SOD 
from the culture medium in pure form. 
The hMn-SOD according to the invention purified by these methods has a 
biological activity spectrum identical to the genuine enzyme both in vivo 
and in vitro. 
These activities include both immunological properties (e.g. cross-reaction 
with antibodies of genuine hMn-SOD against the hMn-SOD according to the 
invention) and also biochemical and enzymatic activities. In order to 
characterise hMn-SOD biochemically and enzymatically, the method described 
by Marklund, S. (Marklund, S. & Marklund, G., Eur. J. Biochem. 47, 
469-474, 1974) may be used, for example, according to which a strict 
distinction must be drawn between enzymes containing Cu/Zn and those 
containing Mn, for example by the addition of KCN (which inhibits 
Cu/Zn-SOD but not Mn-SOD) or using the different pH dependencies of their 
activities (see particularly Ysebaert-Vanneste, M., Vanneste, W.H., Anal. 
Biochem. 107, 86-95, 1980). 
The polypeptide according to the invention includes not only the mature 
hMn-SOD which is described in detail but any modification of this enzyme. 
These modifications include, for example, shortening of the molecule at 
the N- or C-terminal end, and the substitution of amino acids by other 
groups, which do not substantially affect the enzyme activity. 
The invention relates not only to genetic sequences which code specifically 
for the hMn-SOD which is described and demonstrated in the examples, but 
also to modifications which are easily and routinely obtainable by 
mutation, degradation, transposition or addition. Any sequences which code 
for the hMn-SOD according to the invention (i.e. which have the 
corresponding, known biological activity spectrum) and which are 
degenerate compared with those shown, are also included; experts in this 
field will be able to degenerate DNA sequences, particularly in the coding 
regions. Similarly, any sequence which codes for a polypeptide with the 
activity spectrum of the authentic hMn-SOD and which hybridises with the 
sequences shown (or parts thereof) under stringent conditions is also 
included. 
The particular conditions which constitute stringent conditions under which 
hybridisation (including pre-washing, pre-hybridisation, hybridisation and 
washing) should be carried out are defined in the prior art. For 
hybridising oligonucleotides against a gene bank ("gene bank screening") 
the conditions described by Wood, I.M. et al. should preferably be used 
(Proc. Natl. Acad. Sci. USA 82, 1582-1588, 1985). To test whether a 
specific DNA sequence hybridises with one of the DNA sequences according 
to the invention which code for hMn-SOD--either via in situ hybridisation 
against plaques or colonies of bacteria or via Southern Blotting--the 
methods and conditions described in detail by Maniatis, T. et al. should 
be adopted (Maniatis T. et al., Molecular Cloning, Cold Spring Harbor 
Laboratory, 1982, particularly pages 326-328 and 387-389). All signals 
which are clearly distinguishable against the background therefore 
indicate a positive hybridisation signal. 
More specifically, the problems described above are solved by preparing the 
RNA from human tissue, preferably from human placenta tissue. Whereas 
tissue culture cells can be disintegrated directly with hot phenol, tissue 
for this type of extraction first has to be broken up in deep-frozen 
condition, advantageously in the presence of powdered or granular dry ice 
or in liquid nitrogen (e.g. Starmix). 
Aggregates of mRNA and other RNAs formed by phenol may be broken up again 
using formamide or by heating (e.g. to 65.degree. C.). A preferred method 
of isolating RNA is the Chirgwin method (Chirgwin, J.M. et al., 
Biochemistry 18, 5294-5299, 1979). The poly(A).sup.+ RNA may be 
conveniently purified from the isolated protein and DNA preparation by 
affinity chromatography, e.g. poly(U) Sepharose or oligo(dT) cellulose, 
since eukaryotic mRNA populations generally have a poly(A) tail at their 
3' end (Aviv, H., Leder, P., Proc. Natl. Acad. Sci. USA 69, 1409-1412, 
1972; Lindberg, U., Persson, T., Eur. J. Biochem. 31, 246-254, 1972). 
Isolation of the poly(A).sup.+ RNA may preferably be carried out using the 
method described by Auffray (Auffray, C., Rougeon, F., Eur. J. Biochem. 
107, 303-314, 1980). 
The purified mRNA may be concentrated by dividing up the entire mRNA 
fraction according to size (e.g. by centrifuging in a sucrose gradient. 
The desired mRNA may be detected, for example, using known in vitro 
protein biosynthesis systems (reticulocytes, oocytes of Xenopus laevis). 
The purified mRNA or the concentrated fraction is used as a template for 
synthesising the first strand of the cDNA, which is done using reverse 
transcriptase and a primer. The primers used may be either oligo (dT) or 
synthetic primers; the latter may be obtained using the known amino acid 
sequence of hMn-SOD and make it possible to carry out repeated priming of 
reverse transcription (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 primer in the presence of dNTPs. 
The second strand of the cDNA may be synthesised by various known methods, 
of which priming with a complementary primer (Rougeon, F., Mach, B., J. 
Biol. Chem. 252, 2209-2217, 1977), self-priming with the aid of a 
"hairpin" structure located at the 3' end of the cDNA (Efstratiadis, A. et 
al., Cell 7, 279, 1976) or with an Okazaki fragment-like primer formed by 
RNaseH (Gubler, U., Hoffmann, B.J., Gene 25, 263, 1982) may be mentioned 
in particular. The preferred method according to the present invention is 
the one described by Huynh, T.V. (Huynh, T.V. et al., in DNA Cloning Vol 
I, (D.M. Glover ed.), chapter 2, pages 49-78, 1985). The double-stranded 
cDNA obtained by this method can be cloned or packaged directly in a 
suitable vector, e.g. in a cosmid, insertion or substitution vector, more 
particularly in a lambda vector, preferably in .lambda.gt10 (Huynh, T.V. 
et al., 1985). There are a number of known methods of cloning in lambda, 
of which "homopolymer tailing" using dA-dT or dC-dG or the linker method 
with synthetic linkers should be mentioned by way of example (Maniatis, T. 
et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982; Huynh, 
T.V. et al., DNA Cloning Vol. I (D.M., Glover ed.) 1985, 1980; Watson, 
C.J., Jackson, F. dto, 1985, chapter 3). In the cloning of the cDNA 
according to the invention, this is inserted into the EcoRI site of 
.lambda.gt10. The in vitro packaging and cloning of the cDNA according to 
the invention and the construction of the cDNA gene bank were carried out 
according to Huynh, T.V. et al. 1985, pages 49-78. 
Using the phage population obtained, which represents a cDNA gene bank from 
placental tissue, amplification and plaque purification were carried out 
by infecting a suitable host, particularly E. coli, preferably E. coli C 
600, and, respectively, by securing the lytic replication cycle of lambda. 
The cDNA gene bank was investigated under stringent hybridisation 
conditions with radioactively labelled synthetic oligonucleotides which 
had been obtained using the published amino acid sequence (Barra, D. et 
al., Oxy Radicals and their scavenger Systems, Vol. 1, 336-339, 1983). In 
the present invention, the method of hybridisation in situ described by 
Benton and Davis (Benton, W.D., Davis, R.W., Science 196, 180-182, 1977) 
was used. Preferably, two mixtures, each consisting of eight synthetic 
23-mer oligonucleotides of formulae Va and Vb were used, which are 
colinear with amino acids 39 to 46 and 200-207, respectively, of the amino 
acid sequence published by Barra, D. et al. (see above) and which take 
into account the degeneracy of the genetic code. The last base at the 5' 
end of these DNA probes lacks the wobble base for the entire codon for Gln 
(amino acid 46) or Glu (amino acid 207). A, G, C and T represent the 
corresponding nucleotides whilst I represents inosine. 
##STR6## 
The oligonucleotide probes may be prepared by known chemical methods of 
synthesis. For the present invention, a Model 381A DNA Synthesizer 
(Applied Biosystems) was used. 
The synthesis of all possible combinations of these two DNA probes ensures 
that at least one of the oligonucleotides present forms an optimum pair 
with the single-stranded DNA region of the desired hMn-SOD gene, 
complementary to the probe. The use of two independent pools of 23-mer 
oligonucleotides reduces the possibility of selecting "false" positives. 
After isolation of inherently homogeneous plaques which have been 
identified by positive signals after hybridisation with the two 23-mer DNA 
probes, it was possible to isolate seven recombinant phages and to 
sequence 500 to 1000 bp long EcoRI fragments of their DNA. After sequence 
analysis of these EcoRI fragments by 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 into the EcoRI site of the 
M13 vector (Bluescribe, Vector Cloning Systems) and transformation in E. 
coli, for example E. coli JM101, it was discovered that the EcoRI 
fragments contain cDNA inserts which code for hMn-SOD from amino acid 22 
(clones BS5, BS8, BS9, BS13, BSXIII) or from amino acid 26 (clones BS3, 
BS12). 
However, it was also found, surprisingly, that some deviations from the 
amino acid sequence described by Barra, D. et al. (1984, loc.cit.) also 
arose from the DNA sequences obtained: 
______________________________________ 
Amino acid 
according 
Amino Amino acids 
to Barra, 
Clone acid Codon derived D. et al., 1984 
______________________________________ 
BS3, BS12, 29 CAG Gln Lys (29) 
BS5, BS9, BS13 
29 AAG Lys Lys (29) 
BSXIII 
BS3, BS12, BS13, 
42 GAG Glu Gln (42) 
BS5, BS9 
BSXIII 88 GAG Glu Gln (88) 
BS8 29 AAG Lys Lys (29) 
42 GAG Glu Gln (42) 
88 GAG Glu Gln (88) 
109 GAG Glu Gln (109) 
124 GGT Gly .DELTA. 
125 TGG Trp .DELTA. 
139 GAA Glu Gln (129) 
______________________________________ 
The DNA sequence of a 617 bp long EcoRI fragment which could be isolated 
from one of the clones obtained, e.g. BS8, 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).sub.30 tail. Sections of 
linker sequences are also shown, up to the (complete) EcoRI sites. 
