GB2188933A - Expression vectors for production of polypeptides, method for enhanced expression of polypeptides, hosts containing the expression vectors, products manufactured thereby - Google Patents

Description

SPECIFICATION Expression vectors for production of polypeptides, method for enhanced expression of polypeptides, hosts containing the expression vectors, products manufactured thereby Field of the invention This invention relates to proteins and polypeptides, pharmaceutical compositions, promoters, operators, genes, vectors, host organisms and processes for their production.

Background of the invention This invention relates to proteins and polypeptides, especially Met-aprotinin and Met-aprotinin homologues, their production via recombinant DNA technology, a novel organism expressing genes, novel expression vectors including promoters, operators, amplified genes and to a procedure for construction of oligomeric genes.

The chemically synthesized DNA molecules as disclosed herein are characterized by the DNA sequence.

In case of genes for Met-aprotinin and Met-aprotinin homologues the DNA sequence is coding for new polypeptides or polypeptides substantially agreeing in the amino acid sequence and composition to that of aprotinin or aprotinin homologues and having the biological activity of aprotinin or of aprotinin homologues.

Aprotinin is a well known peptide comprising 58 amino acids and having an action of inhibiting trypsin, chymotrypsin, plasmin and kallikrein. It is a basic proteinase inhibitor from bovine organs and has become a valuable drug, named Trasylol, for the treatment of various diseases like e.g. hyperfibrinolytic hemorrhage and traumatic-hemmorrrhagic shock (for review see H. Fritz and G. Wunderer, 1983, Drug Res. 33,479494).

Recently it has been shown, that homologues of aprotinin with other aminoacids in position 15, instead of lysine, are valuable proteinase inhibitors having modified effects and efficacies in comparison to aprotinin (DE-OS 33 39 693; H.R. Wenzel et al 1985 in Chemistry of Peptides and Proteins, Vol.3). These aprotinin homologues have strong inhibitory effects on the elastases from pancreas and leukocytes.

Such homologues of aprotinin can be used therapeutically in diseases in connection with excessive release of pancreatic elastase (pancreatitis), serum elastase (artherosclerosis), leukocyte elastase in acute and chronic inflammations with damage to connective tissue, in damage to vessel walls, in necrotic diseases and degeneration of lung tissue. Equally important is the part played by lysosomal enzymes, in particular leukocyte elastase, in inflammatory reactions due to immunological processes, for example rheumatoid arthritis, or as myocardial depressant factor and in shocksyndroms.

Although aprotinin and aprotinin homologues can be obtained from bovine organs and by semisynthetic conversion of the bovine trypsin inhibitor (Tschesche, H., Wenzel, H., Schmuck, R., Schnabel, E. DE-OS 3339 693 of 15 05 1985) the yields are relatively small.

It was perceived that the application of recombinant DNA and associated technologies would be a most effective way of providing the requisite large quantities of high quality aprotinin homologues. The goal was to produce Met-Aprotinin and Met-Aprotinin homologues, biologically active, as unfused products of recom binant DNA technology from a host organism.

DNA coding for polypeptides of known amino acid sequences may be prepared by using the genomic DNA sequence, the cDNA sequence which is complementary to the mRNA or by choosing codons according to the genetic code and preparation of a synthetic gene.

A partial DNA sequence of a bovine genomic clone from bovine pancreatic trypsin inhibitor gene was cloned by S. Anderson and I.B. Kingston, 1983, Proc. Natl. Acad. Sci. USA, 80, 683 & 842 to characterize a genomic clone for aprotinin. Recently two approximately 4 kilobase pair segments of the bovine genome containing a coding region for bovine pancreatic trypsin inhibitor (BPTI=aprotinin) and for bovine spleen inhibitor II have been published by Kingston, l.B. and Anderson, S. 1986, Biochem. J. 233, 44350.

It was shown recently that homologues of aprotinin could be prepared by DNA technology using a construction where the gene was fused to p-galactosidase.

It is normally much easier to cause the expression of mammalian proteins in a bacterial host by a fused gene approach combining the information for an endogeneous (homologue) polypeptide, that exists naturally in a certain genus or species of host cells, with the information of an heterologues polypeptide than to cause a bacterial host cell to express an unfused heterologues polypeptide. This is due to a variety of complex processes which are not completely understood. Some of the factors which are believed to impede the expression or accumuiation of a heterologous polypeptide in a host cell include the following: 1. The heterologous polypeptide may be degraded by the host cell.

2. The heterologous polypeptide may have toxic effects upon the cell.

The problem of degradation is believed to be more severe with regard to small heterologous polypeptides than with regard to large heterologous polypeptides.