Positions 30 to 33 show a ThaI cutting site whilst at positions 367 to 372 
there is a BamHI site. Surprisingly, there are codons at positions 53 to 
61, 155 to 163, 176 to 184 and 500 to 508, which are colinear for 
potential N-glycosylation sites of the corresponding amino acids according 
to the general amino acid arrangements Asn-X-Thr and Asn-X-Ser 
characteristic thereof, wherein X represents valine, histidine or leucine, 
for example, whereas the Cu/Zn-SOD of the cytosol has only one such amino 
acid combination. 
The amino acid differences from the amino acid sequence of Barra, D. et al. 
(Barra, D. et al., J. Biol. Chem. 259, 12595-12601, 1984), which were 
derived from the EcoRI fragment obtained, have already been discussed 
hereinbefore. 
Various strategies may be adopted in order to obtain the missing bases at 
the 3' and/or 5' termini of the hMn-SOD DNA partial sequence from the cDNA 
gene bank to prepare a complete hMn-SOD gene. For example, the cDNA 
obtained may be used as a hybridisation probe against a genomic gene bank, 
in order to obtain the sequence coding for the entire enzyme, or the 
method described by H. Kakidani may be used, for example, using synthetic 
oligonucleotides complementary to the mRNA as specific primers for the 
reverse transcription (Kakidani, H. et al., Nature 298, 245-249, 1982). 
However, it is also possible to synthesise the missing end of the cDNA 
sequence chemically by means of the known amino acid sequence (Barra, D. 
et al., J. Biol. Chem. 259, 12595-12601, 1984) and to link it to the cDNA 
found, thereby obtaining a defined end. 
In the latter method, in order to prepare the complete DNA sequence 
according to the invention for hMn-SOD, the 5' end was completed by two 
oligonucleotides of formulae VIa and VIb which advantageously had 
XhoI/XbaI--or XbaI/NcoI--projecting ends. According to the invention, the 
3' end of the ADHI promoter was taken into consideration at the 5' end of 
the coding strand (Formula VIa) 
##STR7## 
After Combination of the two synthetic oligonucleotides of formulae VIa and 
VIb, cloning into a suitable vector, for example a correspondingly 
modified pUC18 derivative and addition of the ThaI/EcoRI fragment of the 
cDNA according to the invention from one of the clones obtained, the 5' 
end of which has at least the ThaI site, it is possible to obtain a 
plasmid which contains a complete cDNA of the hMn-SOD gene in the correct 
reading frame corresponding to formulae VIIa and VIIb, without the ThaI 
sites. 
##STR8## 
Sequencing of the clones BS5, BS9, BS13, BSXIII and clones BS3 and BS12 
showed that the sequences of clones BS5, BS9, BS13 and BSXIII are 
identical with clone BS8. As already stated, clones BS3 and BS12 differ 
from clone BS8 in amino acid 29 (CAG instead of AAG or Gln instead of Lys, 
formula Ib, IIIb and IVb). Otherwise, there is 100% homology with clone 
BS8 up to base 573 of the EcoRI fragment shown in FIG. 1 (. . . TA*A ACC 
ACG ATC GTT ATG CTG.sup.573). Apart from this base, the two clones BS3 and 
BS12 are identical with respect to the 5-ut (untranslated) region shown in 
Formula VIII. 
##STR9## 
Furthermore, a number of cDNA clones were isolated from a cDNA gene bank 
(placenta) using .lambda.gt11. This cDNA gene bank was prepared in the 
same way as the cDNA gene bank described in the Examples from placenta DNA 
in .lambda.gt10. One of the clones isolated from .lambda.gt11, namely 
clone 4, was subcloned in Bluescribe M13+ in the manner described. 
Sequencing was carried out by repeated priming with the synthetic 17 mer 
oligonucleotides 
EBI 760: 5' AGATACATGGCTTGCAA 3' 
EBI 765: 5' CTCTGAAGAAAATGTCC 3' 
EBI 782: 5' GGAGATGTTACAGCCCA 3' 
EBI 785: 5' AAGGAACGGGGACACTT 3' 
Clone 4 is identical to clones BS3 and BS12 from .lambda.gt10 apart from 
amino acid 29 (AAG or Lys) and a . . . TCTA . . . sequence at the 3' end 
adjoining the multicloning site. Although the analysed DNA sequence of the 
remaining 61 bases of the 5' end (before formula Ia, clone BS8, 
corresponding to codons +1 to +21 corresponding to Lys to Glu) shows some 
base changes compared with the derived DNA sequence (Formula II, contained 
in Formula IIIb), the translation of this DNA section does not produce any 
differences from Barra et al., 1984. A leader sequence in front of the ATG 
was also analysed. Formula IX shows the sequence of clone 4 found. 
##STR10## 
Other clones have 5'ut regions of different lengths. 
The DNA sequences according to the invention may be incorporated in various 
expression vectors and expressed with the aid of the control elements 
described, for example in pES103 with the ADHI promoter (DSM 4013). pES103 
is obtained by incorporating the 1500 bp long BamHI/XhoI fragment of the 
ADHI promoter (e.g. Ammerer, G., Methods in Enzymology 101, 192-201, 1983) 
in the pUC18 derivative pES102, which contains an Xho linker in the HincII 
cutting site. 
Instead of this ADHI promoter sequence originally of 1500 bp, it is also 
possible to use an ADHI promoter shortened to a length of about 400 bp as 
the BamHI/XhoI fragment. The shortened ADHI promoter (ADHIk) is obtained 
by digesting plasmid pWS323E (DSM 4016) with BamHI/XhoI and isolating the 
ADHIk promoter. 
For the correct termination, a suitable terminator sequence, conveniently 
an ADH terminator, preferably the ADHII terminator is ligated behind the 
hMn-SOD. The ADHII terminator (Beier, D. R., Young, E. T., Nature 300, 
724-728, 1982) can be obtained by SphI digestion of pMW5-ADHII (Washington 
Research Foundation) as a fragment 1530 bp long and, after subsequent 
HincII digestion, as a final ADHII terminator (329 bp), or from plasmid 
pGD2 (DSM 4014) as a HindIII/XbaI fragment 336 bp long. 
For expression in yeast, there are various yeast vectors available 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, 121-133, 1979; ATCC 37115), pJDB 207 (DSM 3181, filed on 
28.12.1984), YIp5 (Struhl, K. et al., Proc. Natl. Acad. Sci USA 76, 
1035-1039, 1979; ATCC 37061), pEAS102 (pEAS102 can be obtained by 
digesting YIp5 partially with PstI and completely with BamHI and ligating 
the isolated 4.3 kb fragment which contains the URA3 gene with the 4.4 kb 
BamHI/PstI fragment of pJDB207). 
With these yeast vectors which carry an expression cassette with the 
hMn-SOD gene according to the invention it is possible to transform 
suitable yeast cells by known methods. Suitable yeast cells for expression 
are preferably all those which are deficient for their own yeast-specific 
Mn-SOD and which contain a selectable yeast gene, such as HIS3, URA3, LEU2 
and SUP, to name but a few. Mutants of this kind which contain, for 
example, mutated genes constructed in vitro or in vivo and contain them 
via a "transplacement" may be obtained by integrative transformation (e.g. 
Winston, F. et al., Methods in Enzymology 101, 211-228, 1983). The Mn-SOD 
gene of the yeast which is to be mutated is contained, for example, in 
plasmid pL41 as a BamHI fragment (van Loon et al., Gene 26, 261-272, 
1983). Since the entire sequence of this BamHI fragment is known (Marres, 
C.A.M. et al., Eur.J.Biochem. 147, 153-161, 1985), the Mn-SOD gene of the 
yeast is obtainable even without pL41. 
The hMn-SOD produced by such transformants can be obtained by known methods 
of protein isolation and protein purification. The cell decomposition may 
be carried out, for example, according to van Loon et al. (Proc. Natl. 
Acad. Sci. USA 83, 3820-3824, 1986). 
For the expression of hMn-SOD in bacteria, preferably E. coli, more 
specifically E. coli HB101, C600 and JM101, it is possible to use the 
established expression systems mentioned hereinbefore. For this purpose, 
the DNA sequences according to the invention must be brought under the 
control of a powerful E. coli promoter (loc.cit.), not under a eukaryotic 
promoter. Examples of these known promoters are lac, lacuv5, trp, tac, 
trp-lacuv5, .lambda.P.sub.L, ompF and bla. The obligatory use of a 
ribosomal binding site to ensure efficient translation in E. coli has 
already been described in detail earlier. 
In order to demonstrate the expression of the hMn-SOD activity by E. coli, 
the bacteria are disintegrated in a suitable conventional culture medium 
after incubation and the supernatant is tested for 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 expression of the hMn-SOD gene may also be detected by labelling the 
proteins in maxicells. Plasmid-coded proteins may be labelled selectively 
in vivo using the maxicell technique (Sancar, A. et al., J. Bacteriol, 
137, 692-693, 1979). The E. coli strain CSR603 (CGSC 5830) has no DNA 
repair mechanisms. A suitable dose of UV radiation destroys the bacterial 
chromosome, but some of the substantially smaller plasmid DNAs which are 
present in several copies per cell remain functional. After all the 
undamaged, replicating cells have been killed off by means of the 
antibiotic D-cycloserine and the endogenous mRNA has been consumed, only 
plasmid-coded genes are transcribed and translated in the remaining cells. 
The proteins formed may be radioactively labelled and detected by the 
incorporation of .sup.35 S-methionine. E. coli CSR603 is transformed with 
the expression plasmids by conventional methods and selected for 
transformed bacteria on ampicillin-containing agar plates. The preparation 
of the maxicells and the labelling of the proteins are carried out by the 
method of A. Sancar (1979, loc. cit.) A .sup.14 C-methylated protein 
mixture (Amersham) is used as the molecular weight standard. The plasmid 
containing only the promoter without the hMn-SOD gene is used as control. 
After transformation of the host, expression of the gene and fermentation 
or cell cultivation under conditions in which the proteins according to 
the invention are expressed, the product can usually be extracted by known 
chromatographic methods of operation, so as to obtain a material which 
contains proteins with or without leader and tailing sequences. The 
hMn-SOD according to the invention can be expressed with a leader sequence 
at the N-terminus, which may be removed from some host cells as already 
described. If not, the leader polypeptide (if present) must be cleaved, as 
described hereinbefore, to obtain mature hMn-SOD. Alternatively, the 
sequence can be modified so that the mature enzyme is produced directly in 
the microorganism. The precursor sequence of the yeast mating pheromone 
MF-alpha-1 may be used for this case, to ensure correct "maturation" of 
the fused protein and the secretion of the products into the growth medium 
or the periplasmic space. The "secretion" of the hMn-SOD in yeast 
mitochondria may be effected by placing the leader sequence for the yeast 
Mn-SOD gene directly before the hMn-SOD gene. 
Suitable leader sequences, for example those described by Marres C. A. M. 
et al., Eur. J. Biochem. 147, 153-161 (1985) or derivatives thereof, may 
either be of natural origin or may be isolated from corresponding 
eukaryotic cells (for example S. cerevisiae) or they may be produced 
synthetically. For example, a yeast-specific DNA presequence which is 
necessary for importing the hMn-SOD into the yeast mitochondrium may be 
obtained by ligating individual synthetic oligonucleotides. According to 
the invention, the complete presequence may be inserted between the start 
codon ATG and the first codon for the first amino acid of the mature 
hMn-SOD (Lys, e.g. AAG) or any desired portion of an N-terminal end 
thereof, for example in formulae II, IIIa, IIIb, VIa, VIIa, VIIb, VIII or 
XI. Similarly, a presequence of this kind may be incorporated directly 
after the ATG start codon and directly before the first codon of a DNA 
which is mutated from the genuine DNA sequence of hMn-SOD by sequence 
modifications and which codes for a protein with hMn-SOD activity. 
A leader sequence which can be used according to the invention for the 
purpose of importing an hMn-SOD into the yeast mitochondrium is shown in 
formula X which follows, in which the known sequence GCA GCT (Marres, C. 
A. M. et al., 1985, loc. cit.) is substituted for GCT GCA (both triplets 
code for alanine) and a PvuII recognition site is created. 
##STR11## 
Preferably, the leader sequence, for example as in formula X, may be 
contained in the XhoI/XbaI fragment of formula VIa. This ensures that this 
128 bp linker with the leader can be linked to the remaining hMn-SOD gene 
via the XhoI and XbaI sites in such a way that the leader sequence is 
located immediately after the start ATG and immediately before the first 
amino acid (lysine) of the hMn-SOD (formula XI). 
##STR12## 
Purification of the hMn-SOD from cells may be carried out by known methods. 
The hMn-SOD according to the invention prepared by genetic engineering are 
suitable, owing to their biological/enzymatic spectrum of activity on the 
one hand and on account of the quantity of highly purified enzyme now 
available which has maximum possible immunological identity with genuine 
hMn-SOD, on the other hand, for every type of prevention, treatment and/or 
diagnosis in inflammatory, degenerative, neoplastic or rheumatic diseases, 
for wound healing, in autoimmune diseases and in transplants, and for the 
prevention and treatment of diseases which are accompanied by a deficiency 
of hMn-SOD or are causally linked thereto. For example, the clinical 
applications include those which may be inferred from Bannister W. H. and 
Bannister J. V. (Biological and Clinical Aspects of Superoxide and 
Superoxide Dismutase, Vol. 11B, Elsevier/North-Holland, 1980) and 
Michelson, A. M., McCord, J. M., Fridovich (Superoxide and Superoxide 
Dismutases, Academic Press, 1977). Furthermore, the following clinical 
applications should be considered: for perfusion wounds, strokes, 
alcohol-damaged livers, premature babies, possibly pancreatitis, acute 
respiratory diseases, (ARDs), emphysema, dialysis-damaged kidneys, 
osteoarthritis, rheumatoid arthritis, radiation-induced damage, 
sickle-cell anaemia. 
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 may be administered either 
systemically or topically, whilst in the former case conventional 
parenteral routes of administration (e.g. i.v., i.m., s.c., i.a.) and for 
the latter case the known preparations may be used (e.g. pastes, 
ointments, gels, tablets for sucking or chewing, powders and other galenic 
formulations which permit local resorption of the hMn-SOD preparations and 
pharmaceutically acceptable carriers). A therapeutically effective dosage 
range of around 4 mg, for example, per day may be used depending on 
individual criteria (e.g. the patients, the severity of the illness, etc).

The following examples, which are not intended to restrict the invention, 
illustrate the invention in detail. 
Materials used 
Unless otherwise stated in the Examples which follow, the following 
materials, solutions, plasmids, vectors and microorganisms are used: 
______________________________________ 
ADHI promoter: DSM 4013 (pES103), deposited on 
(1500 bp BamHI/XhoI) 
27.2.87 
ADHI promoter, DMS 4016 
abbreviated to: (400 bp BamHI/XhoI) 
(pWS323E), filed on 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 MgCl.sub.2, 
100 mcg/ml BSA 
Core buffer: 50 mM Tric-HCl pH 8.0, 10 mM 
MgCl.sub.2, 50 mM NaCl 
Denaturing solution: 
0.5 M NaOH, 1.5 M NaCl 
Denhardt solution: 
1 g polyvinylpyrrolidone, 
(50x) MW 360,000, 1 g Ficoll, 
1 g bovine serum albumin (BSA) 
ad. 100 ml H.sub.2 O 
E. coli C600: F.sup.--, supE44, thi1, thr1, leuB6, 
lacY1, tonA21, .lambda..sup.-- (ATCC 23724) 
E. coli JM101: supE, thi, .DELTA.(lac-pro AB), 
[F', traD36, proAB, lacI.sup.q Z, .DELTA.M15] 
High buffer: 100 mM NaCl, 50 mM Tris-HCl 
pH 7.5, 10 mM MgCl.sub.2, 1 mM 
Dithiothreitol (DTT) 
HincII buffer: 10 mM Tris-HCl pH 7.5, 60 mM 
NaCl, 10 mM MgCl.sub.2, 
1 mM 2-mercaptoethanol, 
100 mcg/ml BSA 
Hybridising solution: 
like pre-hybridising solution 
but without salmon sperm DNA 
Klenow reaction 22 mcl DNA/H.sub.2 O, 2.5 mcl 10 .times. 
solution: NTR buffer (0.5M Tris-HCl 
pH 7.2, 0.1M MgSO.sub.4, 1 mM DTT, 
500 mcg/ml BSA) per 1 mcl 
2 mM dATP, dGTP, dCTP, dTTP, 
2.5 U Klenow fragment (0.5 mcl) 
Lambda buffer: 100 mM Tris-HCl pH 7.5, 10 mM 
MgCl.sub.2, 1 mM EDTA 
LB agar: LB liquid medium, 15 g/l Bacto- 
Agar (Difco) 
LB liquid medium: 
10 g/l Bacto-Tryptone (Difco), 
5 g/l yeast extract (Difco), 
5 g/l NaCl, 10M NaOH ad. pH 7.4 
Ligation solution: 
66 mM Tric-HCl pH 7.6, 10 mM 
MgCl.sub.2, 5 mM DTT, 1 mM ATP, 
1U T4-DNA ligase 
Neutralising solution: 
0.5M Tris-HCl pH 7.5, 1.5M 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 MgCl.sub.2 
Prehybridising 5 .times. SSC, 5 .times. Denhardt solution, 
solution: 50 mM Na-phosphate buffer pH 
6.8, 1 mM Na.sub.2 P.sub.4 O.sub.7, 100 mcM 
ATP, 0.1% SDS, 30-100 (50) 
mcg/ml denatured, ultrasound- 
treated salmon sperm DNA 
pUC18: Pharmacia 
pURA3: DSM 4015, deposited on 27.2.87 
S. cerevisiae DBY747: 
a, leu2, his3, trp1, ura3 
(Yeast Genetic Stock Center, 
Berkeley) 
SC-URA medium: 0.67% BYNB (Difco), 2% glucose, 
2% 50 .times. AS mix (containing 
per liter: 1 g histidine, 6 g 
leucine, 2.5 g tryptophan, 
4 g lysine, 1.2 g adenine, 
2 g arginine, 1 g methionine, 
6 g phenylalanine, 5 g threonine, 
6 g isoleucine) 
SmaI buffer: 10 mM Tric-HCl pH 8.0, 20 mM 
KCl, 10 mM MgCl.sub.2, 10 mM 2- 
mercaptoethanol, 100 mcg/ml BSA 
SphI buffer: 10 mM Tric-HCl pH 7.5, 100 mM 
NaCl, 10 mM MgCl.sub.2, 10 mM 2- 
mercaptoethanol, 100 mcg/ml BSA 
SSC (20x): 3.0M NaCl, 0.3M Na.sub.3 -citrate, 
pH 7.0 
SSPE (20x): 3.6M NaCl, 0.2M Na.sub.2 HPO.sub.4, 
20 mM EDTA, with NaOH (10N) 
ad. pH 7.4 
TE buffer: 10 mM Tric-HCl pH 8.0, 1 mM EDTA 
ThaI buffer: 50 mM Tric-HCl pH 8.0, 
10 mM MgCl.sub.2 
Top agarose: LB liquid medium, 0.7% agarose 
(Seaken FM-agarose) 
Prewash solution: 