Although a wide variety of heterologous polypeptides with more than about 100 aminoacid residues have been expressed in E. coli relatively few small heterologous polypeptides have been expressed in E.coli, despite numerous attempts to do so.

A number of efforts to overcome the problems of causing heterologous polypeptides to be expressed in bacterial cells have utilized fusion polypeptides.

The disadvantages of such approaches include the following: 1. The inserted structural sequence must be in the proper reading frame relative to the AUG start codon of the E. coli gene.

2. The heterologous polypeptide must be cleaved out from the fusion polypeptide by chemicals which modify and/or destroy certain aminoacid residues (for example methionine by cyanogen bromide; tryptophane by succinimide) and therefore the heterologous polypeptide must be free of such amino acid residues.

The copy number of the gene to be expressed is normally one for each expression vector. Theoretically it is possible to regulate the expression of several genes with one gene expression control system (N. Lee et al 1984, Nucl. Acids Res. 12, 6797-6812).

To increase the expression level of heterologous polypeptides orto stabilize an expressed polypeptide can be achieved by a multidomain polymer strategy as was shown by S-H Shen. 1984 Proc. Nat. Acad. Sci. USA 81, 46274631. He showed that multiple copies of the proinsulin coding sequence could increase the stability of products expressed with fused and unfused expression systems. His product was a multidomain polypeptide which must be cleaved by cyanogen bromide. The best expression level was achieved with three domains.

The mechanisms by which three abnormal proteins including heterologous polypeptides, are degraded in E. coli are not fully understood. Selection of certain host strains can stabilize some otherwise unstable polypeptides (Gottesmann, S. and Zipser, D, 1970 J. Bacteriol. 133, 844-851). However, these mutants do not stabilize each heterologous polypeptide expressed in E. coli.

One object of the present invention is to provide Met-aprotinin, Met-aprotinin homologues, nucleic acids encoding them, vectors incorporating the nucleic acids, novel modified cells transformed therewith and methods of obtaining Met-aprotinin and Met-aprotinin homologues. The term Met-aprotinin refers to an aprotinin wherein the amino acid in the zero (0) position is methionine.

For the gene construction it was most advantageous to choose codons for preparing synthetic genes with a proper design and which promise widespread application.

This is especially the case by constructing a synthetic master gene comprising of DNA blocks or cassettes terminated by unique recognition sites of restriction enzymes. Such a gene design allows easy modification or mutation of all DNA sequences within such DNA blocks and is enclosed herewith.

Homologues of aprotinin substituted with an additional methionine at the amino terminus were prepared by such a recombinant DNA technology. Such homologues of aprotinin as for example Val-15-, lIe-I 5-, Leu-15-, Phe-15-, Ala-15-, Arg-15-, Gly-15-, Ser-15-, Thr-15-, Trp-15-, Met-15- and Tyr-15 aprotinin have been found to be equivalent to the known aprotinin and its homologues.

A pharmaceutical composition in unit dosage or liquid form is described.

The present invention provides additionally a novel expression vector comprising regulating sequences as promoters, operators, ribosome binding sites, sequences for ribosome pooling, transcription terminators, further typical plasmid functions and genes or oligomers of genes causing bacterial host cells to express unfused heterologues polypeptides.

In a further aspect of the invention we provide a process for the production of gene products comprising oligomers of the gene, which are combined with DNA sequences for ribosome binding and coding for single (unfused) polypeptides by causing a bacterial host to express the oligomerised genes.

According to this aspect we have disclosed a procedure of oligomerisation of single genes with a special construction vector and we have disclosed a bacterial host which was modified genetically.

Briefdescription ofthe invention The present invention is related to microbially produced Met-aprotinin and Met-aprotinin homologues.

The microbially produced Met-aprotinin can be substituted at position 15 by any naturally occurring amino acid in particular with Arg, Val, lle, Leu, Phe, Gly, Ser, Met, Trp, Tyr and Ala.

The microbially produced Met-jrotinin can also be substituted additionally or alone in position 52 by Glu, Leu, Val, Thr or Ser. So one can obtain microbially produced Glu-52-Met-aprotinin or Val-1 5-Glu-52-Metaprotinin, or lle-1 5-Glu-52-Met-aprotinin or Leu-1 5-Glu-52-Met-aprotinin.

The DNA coding for Met-aprotinin can be substituted at codon 15 and/or codon 52 by a codon which is coding for any natural occurring aminoacid. The DNA may be substituted at codon 15 by a codon which is coding for an aminoacid selected from the group Arg, Val, Thr, lle, Leu, Phe, Gly, Ser, Trp, Tyr, Met and Ala. At codon 52 the DNA can be substituted by a codon which is coding for an aminoacid selected from the group Glu, Leu, Val, Thr and Ser.