1M NaCl, 50 mM Tric-HCl pH 
8.0, 1 mM EDTA, 0.1% SDS 
______________________________________ 
EXAMPLE 1 
Construction of a cDNA gene bank 
Dice-sized pieces of fresh human placenta tissue were shock-frozen in 
liquid nitrogen and the tissue was powdered at below -80.degree. C. The 
RNA was then extracted from the powdered tissue material using the 
procedure described by Chirgwin, J. M. et al. and then prepared (Chirgwin, 
J. M. et al., Biochemistry 18, 5294-5299, 1979). 
The poly(A) .sup.+ RNA was prepared from the resulting RNA using the method 
of Aviv, H. and Leder, P. (Proc. Natl. Acad. Sci. USA 69, 1409-1412, 
1972). The cDNA was synthesised using a "cDNA synthesis system" (Amersham 
RPN 1256). 
Packaging was carried out with Gigapack (vector cloning systems). All other 
procedural steps for cloning into the EcoRI site of .lambda.gt10 were 
carried out as prescribed by Huynh T. V. et al. (DNA Cloning Vol. 1, D. M. 
Glover ed., IRL Press, Chapter 2, 1985) except that E. coli C 600 was used 
as the "plating bacteria". The titre of the .lambda.gt10 phage 
representing the cDNA gene bank was 1.2.times.10.sup.10 pfu/ml, the number 
of independent clones 1.times.10.sup.6. 
EXAMPLE 2 
Amplification of the .lambda.gt10 gene bank 
A suitable E. coli strain (C600, genotype F-, supE44, thi1, thr1, leuB6, 
lacY1, tonA21, lambda(M. A. Hoyt et al., 1982, cell 31, 5656) was 
precultivated overnight at 37.degree. in LB medium supplemented with 0.2% 
maltose. 
This overnight culture was centrifuged for 5 min at 3000 rpm and suspended 
in ice cold 10 mM MgSO.sub.4 solution so that the OD600 nm was 4.0. The Mg 
cells thus prepared were stored at 4.degree. C. and could be used for a 
week. 
12.times.200 mcl of Mg cells were mixed, in sterile test tubes, with a 
phage suspension (50000 pfu of the cDNA gene bank per plate) and incubated 
at 37.degree. C. for 20 min. Then 6-7 ml of molten top agarose adjusted to 
a temperature of 42.degree. C. (containing 10 mM MgSO.sub.4, final 
concentration) were pipetted into each test tube, mixed and poured out 
onto 12 agar plates (13.5 cm in diameter) preheated to 37.degree. C. with 
10 mM MgSO.sub.4 and the plates were incubated at 37.degree. C. for 6-12 
hours. 
EXAMPLE 3 
Primary screening to identify recombinant .lambda.-phages 
a. Preparation of the nitrocellulose filters 
After incubation the plates thus prepared were cooled to 4.degree. C. 
Nitrocellulose filters numbered with a pencil were placed on the surface 
of the plates and their positions on the plates were marked with pin 
pricks. About 1 min after being thoroughly wetted, the filters were 
carefully removed again, placed in denaturing solution and incubated for 1 
min at room temperature (RT). They were then neutralised in neutralising 
solution for 5 min at RT and incubated for 30 sec in 2.times.SSPE, again 
at RT. 
Up to 3 further extracts were prepared from each plate, with the filters 
being left on the plate 30 sec longer each time. The positions of the pin 
pricks were transferred accurately to the next filters. 
The filters were dried in air, lying on filter paper, and the DNA was fixed 
at 80.degree. C. by baking for 2 hours. The plates were kept until the 
results of the following hybridisation were obtained. 
b. Preparation of the .sup.32 P-labelled probes 
The synthetic oligonucleotide mixtures were prepared using a 381A DNA 
synthesiser (Applied Biosystems), purified by polyacrylamide gel 
electrophoresis (20% in 8M urea, T. Maniatis et al., Molecular Cloning, 
Cold Spring Harbor Laboratory, 1982, page 173 ff) and desalinated over 
Sephadex G50 (Pharmacia). The DNA probes thus synthesised are 
complementary to RNA base sequences which code a) for amino acids 39-46 or 
b) for amino acids 200-207 (D. Barra et al., Oxy Radicals and their 
scavenger Systems, Vol. 1, 336-339, 1983) and have the following base 
sequences: 
##STR13## 
wherein A, G, C and T represent the corresponding nucleotides and I 
represents inosine. 
The chemically synthesised DNA probe mixtures were each dissoved in water 
at a concentration of 20 pM/mcl. 
Reaction mixture: 
20-100 pM gamma.sup.32 -PATP (&gt;3000 Ci/mmol, Amersham), lyophilised from 
ethanolic solution, 20-100 pmol oligonucleotide, 1 mcl 10.times.kinase 
buffer (0.7M Tris-HCl pH 7.6, 0.1M MgCl.sub.2, 50 mM dithiothreitol, 10 
units T4 polynucleotide kinase (BRL), water ad. 10 mcl. 
The reaction lasted 60 min at 37.degree. C. and was stopped by the addition 
of 25 mM EDTA. Any radioactivity not incorporated was removed by exclusion 
chromatography using a 1 ml Biogel P6-DG (Biorad) column produced in a 1 
ml one-way syringe. TE buffer was used as eluant. 
c. In situ hybridisation 
In order to remove any residual agarose and bacteria from the 
nitrocellulose which will cause considerable background radiation during 
hybridisation, the filters were incubated in a sufficient volume of 
prewash solution at 65.degree. C., whilst being tilted for a period 
ranging from some hours to overnight. In order to saturate non-specific 
binding sites for DNA on the nitrocellulose filters, these filters were 
incubated for 1-12 hours at 37.degree. C. in the prehybridising solution 
which had earlier been degassed in vacuo. 
The radioactively labelled DNAs used for hybridisation (about 
1.times.10.sup.9 cpm/mcg) were added to the required quantity of degassed 
hybridising solution which was preheated to 37.degree. C. In order to keep 
the concentration of the DNA probe as high as possible in the hybridising 
solution, only just enough hybridising liquid to keep the filters just 
covered with liquid was used. Hybridisation lasted for 12-18 hours at 
37.degree. C. 
The nitrocellulose filters were then rinsed three times in 6.times.SSC and 
0.05% SDS (4.degree. C.) by the method of Wood et al., (Proc. Natl. Acad. 
Sci. Vol 82, 1585-1588, 1985) and similarly washed at 4.degree. C. for 
2.times.30 min. The filters where then rinsed three times at room 
temperature (RT) in a freshly prepared solution containing 3M 
tetramethylammonium chloride (Me4NCl), 50 mM Tris-HCl pH 8, 2 mM EDTA and 
0.05% SDS, washed 2.times.30 min at RT and finally washed 3.times.30 min 
at 49.degree. C. (oligonucleotide mixture a)) or at 52.degree. C. 
(oligonucleotide mixture b)), dried in air (oligonucleotide mixture b)) 
and stuck to paper. X-ray films were exposed for 2-8 days at -70.degree. 
C. using an "intensifying screen". 
EXAMPLE 4 
Plaque purification 
Since no individual plaques could be isolated in the first search, with the 
high density of plaques used, the recombinant lambda phage were purified 
by several successive searches whilst the plaque density was 
simultaneously reduced. After development of the autoradiograms, regions 
were isolated from the agar plate (of 3 primary screenings carried out, of 
28 regions, 2 were positive, of 35 regions 1 was positive and of 15 
regions 5 were positive), which yielded a positive hybridising signal on 
the two nitrocellulose filters which had been hybridised in duplicate. The 
desired site was pricked out of the agar using the sharp end of a sterile 
Pasteur pipette and transferred into 0.3-0.6 ml of lambda buffer (100 mM 
Tris-HCl pH 7.5, 10 mM MgCl.sub.2 and 1 mM EDTA). However, SM buffer may 
also be used (Maniatis T., Molecular Cloning, 1982, page 70). After the 
addition of one drop of chloroform, the phages were left to diffuse out of 
the agar overnight at 4.degree. C. and each individual phage suspension 
was plated out again in several dilutions. Another nitrocellulose filter 
was prepared from plates having 300-100 plaques and this extract was then 
hybridised against both DNA probes. This procedure was repeated, and 
individual plaques were followed up, until all the plaques on a plate gave 
a positive hybridisation signal. 
EXAMPLE 5 
Analysis of the phage clones obtained 
a. Titration of .lambda.-phage 
The phage suspensions were diluted with lambda buffer in dilution steps of 
1:10, mixed by tilting several times, and plated out. After incubation at 
37.degree. C. the plaques formed on the bacterial lawn were counted and 
the titre (plaque forming units (pfu)) was determined. The titre for the 
purified phage suspensions was 2.2-8.6.times.10.sup.10 pfu/ml. 
b. Preparation of lambda phage DNA 
After isolation and titration of the inherently homogeneous phage clones, 
they wrere plated in a density of 2.times.10.sup.6 pfu/13.5 cm of 
Petridish (with culture medium of composition: 1.5% agarose, 10 g/l 
tryptone, 5 g/l yeast extract, 5 g/l NaCl, 10 mM MgSO.sub.4, and 0.2% 
glucose) with 200 mcl of C600 Mg cells (4 OD.sub.600), incubated for 5 
hours at 37.degree. C. and then cooled to 4.degree. C. Elution of the 
phage was effected by covering the plates with 8 ml of lambda buffer and a 
few drops of chloroform and tilting gently at 4.degree. C. overnight. The 
supernatant purified by centrifuging (15000 rpm, 15 min, 4.degree. C.) was 
finally removed and the phage were pelleted by centrifuging at 50000 rpm 
(Beckman Ti50 rotor) for 30 min at RT. After the addition of 500 mcl of 
lambda buffer and incubation with ribonuclease A (RNase A, 10 mcg/ml) and 
deoxyribonuclease (DNase, 1 mcg/ml), for 30 min at 37.degree. C., the salt 
concentration was increased by the addition of 25 mcl of 0.5M EDTA, 12 mcl 
of 1M Tris-HCl pH 8.0 and 6.5 mcl of 20% SDS and the enzymes present were 
deactivated by incubating at 70.degree. C. for 15 min. After extracting 
once with phenol and twice with chloroform/isoamyl alcohol (24:1) in equal 
volumes the DNA was precipitated by the addition of 0.1 vol. 3M sodium 
acetate, pH 5.2, and 2 vol. of alcohol, then centrifuged off, washed with 
70% alcohol, dried and taken up in 50 mcl of TE buffer. 
c. Restriction analysis 
2 mcl of DNA solution were incubated with 5 units of EcoRI in HIGH buffer 
for 2 hours at 37.degree. C., the fragments obtained were separated on a 
1% agarose gel (T. Maniatis et al., 1982, p149ff) under a voltage of 1-5 
volts per cm, the fragments with lengths ranging from 500 to 1000 base 
pairs were eluted from the gel (G. M. Dretzen et al., Anal. Biochem. 112, 
295-298, 1981) and finally subjected to sequence analysis. 
d. Sequence analysis 
Subcloning of the restriction fragment into a vector (Bluescribe M13+ or 
M13-, vector cloning systems (C. Yanisch-Perron et al., Gene 33, 103-119, 
1985)) suitable for 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) was carried out by the usual methods for 
effecting the 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). In this way 100 ng of 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 to 12 hours 
at 14.degree. C. in 10 mcl of ligation solution) and with this recombinant 
construction (entitled BS3, BS5, BS8, BS9, BS12, BS13, BSXIII) competent 
E. coli cells (strain JM 101) were transformed. The single strand DNA of 
the recombinant phages was isolated and sequenced according to Sanger. The 
sequences read were processed using suitable computer programmes (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). 
EXAMPLE 6 
Construction of an expression cassette 
In order to express the hMn-SOD in yeast, it is necessary to complete the 
isolated cDNA and to construct an expression cassette, the ADHI promoter 
being used in its original length (about 1500 bp, Methods in Enzymology, 
Vol. 101, Part C, 192-201, 1983), in shortened form (ADHIk about 400 bp) 
and the ADHII terminator (Dr. R. Beier and E. T. Young, Nature 300, 
724-728, 1982) being used as well. 
a. Completion of the gene 
Since the isolated cDNA clone 8 lacks the bases corresponding to the 21 
amino acids (AA) at the N terminus, in order to complete the gene 
according to the reported amino acid sequence (D. Barra et al., J. Biol. 
Chem. 259, 12595-12601, 1984) taking into account the yeast codon 
selection (P. M. Sharp et al., Nucl. Acids. Res. 14, 5125-5143, 1986) 2 
pairs of oligonucleotides were constructed and synthesised (381A DNA 
synthesiser, Applied Biosystems) as the XhoI-XbaI fragment (OP1, 
corresponding to formula VIa) or the XbaI-NcoI fragment (OP2, 
corresponding to formula VIb). OP1 was inserted via XhoI/XbaI into the 
plasmid V 17 (obtained from pUC18 (J. Vieira and J. Messing, Gene 19, 259, 
1982) after HincII restriction and insertion of XhoI linkers (New England 
Biolabs, d(CCTCGAGG) and SmaI restriction of the resulting plasmid pES102 
with subsequent insertion of NcoI linkers (New England Biolabs, 
d(CCCATGGG)) (FIG. 2), whilst OP2 was inserted via XbaI/NcoI. In order to 
do this, 4 mcg of V 17 DNA were digested with 10 units of XbaI and NcoI or 
XhoI and XbaI in 40 mcl of CORE buffer for 2 hours at 37.degree. C. and 
purified by gel electrophoresis (0.7% agarose, see above). 5 mcl portions 
of the synthesised single strands of OP1 or OP2 (10 pM/mcl in each case) 
were mixed together, incubated for 10 minutes at 65.degree. C. and slowly 
cooled to RT. 1/10 thereof was ligated with 50 ng of doubly cut vector 
(XhoI/XbaI for OP1 and XbaI/NcoI for OP2) under the conditions described 
above (plasmids HSOD2 and HSOD3, FIG. 2). Finally, HSOD2 and HSOD3 were 
combined to form plasmid HSOD4 via ScaI/XbaI (i.e. after double digestion 
with ScaI and XbaI in CORE buffer for 2 hours at 37.degree. C.) after 
purification and isolation of the cut vectors by gel electrophoresis and 
ligation under the conditions described above (cloning of the oligo pairs 
OP1 and OP2) (FIGS. 2, 3). This plasmid HSOD4 was prepared to receive the 
ThaI/EcoRI cDNA fragment by NcoI restriction, followed by Klenow fill-in 
and EcoRI restriction: 5 mcg of DNA were incubated for several hours at 
37.degree. C. in 50 mcl of high buffer with 18 units of NcoI, the cut DNA 
was purified by gel electrophoresis, then isolated and half of it was 
incubated in 30 mcl of Klenow reaction solution for 1 hour at RT. 
After the reaction had been ended by the addition of 2 mcl of 0.5M EDTA and 
the reaction solution had been incubated at 70.degree. C. for 10 minutes 
the DNA was purified by gel electrophoresis, isolated and re-cut with 7.5 
units of EcoRI in 20 mcl of HIGH buffer, purified again and isolated. 
(FIG. 5) 
The ThaI/EcoRI cDNA fragment was prepared as follows: 
Competent E. coli host cells (strain JM 101) 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, 
page 368). 
After restriction with ThaI (10 mcg of plasmid were digested in 40 mcl of 
ThaI buffer with 25 units of ThaI for 8 hours at 60.degree. C.), recutting 
the 759 bp ThaI fragment with EcoRI (see above), followed by purification 
by gel electrophoresis and isolation of the corresponding fragment, the 
ThaI/EcoRI fragment thus obtained (FIG. 4) was combined with the 
correspondingly prepared plasmid HSOD4 to form HSOD6 (FIG. 5) (about 100 
ng of fragment were ligated with 50 ng of cut vector in 10 mcl of ligation 
solution (see above)). Plasmid HSOD6 thus contains the complete cDNA for 
hMn-SOD including Met. The reading frame is retained. 
b. Construction of the expression cassette 
Plasmid HSOD6 was doubly digested with XhoI and EcoRI (5 units/mcg of DNA) 
in CORE buffer, the XhoI fragment (gene) was isolated and inserted into 
the plasmid PKH1 or PKH2 via XhoI/EcoRI. The plasmids PKH1 and PKH2 were 
prepared as follows (FIGS. 6, 7, 8): after SmaI restriction (1 mcg of 
plasmid was digested with 5 units of SmaI in SmaI buffer for 2 hours at 
37.degree. C.), purification and isolation, BgIII linkers were inserted in 
plasmid PES 103, which contains the ADHI promoter as a 1500 bp BamHI-XhoI 
fragment in PES 102 (PES 102 is a pUC18 derivative which contains in the 
HincII cutting site an XhoI linker, the construction of the BamHI-XhoI 
fragment being described in "Methods in Enzymology" 101, 192-201) (T. 
Maniatis et al., 1982, page 396). The plasmid thus obtained (P154/1, FIG. 
6) was changed into plasmid 154/2 by EcoRI restriction (see above), Klenow 
fill-in (see above) and religation (1 mcg of DNA was incubated in 40 mcl 
of ligation solution (see above) overnight at 14.degree. C.) (FIG. 6). 
Also starting from plasmid pES103, the linker -XhoI.EcoRI. XbaI.HindIII- 
(FIG. 7, synthesised using a 381A DNA synthesiser) was inserted after 
double digestion with XhoI and HindIII in CORE buffer. This linker 
contains the sequence 
##STR14## 
The ADHII terminator was inserted in the resulting plasmid 150/1 via 
XbaI/HindIII (double digestion in CORE buffer) (plasmid 150/2 (FIG. 7)). 
The ADHII terminator was obtained as follows: plasmid pMW5 ADHII 
(Washington Research Foundation) was digested with HindIII (core buffer) 
then with SphI (in SphI buffer) and the isolated 605 bp fragment was 
cloned into the vector V18 and an XbaI linker (Biolabs, CTCTAGAG) was 
incorporated in the HincII cutting site (for ligation see above). A 335 bp 
long XbaI/SphI fragment was ligated into pUC18 (XbaI/SphI) (pGD2). 