The invention further relates to a DNA coding for a protein or a polypeptide provided upstream with DNA having the sequence

or functional equivalents thereof and also to a DNA coding for a protein or a polypeptide provided downstream with DNA having the sequence

or functional equivalents thereof.

In particular the invention relates to a DNA coding for a protein or a polypeptide provided upstream with DNA having the sequence

and downstream with DNA having the sequence

or functional equivalents thereof.

For example a polypeptide the DNA is coding for may be insulin, proinsulin, vasopressin, oxytocin, ribonuclease, growth hormone, Met-aprotinin and homologues of Met-aprotinin.

Another object of the present invention is an expression system for a protein or for a polypeptide comprising of one or more copies of a DNA coding for a protein or a polypeptide wherein any copy of the coding DNA is provided upstream with DNA having the sequence

and downstream with DNA having the sequence

and functional equivalents of said sequences.

A DNA having the sequence

is described. This DNA is a DNA sequence having a function as adaptor.

Further a DNA having the sequence

is described. This DNA is a DNA sequence having a function as linking sequence. An another object of the invention is a DNA having the sequence

This DNA is coding for a short peptide and has a function as expression enhancer.

A DNA is described having a function as operator with the sequence

and a DNA having a function as promoter with the sequence

It is known to a skilled artisan in this field, that the substitution of one or more bases in the described sequence is not necessarily related to a loss or alteration of function. Therefore the invention is also related to functional equivalents of said sequences.

Both DNA sequences may be combined to a DNA having a function as promoter and operator with the sequence

or functional equivalents of said sequence.

The expression system described above may be provided upstream with the DNA having a function as promotor and operator and then incorporated into a plasmid.

The term "expression system" refers to a DNA sequence comprising a structural sequence which is coding for a protein or polypeptide and comprising regulatory sequences such as promotor- and operator gene.

This plasmid is useful for transforming a microorganism in particular an E. coli microorganism.

Most preferred is an E. coli BR 17 microorganism which is transformed with the plasmid and having the DSM designation DSM 3685 and DSM 3686.

The invention is related to plasmids having the restriction cleavage map of piWiT 10 wL1 and piWiT 11 and also to the plasmids piWiT 10 wi7 and piWiT 11.

The plasmids piWiT 10 wL1 and piWiT 11 are useful in a process for production of proteins and polypeptides in particular in a process for production of a polypeptide selected from the group insulin, proinsulin, vasopressin, oxtocin, ribonuclease, growth hormone, Met-aprotinin and homologues of Met-aprotinin.

The microorganisms used in the present invention were deposited with Deutsche Sammlung von Mikroorganismen, Griesbachstrasse 8, D-3400 Göttingen, The DSM-designation of the host microorganism E.

coliBR 17is DSM 3684.

The DSM-designation of the host microorganism E. coli BR 17transformed with the plasmids piWiT10 wil 1 and piWiT10 wi7 is DSM 3685 and DSM 3686.

Brief description ofthe drawings Figure la. Design of the synthetic gene ib. DNA sequence of DNA Fragments Figure 2a. Construction vector piWi T9 2b. Partial DNA sequences of piWi T9 Figure 3. Amplification scheme for oligomerisation of the synthetic gene Figure 4a. Expression vector piWi T 11 4b. Partial DNA sequence of piWi T 11 Figure 5a. Plasmid PiWi T10 wL1 5b. Partial DNA sequences of piWi T10 wL1 Figure 6. SDS Protein Gel Electrophoresis Figure 7. SDS Protein Gel Electrophoresis Figure 8. Renaturation of Met-Aprotinin Figure 9. Western Blot Figure 10. Chromatography of Met-Aprotinin.

In the following the strategy for construction and selection, the preparation of DNA fragments coding for Met-aprotinin, Met-aprotinin homologues, construction vectors, expression vectors, expression plasmids, microorganisms transformed therewith and the microbial production of Met-aprotinin and Met-aprotinin homologues, pharmaceutical compositions are disclosed.

Standard methods for recombinant DNA work were used as described by Maniatis et al, 1982, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, sometimes with modifications as described hereinafter.

Strategy for construction and selection of DNA fragments coding for a synthetic inhibitor gene The known protein sequence and the genetic code of aprotinin and aprotinin homologues were used to determine a DNA sequence coding for such polypeptides.

The degeneracy of the genetic code permits substantial freedom in the choice of codons for any given amino acid sequence.