The vector V18 was obtained by incorporating a HindIII linker in pUC18 in 
the SmaI site and the HindIII site is missing from its original location, 
so that the multicloning site in V18 runs as follows: 
EcoRI.SstI.KpnI.HindIII.BamHI.XbaI.SalI.PstI.SphI 
Finally, after double digestion with XbaI/HindIII in CORE buffer the ADHII 
terminator was isolated by the usual methods (see above). Plasmid 150/2 
thus contains the units necessary for gene expression, apart from the gene 
which is to be inserted via XhoI/EcoRI, namely approximately 1500 bp 
(promoter), 7 bp (XhoI linker), 6 bp (EcoRI linker), 7 bp (XbaI linker), 
329 bp (terminator). These units were then inserted into the vector 154/2 
(FIG. 8) via BamHI/HindIII (double digestion in CORE buffer). In the 
resulting plasmid PKHI (FIG. 8) the ADHI promoter was analogously replaced 
by the shortened promoter ADHIk as the BamHI/XhoI fragment (412 bp) (pKH2, 
FIG. 9). 
Finally, the complete cDNA gene (see above) cut out of HSOD6 was inserted 
into both plasmids via XhoI/EcoRI (see above). The resulting plasmids 
HSOD7/1 and HSOD7/2 (FIG. 9 shows only HSOD7/2) differ from one another 
only in the different promoters ADHI and ADHIk (see above). The expression 
cassettes thus prepared were inserted into the correspondingly prepared 
and freely obtainable yeast transformation vectors YEp13 (J. R. Broach et 
al., Gene 8, 121-133, 1979, ATCC 37115), pJDB207 (DSM 3181, deposited on 
28.12.84), pEAS102 (see above), YIp5 (K. Struhl et al., Proc. Natl. Acad. 
Sci. USA 76, 1035-1039, 1979, ATCC 37061) via the cutting sites BamHI and 
HindIII, via BglII/HindIII (after double digestion of the plasmids in CORE 
buffer and isolation of the expression cassettes cut out). 
EXAMPLE 7 
Preparation of a yeast Mn-SOD mutant suitable for expression 
The gene for yeast Mn-SOD (A. P. G. M. van Loon et al., Gene 26, 261-272, 
1983) is contained as a BamHI fragment in the vector PL 41 (FIG. 10) and 
the sequence has been published in full (C. A. M. Marres et al., Eur. J. 
Biochem. 147, 153-161, 1985). After restriction with BamHI (2 mcg plasmid 
were digested with 5 units in 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM 
MgCl.sub.2, 100 mcg/mcl bovine serum albumin for 2 hours at 36.degree. C.) 
the 2045 bp long BamHI fragment which contains the gene was purified as 
usual by gel electrophoresis and isolated and subcloned via BamHI into the 
vector VO (pUC18, but with no HindIII cutting site). 
The vector VO was obtained by cutting 1 mcg of pUC18 with HindIII (CORE 
buffer), isolating the linearised fragment from the gel by known methods, 
filling in the projecting ends with 2 U Klenow polymerase (ligase 
buffer+0.2 mM dNTP) and religating after 30 minutes at RT by the addition 
of 2 U T4-DNA ligase overnight at 14.degree. C. 
The plasmid SODY1 (FIG. 10) was purified by NruI restriction (1 mcg of 
plasmid were digested with 5 units of NruI in NruI buffer for 2 hours at 
36.degree. C.) by gel electrophoresis and changed to SODY3 (FIG. 10) by 
the insertion of a HindIII linker (CAAGCTTG) (FIG. 10). Finally, the URA3 
gene (obtained from pURA3) was inserted into the HindIII cutting site: 4 
mcg of SODY3 were digested with 20 units of HindIII for 2 hours at 
37.degree. C. in CORE buffer and dephosphorylated: 40 mcl of H.sub.2 O, 10 
mcl of 1 mM EDTA, 5 mcl of 1M Tris-HCl pH 9.5, 1 mcl of 100 mM spermidine, 
1 mcl of calf intestinal alkaline phosphatase (CIAP, 1 mg/ml H.sub.2 O) 
were added to 40 mcl of digestion mixture and the whole was incubated at 
36.degree. C. After 15 minutes, a further 1 mcl of CIAP were added and the 
mixture was incubated for another 15 minutes. The dephosphorylated vector 
was also purified by agarose gel electrophoresis. 2 mcg 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-SOD 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 can be 
determined, since the URA3 gene contains an asymmetric PstI site. 
A "gene transplacement" was carried out (Methods in Enzymology 101, 202-211 
and 211-228) with the plasmid SODY7 and SODY8 in the strain DBY 747 
(genotype a, leu2, his3, trp1, ura3, Yeast Genetic Stock Centre, 
Berkeley). The strain DBY 747 was transformed with the BamHI fragment from 
SODY7 and SODY8 (J. D. Beggs, Nature 275, 104, 1978). To do this, 20 mcg 
of SODY7 or SODY8 were cut with 50 U BamHI in 200 mcl of BamHI buffer (150 
mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl.sub.2, 1 mM DTT) and the entire 
digestion mixture (without separating off the pUC portion) was extracted 
with phenol (Maniatis, T. et al., Molecular Cloning, 1982, page 458ff) and 
concentrated by ethanol precipitation (addition of 20 mcl of 3M sodium 
acetate pH 5.5, 500 mcl of ethanol). The DNA was taken up in 10 mcl of 
water and used directly for the transformation of yeast. 
The transformants were selected for uracil prototrophy. 
Individual transformants were cultivated overnight in 5 ml of SC-URA medium 
at 28.degree. C. The cells were harvested by centrifuging, broken by the 
method of van Loon et al. (Proc. Natl. Acad. Sci. USA 83, 3820-3824, 1986) 
and tested for their content of Mn-SOD. The measurement of Mn-SOD and 
Cu/Zn-SOD by gel electrophoresis were carried out by existing methods (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 Vol. 121, 404-427, 1964). The method 
which proved best was the separation of the proteins followed by negative 
staining with nitroblue tetrazolium (B. J. Davis, 1964; Ch. Beauchamp and 
I. Fridovich, 1971). It is possible to increase the sensitivity by 
staining with dianisidine (H. P. Misra and I. Fridovich, 1977). A 
spectrophotometric assay (Hyland, K. et al., Anal. Biochem. 135. 280-287, 
1983) with alkaline dimethylsulphoxide as the O.sub.2.sup.- --generating 
system and with cytochrome c as "scavenger". 
Mn-SOD on the one hand and Cu/Zn-SOD on the other hand are distinguished by 
the addition of KCN (see above and M. Ysebaert-Vanneste and W. H. 
Vanneste, Anal. Biochem. 107, 86-95, 1980). The strains SODY7/2, SODY7/6, 
SODY7/8 and SODY7/10 contained no Mn-SOD activity. 
EXAMPLE 8 
Preparation of the expression vectors 
The expression cassettes described in Example 6b were cut out of the 
plasmids HSOD7/1 and HSOD7/2, respectively, as BglII/HindIII fragments (in 
each case, 2 mcg of plasmid DNA in the CORE buffer, 2 hours at 37.degree. 
C. with 10 U of enzyme). Similarly, 1 mcg of YEp13, pJDB207 and pEAS102 
were each cut with HindIII-BamHI (digestion conditions as described 
above). 
50 mcg of vector DNA and 200 mcg of insert were ligated in ligase buffer 
(as described) with 1 U ligase overnight at 14.degree. C. and used to 
transform the E. coli strain HB101. The following Table contains the names 
of the corresponding plasmids. 
TABLE 1 
______________________________________ 
Names of the expression vectors 
Vector Insert: HSOD7/1 
HSOD7/2 
______________________________________ 
YEp13 pWS550A pWS371A 
pJDB207 pWS490A pWS372A 
pEAS102 pWS491A pWS373A 
______________________________________ 
EXAMPLE 9 
Preparation of a yeast strain (WS30-5 g) suitable for transformation 
A yeast strain was prepared which contains, in addition to the genetic 
markers described for the yeast strain SODY7/2, a mutation in one of the 
lysosomal chief proteases (which can activate other lysosomal proteases by 
their activity) and thus releases fewer proteases when the yeast cells are 
broken up (mutation pep4) (E. W. Jones et al., Genetics 102, 665-677, 
1982). 
The Mn-SOD-deficient strain SODY7/2 was crossed with the protease-deficient 
strain WS20-25 (.alpha. leu2 his3 trp1 ura3 pep4) and the resulting 
haploids were investigated for their genetic markers (F. Sherman et al., 
Methods in Yeast Genetics, Cold Spring Harbor, N.Y., 1972). 
The resulting strain WS30-5 g (leu2 his3 trp1 pep4 sod1) is readily 
transformable and fulfils the desired conditions. 
Such crossing may also be carried out with equally good results with other 
well known and easily obtainable yeast strains, for example with 20 B-12 
(Yeast Genetic Stock Center, Berkeley). 
EXAMPLE 10 
Yeast transformation and expression in yeast 
The yeast strain SODY7/2 was transformed with the plasmids pWS371A, pWS372A 
and pWS373A (J. D. Beggs, Nature 275, 104-109, 1978) and the transformants 
were investigated for their expression. 
To achieve this, a pre-culture of the transformants was prepared in SC-Leu 
liquid medium (analogous to the SC-URA medium described, except that it 
additionally contains 2.4 g of uracil but no leucine) (shaking at 300 rpm 
at 28.degree. C. overnight). 100 mcl thereof were inoculated into 4 ml of 
YP5% D (1% Bacto yeast extract, 2% Bacto peptone, 5% glucose) and 
cultivated overnight (like the pre-culture). The cells were harvested and 
broken as already described in Example 7. The quantity of crude cell juice 
corresponding to 1 ml of culture was transferred to the activity gel. The 
activity test was carried out as described in Example 7. 
The yeast strain WS30-5 g (leu2 his3 trp1 pep4 sod1) was transformed with 
the plasmids pWS550A, pWS490A, pWS491A. The preparation of the pre-culture 
and culture and the measurement of the hMn-SOD activity were carried out 
as described above. 
The expression of the plasmids pWS490A, pWS491A in yeast strain WS30-5 g is 
documented by FIG. 11. 
The quantity of MnSOD measured in the yeast under these conditions 
corresponded to approximately 0.5 mg/liter of culture. 
EXAMPLE 11 
Synthesis of a linker containing the yeast leader DNA sequence 
Six different oligonucleotides EBI 656, EBI 636, EBI 643, EBI 646, EBI 660 
and EBI 638 of the following sequences and lengths 
##STR15## 
were prepared using a 381 A DNA synthesiser (Applied Biosystems), as 
described in 3b. 
The oligonucleotides EBI 636, EBI 643, EBI 646 and EBI 660 were 
phosphorylated for the subsequent ligase reaction at their 5' ends under 
the following conditions: 
______________________________________ 
Reaction mixture No. 1 
2 mcl EBI 636 (= 100 pmol) 
1 mcl 10 .times. linker kinase buffer 
3 mcl 10 mM ATP 
1 mcl T4 polynucleotide kinase, 
Biolabs 10U/mcl 
3 mcl of water 
Reaction mixture No. 2 
Analogous to No. 1 but with 
2 mol (100 pmol) of EBI 660 
Reaction mixture No. 3 
2 mcl oligonucleotide EBI 643 
(= 100 pmol) 
2 mcl oligonucleotide EBI 646 
(= 100 pmol) 
1 mcl 10 .times. linker kinase buffer 
3 mcl 10 mM ATP 
1 mcl T4 polynucleotide 
kinase (10 units) 
1 mcl water 
10 .times. linker kinase 
0.7M Tris-HCl pH 7.6 
buffer: 0.1M MgCl.sub.2 
0.05M DTT (dithiothreitol) 
______________________________________ 
The reaction lasted 30 minutes at 37.degree. C. The T4 polynucleotide 
kinase was then deactivated by heating to 100.degree. C. 
The oligonucleotides EBI 656 and EBI 638 which are intended to form the 5' 
ends of the finished 128 bp long DNA insert (formula XI) were not 
phosphorylated, in order to avoid the formation of multimeric DNA inserts 
in the subsequent ligase reaction. 
A composition of the desired linkers from the individual oligonucleotides 
was achieved according to the following plan: 
##STR16## 
2 mcl (=100 pmol) of EBI 656 were added to reaction mixture No. 1 and 2 mcl 
of EBI 638 (=100 pmol) were added to reaction mixture No. 2 for the 
annealing reaction (hybridisation of the complementary oligonucleotides 
with each other). Reaction mixture No. 3 already contains 2 complementary 
oligonucleotides (EBI 643, EBI 646). All 3 reaction mixtures were heated 
to 100.degree. C. for 2 minutes and slowly cooled in a water bath. 
The short double-stranded DNA fragments produced in reactions Nos. 1 to 3 
were ligated together as follows: 
______________________________________ 
10 mcl of reaction mixture No. 1 (EBI 636 + EBI 656) 
10 mcl of reaction mixture No. 2 (EBI 660 + EBI 638) 
10 mcl of reaction mixture No. 3 (EBI 643 + EBI 646) 
3 mcl 10 mM ATP 
1 mcl DNA ligase, Boehringer Mannheim, 7 Units/mcl 
______________________________________ 
The reaction lasted for 15 hours at 4.degree. C. 
The DNA was separated according to size on 1% agarose gel and the desired 
DNA fragment of formula XI 128 bp long was eluted from the gel (G. M. 
Dretzen et al., Anal. Biochem. 112. 295-298, 1981). 
EXAMPLE 12 
Construction of the expression vectors containing the leader DNA sequence 
Plasmid HSOD6 was doubly digested with XhoI and XbaI (5 units/mcg of DNA) 
in CORE buffer in the usual way and the 128 bp long linker 
(XhoI--mitochondrial leader--XbaI) was inserted therein by known methods 
(pEO22-A). The hMn-SOD gene now provided with the mitochrondrial yeast 
leader DNA sequence was doubly digested with XhoI-EcoRI (5 units per mcg 
of DNA) in the CORE buffer and inserted via XhoI-EcoRI, in pKH1 (Example 
6b, FIG. 8) (pEO23-A). 
The expression cassette thus prepared was inserted, analogously to Example 
8, via Bg1II/HindIII (after double digestion of the plasmids in CORE 
buffer and isolation of the expression cassette cut out) into the 
correspondingly prepared yeast transformation vector YEp13, pJDB207 and 
pEAS102 via the cutting sites BamHI and HindIII. Table II which follows 
denotes the plasmids thus obtained. 
TABLE 2 
______________________________________ 
Titles of the expression vectors 
Vector Name of plasmid 
______________________________________ 
pJDB207 pEO24-AB 
pEAS102 pEO25-AC 
YEp13 pEO26-AD 
______________________________________ 
EXAMPLE 13 
Yeast transformation and expression in yeast 
The yeast strain WS30-5 g (Example 9) was transformed with the plasmids 
listed in Table 2 and the transformants were tested for their expression 
(Example 10). 
For fermentation of the transformed yeast strain WS30-5g a pre-culture 
having the following composition was cultivated with a magnetic stirrer 
and with aeration, until an optical density OD.sub.546 =0.01 was achieved: 
6.7 g/l yeast nitrogen base w/o amino acids (Difco), 10 g/l glucose, 0.16 
g/l arginine, 0.25 g/l lysine, 0.06 g/l tryptophan, 0.08 g/l methionine, 
0.03 g/l cysteine, 0.10 g/l histidine, 0.16 g/l tyrosine, 0.17 g/l 
phenylalanine, 0.16 g/l threonine, 0.18 g/l isoleucine, 0.21 g/l valine, 
0.40 g/l glutamic acid, 0.21 g/l glycine, 0.02 g/l of cystine, 0.15 g/l 
alanine, 0.20 g/l asparaginic acid, 0.20 g/l proline, 0.15 g/l serine, 
0.10 g/l asparagine, 0.20 g/l glutamine, 25 mg/l adenine, 50 mg/l uracil. 
The subsequent main culture having the composition: 8.0 g/l 
(NH.sub.4).sub.2 SO.sub.4, 2.56 g/l (NH.sub.4).sub.2 HPO.sub.4, 1.16 g/l 
KCl, 0.60 g/l MgSO.sub.4.7H.sub.2 O, 0.56 g/l CaCl.sub.2.2H.sub.2 O, 0.04 
mg/l biotin, 80 mg/l m-inositol, 40 mg/l Ca-pantothenate, 8 mg/l thiamine, 
2 mg/l pyridoxine, 3.1 mg/l CuSO.sub.4.5H.sub.2 O, 19 mg/l 
FeCl.sub.3.6H.sub.2 O, 12 mg/l ZnSO.sub.4.7H.sub.2 O, 14 mg/l 
MnSO.sub.4.H.sub.2 O, 5 mg/l H.sub.3 BO.sub.3, 1 mg/l KI, 2 mg/l Na.sub.2 
MoO.sub.4.2H.sub.2 O, 1 g/l yeast extract, 0.2 g/l uracil, 0.1 g/l 
adenine, 0.5 g/l citric acid, 15 g/l glutamic acid, 0.2 g/l histidine, 0.5 
g/l tryptophan, 100 g/l glucose was produced in the 201 fermenter 
(CHEMAP). For this purpose, 5 % of the quantity of pre-culture was used as 
the inoculum and cultivation was effected with stirring (1000 rpm), 
aeration (0.5 vvm) and at a constant pH (5.0) at 28.degree. C. in a 201 
fermenter. 
After the glucose content had fallen to 50 g/l, a further 50 g/l of glucose 
were added and fermentation was continued until the glucose content was 10 
g/l (which happened after 45 hours). The fermentation liquor was then 
cooled, centrifuged and the biomass was frozen. The yield of biomass was 
18 g/l of the wet cell weight. 
The expression of the plasmid pEO24-AB, pEO25-AC and pEO26-AD in yeast 
strain WS30-5g is documented in FIG. 12. 
EXAMPLE 14 
Yeast mitochondria preparation 
In order to determine whether the insertion of the yeast mitochrondrial 
leader sequence before the hMn-SOD gene causes the protein to be imported 
into the mitochondria, yeast mitochondria were prepared and the Mn-SOD 
activity in the mitochondria and in the cytoplasm was analysed. 
Yeast mitochondria were prepared by a modified form of the method of G. 
Daum et al., Journal Biol. Chem., 257, 13028-13033, 1982. A pre-culture of 
the transformants in SC-Leu liquid medium (Example 10) was cultivated by 
shaking (300 rpm) at 28.degree. C. overnight. 25 ml were inoculated into 
225 ml of YPD medium and cultivated overnight, like the pre-culture. The 
cells were generally measured at an optical density of 5-7 at 600 nm and 
harvested by centrifuging (Sorval, 6500 rpm, 5 min.). The cells were 
washed once with 100 ml of water. The cell pellet was suspended in 1M 
mannitol, 20 mM KP.sub.i (KH.sub.2 PO.sub.4 /K.sub.2 HPO.sub.4) pH 7.4 (1 
ml per 300 mg of cell weight) and 1 mg/ml of zymolase (Miles, MW 500) was 
added. Spheroplasts were produced by slowly shaking for 2 hours (50 rpm) 
at 28.degree. C. 