All possible base substitutions among the codons designating the amino acid sequence of th is protein were determined. According to this, all potential restriction sites located within the possible DNA sequences were determined.

The codon choice for master genes were guided by the following considerations: First, codons and fragments were selected, and fragment assembly was designed, so as to avoid undue complementarity of the fragments.

Secondly, the region upstream of the initiation codon contains a ribosomal binding site, for codons around the region of the initiation codon those were chosen which enhance ribosome binding (Lit G.F.E. Scherer et al, 1980, Nucl. Acids Res. 8,38953907).

Thirdly, restriction sites were chosen necessary for facilitating verification of transformants or base substitutions by replacement of appropriate fragments with other fragments so that one can produce easily modifications of Aprotinin, and for creating a broad range of possibilities for cloning and expression work.

Fourthly, a majority of the codons chosen are those preferred in the expression of microbial genomes (see H. Grosjean and W. Fiers, Gene, 18(1982)192-209; M. Gouy and C. Gautier, Nucleic Acids Research, (1982) 7055-7074).

The principal design for synthetic Aprotinin genes and their homologues are shown in Figure la.

Construction of synthetic genes The synthetic genes for Met-aprotinin homologues were constructed via a master gene by the assembly of 14 purified oligonucleotides. This construction was done as shown in Figure 1 a. The DNA sequences of the fragments for the synthetic inhibitor are shown in Figure 1 b.

The master gene includes a robosome binding site, the initiation codon ATG, the termination codon, TAG, the 5' terminal restriction site for Xba I, the 3' terminal restriction site for Hind Ill, additionally the internal restriction sites for Xho I, Apa I, Stu I, Acc I, Pst I, Sst II and Sph I. These sites especially the internal ones facilitate the cloning of the coding sequence, the modification of the master gene by exchanging DNA fragments which code for other amino acids or which have another codon usage. The total spectra of protein engineering is possible with such a construction.

To construct genes for Met-aprotinin homologues only a restriction fragment with an appropriate DNA sequence has to be exchanged. Sequences for such fragments which will code for amino acid alterations at positions 15, for example Apa l-Stu I fragments, were given in Figure 1 b.

Construction vectorpiWi T9 and amplification of the genes The plasmid chosen for experimental Met-aprotinin cloning was piWi T9 (see also Figure 2a.) The construction vector is composed of two fragments from pBR 322 carrying the origin of replication and the tetracycline resistance genes respectively, a fragment of bacteriophage fd 11 DNA carrying a transcription termination signal, and a fragment from the lac operon of E. coli extending from the C-terminus of the lac gene to lust beyond the lac Z gene, the entire lac promotor-operator region and the first five codons of the lac Z gene have been deleted and replaced by a short stretch of synthetic DNA. The synthetic DNA possesses constituitive promoter activity directed towards the lac Z gene and it contains the cloning sites Xba I, Xma I and Hind Ill between the promoter and the lac Z gene.Any DNA cloned into any of these three sites is transcribed from the synthetic promoter, whether it will be translated, too, depends on the sequence. The lac Z gene of piwi T9 is not translated such that active i3-galactosidase is produced unless additional DNA is cloned into any of the cloning sites which provides an initiation codon in phase for the lac Z gene. The DNA sequences of important regions of construction vector piWi T9 are given in Figure 2b.

For oligomerisation of genes the synthetic gene (see Figure 1 a) has been cloned between the Xba I and Hind Ill sites of the construction vector piWi T9. For a tandem duplication of the synthetic gene a Bam Hl-Hind Ill fragment containing one copy of the gene located upstream of the Hind Ill site is ligated in the presence of an Hind Ill-Xba I adaptor to a Xba l-Bam HI fragment containing one copy of the gene located downstream of the Xba I site (see Figure 3). The Hind llI-Xba I adaptor fits into the sticky ends produced by Hind Ill and Xba I but does not regenerate these sites. The resulting plasmid piWi T9 TL2 has a unique Xba I site upstream of the first gene and a unique Hind Ill site downstream of the second gene.Thus doubling may be repeated by the same series of reactions to yield further plasmids containing 4,8, 16 and 32 tandemly repeated genes, each preceeded by its own ribosomal binding site. Since the lac Z gene is no longer needed the 3 kb Eco RI fragment is deleted from the plasmid.

Expression veoforpiWi T1 > and expression plasmid piWi TlO wL 1 For expression of Met-aprotinin and Met-aprotinin homologues a plasmid was constructed in which the appropriate genes could be expressed under the control of a strong promoter-operator and which comprises further DNA sequences very useful for expression of heterologous proteins and polypeptides. The physical map of the expression vector piWi T1 1 is shown in Figure 4a.

Expression vector piWi T1 1 is very similar to construction vector piWi T9, they share a common 3,3 Kb long Xho 1-Eco RI fragment comprising 36 bp lac I DNA, 883 bp pBR 322 DNA (origine of replication), 2067 (+8) bp pBR 322 DNA (tetracycline resistance genes), 9 bp polylinker, 332 bp fd 11 DNA transcription termination signal, 8 bp Bam HI octalinker and 68 bp lac Z DNA. In order to enhance expression a synthetic DNA fragment was constructed and ligated between the Eco Rl-Xho I sites. The sequence of this synthetic fragment is shown in Figure 4b.The fragment comprises sequences for a strong promoter, a RNA start, a lac repressor binding site (operator), a ribosomal binding site, a protein start codon, a short coding region containing five unique restriction sites for cloning (Hind Ill, Xba I, Pst I, Bgl II and Xma I).

For expression purposes the small Xho I-Xba I fragment of a construction vector with oligomers of the synthetic gene, described above, was than exchanged by the small Xho l-Xba I fragment of piWi T1 1, the latter containing promoter, operator, ribosome binding site, and a coding sequence for a hexapeptide. A resulting recombinant plasmid is for example the expression plasmid piWi T10 wL1 with oligomers of the Met-aprotinin gene.

DNA sequences of important regions of expression plasmid are given in Figure 5b.

The microorganism A large number of various microorganisms are known in the art as being suitable for transformation. That is, those unicellular organisms which are capable for being grown in cultures or fermentation. Preferred organisms for transformation include bacteria, yeasts and fungi.

The organism chosen for the work disclosed here, was E. coli.

Avert suitable strain was E. coli B lamda R-, mal- which was made recA by a method described by J.H.

Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory.

Preparation of Met-aprotinin Many proteins synthesized in large quantities in bacteria accumulate in an insoluble form (D.C. Williams, R.M. van Frank, J.B. Burnett, W.L. Muth, 1982, Science 215, 687).

These insoluble proteins are called inclusion bodies. They may usually be solubilized only with denaturants and therefore could easily be purified from other cell proteins.

E.coli strain BR17 was transformed with the plasmid piWi T10 wL1 which encodes the aprotinin gene fourteen times downstream from an E.coli promoter, operator and ribosome binding site. An E.coli strain BR17 piWi T10 wL1 overnight culture was centrifuged, the pellet was resuspended in a breaking buffer and the cells were lysed by a French press.

The cell-lysate was centrifuged for recovering the inclusion bodies. The pellet was washed two times with the breaking buffer.

The purification steps were checked by SDS-polyacrylamide gel electrophoresis according to Laemmli (U.K.

Laemmli 1970, Nature 277, 680-685); Figure 7.

The inactive inhibitor was refolded by the procedure according to Creighton (T.E. Creighton, Proceedings of Genex-UCLA Symposium 1985, Kingstones); Figure 8. The active inhibitor could be detected by trypsin assay and western blot analysis; Figure 9.

The active inhibitor was further purified by chromatography on a trypsin sepharose column according to Fritz (H. Fritz, M. Gebhardt, R. Meister, K. lllchmann, K. HochstraBer, 1970, Hoppe-Seyler's Z. Physiol. Chem.

351,571-574); Figure 10.

The inhibitor was then characterized by microsequencing according to Hewick (R.M. Hewick, M.W.

Hunkapillar, L.E. Hood, W.l. Dreger 1981, J. Biol. Chem. 256,7970-7997).

The first 20 residues from the N-terminus were determined. Without the methionine at the zero position the amino acid sequence was completely identical with that of aprotinin (table 1). The amino acid composition of the inhibitor shows the expected values (table 2). A comparison of aprotinin and Met-aprotinin by trypsin inhibitory activity shows similar values (table 3).

All these experiments show that it is possible to produce Met-aprotinin in E.coli and renature it to the active inhibitor.

Preparation of Met-lle-15-aprotinin Met-lle-15-aprotinin can be prepared in a similar way in E.coli as described for Met-aprotinin (see also Figure 6).

The inhibitory activity was measured by an elastase inhibitory assay (K. Nakajima, M. Zimmerman, J.C.

Powers, M.J. Castillo, B.M. Ashe 1979, J. Biol. Chem. 254, 4027).

The inhibitor was characterized by amino acid analysis and N-terminal sequencing (table 1,2).

All other derivatives of aprotinin could be prepared in a similar way as described for Met-aprotinin and Met-lle-1 5-aprotinin.