The spheroplasts were harvested by centrifuging (3000 rpm, 5 min., Hereaus 
Christ Bench Centrifuge) and washed once with 1M mannitol, 20 mM KP.sub.i 
pH 7.4, 1 mM PMSF (phenylmethylsulphonylfluoride). The supernatant was 
discarded and 1 to 2 pellet volumes of glass beads (diameter 0.1 mm) were 
added. 
The cells were broken up by stirring for 1 minute and suspended in 2.5 ml 
of 0.65M mannitol, 1 mM EDTA, 1 mM PMSF. Whole cells and cell debris were 
centrifuged at 2000 rpm for 5 minutes (Hereaus Christ Bench Centrifuge). 
The mitochondria were then obtained from the supernatant by centrifuging 
(Sorval, J-21, 12000 rpm, 10 min.). The supernatant contains the cytoplasm 
and was removed in order to be investigated later for hMn-SOD activity. 
The reddish-brown mitochondria pellet was washed with the above-mentioned 
buffer (white cytoplasmic constituents were rinsed away) and the 
mitochondria were suspended in 2.5 ml of the same buffer. Any impurities 
were removed by centrifuging again (Hereaus Christ, Bench Centrifuge, 4000 
rpm, 5 min.) and the mitochondria were pelleted from the supernatant in a 
second centrifugation (Sorval J-21, 12000 rpm, 10 min.). The mitochondria 
were broken up with glass beads, in a manner similar to the method for 
breaking up yeast cells (van Loon et al., Proc. Natl. Acad. Sci. USA 83, 
3820-3824, 1986) and tested for their content of Mn-SOD in activity gel 
(FIG. 13). 
EXAMPLE 15 
Purification of the hMn-SOD according to the invention 
The recombinant hMn-SOD was isolated from the strain WS30-5g/pEO24-AB 
(yeast vector pJDB207) via several steps. 
Step 1: Cell disintegration 
The cell mass (Example 13) was washed in 10 ml of distilled water per gram 
of wet weight and centrifuged for 15 minutes at 16000.times.g. The 
precipitate was resuspended in Na, K-phosphate buffer (50 mM, pH 7.0) in 
the ratio 1:3 (w/v). The cells were then broken up in a continuously 
operating cell mill (Dynomill KDL; Bachofer, Basel, Switzerland; 0.6 l 
grinding container, water-cooled) using glass beads (0.1 mm in diameter) 
at a flow rate of 6 liters per hour. The cell extract was centrifuged for 
15 minutes (16000.times.g, 4.degree. C.) and the precipitate was 
discarded. 
Step 2: Polyethyleneimine precipitation 
A 5% (w/v) aqueous polyethyleneimine solution (pH 8.0) was added with 
stirring to the supernatant from step 1 until a final concentration of 
0.5% was achieved (polyethyleneimine, Serva, Heidelberg). The mixture was 
then stirred for a further 30 minutes and the precipitate was centrifuged 
off at 16000.times.g (30 minutes). 
Step 3: Heat precipitation 
The supernatant from step 2 was heated in steel beakers with stirring in a 
hot water bath (80.degree. C.) to 60.degree. C. and cooled to room 
temperature again in an ice bath. Any protein precipitated was removed by 
centrifuging (10,000.times.g, 10 min., 4.degree. C.). 
Step 4: Ammonium sulphate precipitation 
The supernatant from step 3 was brought to 20% saturation with solid 
ammonium sulphate and the precipitate was removed by centrifuging 
(10,000.times.g, 15 min., 4.degree. C.). The ammonium sulphate 
concentration was then increased to 90% and the precipitate was obtained 
by centrifuging (10,000.times.g, 15 min., 4.degree. C.). The sediment was 
taken up in a little MES buffer (morpholino ethanesulphonate buffer, 50 
mM, pH 6.0; 2-morpholino ethanesulphonic acid of Sigma, Deisenhofen) and 
dialysed overnight against the same buffer. 
Step 5: Cation exchange chromatography 
A Mono S column (Mono S HR 5/5, Pharmacia, Sweden) was equilibrated with 5 
column volumes of MES buffer. After the column had been charged with the 
extract from step 4, any unbound proteins were washed away with 5 column 
volumes of MES buffer. The hMn-SOD according to the invention was then 
eluted in a linear gradient of 0-50 mM NaCl in MES buffer (20 column 
volumes). Fractions which contained Mn-SOD activity were combined and 
dialysed against Na, K phosphate buffer (5 mM, pH 7.0). 
The native yeast SOD enzymes (Mn-SOD, CuZn-SOD) can be separated off in 
this purification step. FIG. 14 shows an elution diagram. 
Step 6: Adsorption chromatography on hydroxylapatite 
A hydroxylapatite column (HA Ultrogel, IBF, Villeneuve-la-Garenne, France) 
equilibrated with phosphate buffer (5 mM, pH 7.0) was charged with the 
dialysate from step 5 and the hMn-SOD according to the invention was 
eluted with a linear gradient (20 column volumes) of 5-300 mM of Na, 
K-phosphate, pH 7.0. 
The degree of purity of hMn-SOD achieved in the individual purification 
steps was monitored by reductive SDS-polyacrylamide gel electrophoresis 
(FIG. 15). 
EXAMPLE 16 
Characterisation of the hMn-SOD according to the invention 
The hMn-SOD according to the invention, purified as in Example 15, was 
analysed by gel permeation HPLC, reverse phase HPLC, N-terminal 
sequencing, SDS-gel electrophoresis, native gel electrophoresis and 
isoelectric focusing and compared with natural hMn-SOD. 
a. Gel permeation HPLC: 
Column: Water protein pack I 125, 2.times.(7.8.times.300 mm), 10 mcm 
particle diameter 
Eluant: 0.5M Na.sub.2 SO.sub.4, 0.02M NaH.sub.2 PO.sub.4, pH 7.0, 0.04% 
Tween 20, 25% propyleneglycol 
Flux: 0.5 ml/min 
Detection: UV absorption, 214 nm 
Natural hMn-SOD or hMn-SOD according to the invention show the main peak of 
the SOD tetramer at a molecular weight of 70,000 and 76,000, respectively, 
calibration being effected by means of four standard proteins. Within the 
experimental degree of error of this method, these values can be regarded 
as identical. 
b. Reverse phase HPLC 
Column: Bakerbond WP C.sub.18, 4.6.times.250 nm, 5 mcm particle diameter, 
30 nm pore diameter 
Eluant A: 0.1% trifluoroacetic acid in water 
Eluant B: 0.1% trifluoroacetic acid in acetonitrile 
Gradient: 20% B for 2 min., 20-68% B in 24 min, 68% B for 10 min., 68-20% B 
in 1 min 
Flux: 1.0 ml/min 
Detection: UV absorption, 214 nm and 280 nm 
Both natural hMn-SOD and hMn-SOD according to the invention show a 
retention time of just 21 minutes (20.7 and 20.9 min respectively). 
c. N-terminal sequencing 
A peak of hMn-SOD according to the invention, desalinated by reverse phase 
HPLC, was sequenced. Sequencing was carried out using a gas phase 
sequenator made by Applied Biosystems (Model 470 A) with the program 
02RPTH. With an initial quantity of 0.8 nM, it was possible to sequence up 
to amino acid 20. 100% agreement was found with the expected sequence (of 
natural protein and cDNA). The leader sequence for transporting into the 
mitochondria had been split off completely. 
d. SDS gel electrophoresis 
Separating gel: 15% acrylamide 
Stacking gel: 4% acrylamide 
Staining: silver staining according to B. R. Oakley et al. (Analyt. 
Biochem. 105, 361-363, 1980). 
Gel measurements: 0.75 mm (8.times.10 cm) 
Running conditions: 60 min, 150 V 
The SDS gel electrophoresis was carried out substantially according to the 
method originally described by U. K. Lammli (Nature 227, 680-685, 1970). 
In the preparation of the samples for hMn-SOD, the samples were mixed with 
DTT as the reducing agent and boiled. hMn-SOD occurred on the SDS gel 
mainly as a monomer with M approximately 25,000. Depending on the 
completeness of the reduction, the tetramer with M approximately 90,000 
can also be detected. FIG. 15 shows a 15% SDS polyacrylamide gel after 
silver staining. 
e. Native gel electrophoresis 
Separating gel: 7.5% native PAGE according to Davis, B. J. (Ann. NY Acad. 
Sci. 121, 404-427, 1964) 
Stacking gel: 2% acrylamide+sucrose 
Gel dimensions: 0.75 mm (8.times.10 cm) 
Running conditions: 75 min, 150 V (const.) 
Staining: Coomassie Blue by known methods and activity staining with 
o-dianisidine according to Misra, H. P., Fridovich, I. (Arch. Biochem. 
Biophys. 183, 511-515, 1977). 
The hMn-SOD according to the invention obtained after hydroxylapatite 
chromatography showed a uniform band located in the same position after 
electrophoresis, both with Coomassie Blue staining (quantity of hMn-SOD 
applied: 0.3 mcg) and also after activity staining with o-dianisidine 
(quantity of hMn-SOD applied: 75, 30 or 15 ng). 
f. Isoelectric focusing 
pH range: 3.5-9.5 
Gel plates: LKB, PAG plate (1 mm.times.(9.times.10 cm)) 
Electrode solutions: 
1M phosphoric acid (anode) 
1M sodium hydroxide solution (cathode) 
Cooling temperature: 7.degree. C. 
Quantity of sample: 4.0 or 6.5 mcg 
Running conditions: 
pre-focusing 500 Vh 
focusing 3000 Vh in all 
Staining: Coomassie Blue, activity staining with o-dianisidine 
pI=8.15 was determined as the isoelectric point.