Pharmaceutical Preparations The present invention includes pharmaceutical preparations which in addition to non-toxic, inert pharmaceutically suitable excipients contain one or more compounds according to the invention or which consist of one or more active compounds according to the invention, and processes for the production of these preparations.

The present invention also includes pharmaceutical preparations in dosage units. This means that the preparations are in the form of individual parts, for example tablets, coated tablets, capsules, pills, suppositories and ampoules, of which the content of active compound corresponds to a fraction or a multiple of an individual dose. The dosage units can contain, for example, one, two, three or four individual doses or one half, one third or one quarter of an individual dose. An individual dose preferably contains the amount of active compound which is given in one administration and which usually corresponds to a whole, a half or a third or a quarter of a daily dose.

By non-toxic, inert pharmaceutically suitable excipients there are to be understood solid, semi-solid or liquid diluents, fillers and formulation auxiliaries of all kinds.

Tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays may be mentioned as preferred pharmaceutical preparations.

Tablets, coated tablets, capsules, pills and granules can contain the active compound or compounds alongside the customary excipients such as (a) fillers and extenders, for example starches, lactose, sucrose, glucose, mannitol and silica, (b) binders, for example carboxymethylcellulose, alignates, gelatin and polyvinylpyrrolidone, (c) humectants, for example glycerol, (d) disintegrants, for example agar-agar, calcium carbonate and sodium carbonate, (e) solution retarders, for example quaternary ammonium compounds, (g) wetting agents, for example cetyl alcohol and glycerol monostearate, (h) adsorbents, for example kaolin and bentonite and (i) lubricants, for example talc, calcium and magnesium stearate and solid polyethylene glycols, or mixtures of the substances listed under (a) to (i).

The tablets, coated tablets, capsules, pills and granules can be provided with the customary coatings and shells, optionally containing opacifying agents, and can also be of such composition that they release the active compound or compounds only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner, examples of embedding compositions which can be used being polymeric substances and waxes.

The active compound or compounds, optionally together with one or more of the abovementioned excipients, can also be in a microencapsulated form.

Suppositories can contain, in addition to the active compound or compounds, the customary water-soluble or water-insoluble excipients, for example polyethylene glycols, fats, for example cacao fat and higher esters (for example C14 alcohol with C16 fatty acid) or mixtures of these substances.

Ointments, pastes, creams and gels can contain the customary excipients in addition to the active compound or compounds, for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, taic and zinc oxide, or mixtures of these substances.

Powders and sprays can contain the customary excipients in addition to the active compound or compounds, for example lactose, talc, silica, aluminium hydroxide, calcium silicate and polyamide powders, or mixtures of these substances. Sprays can additionally contain the customary propellants, for example chlorofluorohydrocarbons.

Solutions and emulsions can contain the customary excipients in addition to the active compound or compounds, such as solvents, solubilizing agents and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cotton seed oil, groundnut oil, maize germ oil, olive oil, castor oil and sesame oii, glycerol, glycerolformal, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances.

For parenteral administration, the solutions and emulsions can also be in a sterile form which is isotonic with blood.

Suspension can contain the customary excipients in addition to the active compound or compounds, such as liquid diluents, for example water, ethyl alcohol or propylene glycol, suspending agents, for example ethoxylated isostea ryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

The formulation forms mentioned can also contain dyestuffs, preservatives and additives which improve the odour and flavour, for example peppermint oil and eucalyptus oil, and sweeteners, for example saccharin.

The therapeuticaliy active compounds should preferably be present in the abovementioned pharmaceutical preparations in a concentration of about 0.1 to 99.5, preferably of about 0.5 to 95, percent by weight of the total mixture.

The abovementioned pharmaceutical preparations can also contain other pharmaceutical active compounds in addition to the compounds according to the invention.

The abovementioned pharmaceutical preparations are manufactured in the usual manner according to known methods, for example by mixing the active compound or compounds with the excipient or excipients.

The active compounds or the pharmaceutical preparations can be administered locally, orally, parenterally, intraperitoneally and/or rectally, preferably orally or parenterally, such as intravenously or intramuscularly.

In general, it has proved advantageous both in human medicine and in veterinary medicine to administer the active compound or compounds according to the invention in total amounts of about 0.5 to about 500, preferably 5 to 100, mg/kg of body weight every 24 hours, optionally in the form of several individual administrations, in order to achieve the desired results. An individual administration contains the active compound or compounds according to the invention preferably in amounts of about 1 to about 250, in particular 3 to 60, mg/kg of body weight.However, it can be necessary to deviate from the dosages mentioned and, in particular, to do so as a function of the nature and body weight of the subject to be treated, the nature and severity of the illness, the nature of the preparation and of the administration of the medicine, and the time or interval over which the administration takes place.

Thus, it can suffice in some cases to manage with less than the abovementioned amount of active compound, whilst in other cases the abovementioned amount of active compound must be exceeded. The particular required optimum dosage and the type of administration of the active compounds can easily be decided by anyone skilled in the art on the basis of his expert knowledge.

EXAMPLES Example 1 Synthesis and Purification of DNA fragments coding for Met-aprotinin and Met4le-15-aprotinin The oligonucleotides which comprise the gene were prepared using solid-phase synthetic methods. The synthetic scheme for the oligomers was as outlined and utilized proton activated, protected 2'deoxyribonucleotide phosphoramidites. All sequential steps were performed in an automated manner on an Applied Biosystems Model 380 DNA Synthesizer using protected nucleotides, solvents, chemicals and reagents obtained from this manufacturer. The solid-phase support, also from the same manufacturer, was controlied pore glass to which the starting 3'nucleotide was already attached. Certain modifications were introduced into the automated reaction cycle in accordance with the Manufacturers Operating Instructions and Users Bulletins.Upon completion of the synthesis, the oligomers were deblocked and cleaved from the solid support within the DNA synthesizer according to the manufacturer's recommendations.

Removal of the blocking groups was completed by heating the aqueous solution containing the oligomer with concentrated ammonium hydroxide at 55"C from 4 to 24 hours in a sealed vial. The resulting solution was evaporated, the residue dissolved in 0.01 M triethylammonium bicarbonate buffer, pH 7.0 (TEAB buffer). This solution was chromatographed over Sephadex-G 509 Gel Filtration Resin. This column was prepared in and eluted with the same TEAB buffer. Material eluting with the void volume was pooled and the solution evaporated.

A portion of the residue (10 to 40% of the absorbance units at 260 nm), dissolved in loading buffer (composition: 0.1% Bromophenol Blue, o.1 % Xylene Cyanol, 10 mM disodium EDTA, in formamide) was further purified by electrophoresis on polyacrylamide gels. The gel size was 18x32 cm with a thickness of 1.5 mm. The weli size for each oligomer purified in this manner was 2 to 5 cm in width and up to five oligomers were purified using a single gel. The concentration of acrylamide in the gel varied from 14to 20%, depending on the chain length of the desired product. For longer oligomers, the 14% acrylamide gel is preferred, while shorter oligomers were purified on up to 20% acrylamide gels. The gels also contained 7 M urea and Tris-borate-EDTA buffer (0.1 M Tris, 0.1 M Borate, 2 mM EDTA pH 8.3).The running buffer was the same Tris-borate-EDTA mixture. Electrophoresis was carried out at 20 to 60 watts, constant power, for from 18 to 6 hours. Such standardized techniques are available in various User information Bulletins available from Applied Biosystems.

Following completion of the electrophoresis, the gel is encased in plastic wrap and the oligomers visualized by shadowing with ultraviolet light. This shadowing is accomplished by placing the wrapped gel on a fluorescent thin layer chromatography plate and viewing the gel with a short wave length ultraviolet light source. The desired product appears as the slowest migrating, major blue DNA fragment by this shadowing technique. The desired band is exised from the gel. The DNA oligomer is eluted from the gel slice onto powdered diethylaminoethyl (DEAE) cellulose using an EpiGene D-Gele electrophoresis apparatus. The oligomer is recovered from the cellulose by elution with 1 M TEAB buffer.The buffer solution containing the oligomer is evaporated, the residue is dissolved in 0.01 M TEAB buffer, and then desalted by passage over a column of Sephadex-G 509 as described previously. The material eluting in the void volume is pooled and lyophilized to give the final product.

Using the procedures outlined above, about 0.5 to 5.0 A260 units of each of the purified oligomers was obtained.

EXAMPLE 2 Construction ofsynthetic genes The synthetic oligonucleotides purified as described in example 1 were incubated with polynucleotide kinase and adenosinetriphosphate (ATP) under standard conditions (Maniatis et al). One tenth of each sample was incubated with 8[32P] ATP instead of ATP. Both samples were mixed afterwards and subjected to electrophoresis as described in example 1. The labeled bands were cut from the gel eluted and purified as before.

The 14 oligonucleotides (Figure 1 a,b) were then mixed in301l1 of doubly concentrated ligase buffer (Maniatis) and allowed to anneal for 20 min at 40 C and 30 min at room temperature. Then 2,al 100 mM ATP pH 7,5,6 ill T4DNA-ligase and 22 FI water were added and ligation allowed to proceed overnight at about 10 C.

The products of the ligation reaction were then digested with Xbal and Hindlll to yield single synthetic gene segments of an overall length of 204 bp comprising an open Xbal site, a ribosomal binding site, an initiation codon ATG, the aprotinin coding sequence, a termination codon TAG, and an open Hindlll site.

EXAMPLE 3 Construction of recombinant plasmids Construction vector DNA (Figure 2a,b) was cut with Xbal and Hindlll (Maniatis), mixed with the synthetic gene segments, prepared as described in example 2, and ligated overnight as before. The sample was then used to transform E.coli Su3 [lac-pro]iX met- argarn supF thi-) according to the method of Hahpahan (D.

Hahnahan, J. Mol. Biol., 1983, 166, 557-580). Cells were spread on rich plates supplemented with 10 Fg tetracycline/ml and 0.005% x-gal (5-bromo-4-chloro-3-indolyl- -D-galactoside J. Miler, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and incubated overnight at 37"C. Plasmid DNA from blue colonies was extracted (Maniatis) and screened for the presence of the restriction enzyme recognition sites present on the synthetic gene segment. Finally, the DNA sequence was determined according to the method of Maxam and Gilbert (A. Maxam & W. Gilbert, Meth. Enzymol., 1983,65, 499-560).

EXAMPLE 4 Construction ofmultimers ofthe gene piWiT9L1 DNA prepared as described in the previous example 3 was used to prepare multimers of the aprotinin gene. Plasmid DNA was digested with Hindlll and BamHI and the smaller fragment containing the 3'end of the tetracycline resistance genes, the origine of replication, the promoter and the met-aprotinin gene was purified (Maniatis). A second sample of plasmid DNA was digested with Xbal and BamHI and the larger fragment containing again the Met-aprotinin gene, the lacZ gene, the transcription termination signal and the 5'end of the tetracycline resistance genes was purified as above.Both fragments were ligated (see example 2) in the presence of the purified adapter molecules

and transformed into E.coli BR17 (see example 5). Since the adaptor molecule possesses protruding ends that fit into a Hindlll site and a Xbal site respectively without regenerating either site the resulting new plasmid with two tandemly repeated aprotinin genes again has only one unique Xbal site upstream of the ribosomal binding site of the first aprotinin gene and also only one unique Hinglll site downstream of the termination codon of the second Met-aprotinin gene. Thus, the same series of reactions can be repeated using this new piWiT9L2 DNA to yield another plasmid carrying fourtandemly repeated Met-aprotinin genes, each preceeded by its own ribosomal binding site.

Prior to a third duplication of the now four approtinin genes the 3Kb EcoRI fragment was deleted from the plasmid in order to reduce its overall size (Figure 3). After the third doubling we happened to pick up plasmid with seven aprotinin genes instead of eight. Plasmids with odd numbers of genes were usually found among the products of such a ligation reaction at low frequency. We attribute their rise to a partial denaturation of the DNA fragments during the melting of the seaplaque agarose (Maniatis) during the purification proceedure.

We did not observe any deviation from a given number of genes after further propagation of these plasmids in E.coli BR17.

Thus, the fourth duplication performed as described above yielded a plasmid with 14 aprotinin genes assembled in an artificial operon that could be transcribed into a polycistronic mRNAwhich could then be translated into 14 separate identical aprotinin peptides.

EXAMPLE 5 Construction ofexpression plasmids The influence of the S'untranslated region of the mRNA the socalled leader sequence of the mRNA on the rate of translation of the gene or genes encoded on that mRNA is scarcely understood. We tested several such leader sequences for their efficiency of translation and obtained different results with different genes. The one leader sequence that gave the best results with our synthetic aprotinin gene is the sequence present in the expression vector piWiT11 (Figure 4a, b).

Afragment containing 14 identical aprotinin genes, each preceeded by its own ribosomal binding site was excised from the construction plasmid with Xbal and EcoRI and inserted between the Xbal and EcoRI sites of the poly linekr of the expression vector piWiT1 1 (Maniatis). Figure 5b shows that a hexapeptide is translated from the mRNA in addition to the 14 aprotinin peptides. Such small peptides have been reported to enhance translation of the following gene(s) (B.E. Schoner et al, Proc. Natl. Acad. Sci. USA, 1984, 81, 5403-5407).

EXAMPLE 6 Construction ofthe E. coli host strain BR17 E. coli B1.8 has been isolated at Berkeley. Its phenotype given as mal and XR was verified. It was subjected to the following procedures: 1 ml saturated culture was centrifuged for a minute in an Eppendorf centrifuge, the pelleted cells were resuspended in 1 ml 0.9% NaCI solution. 0.2 ml of this suspension were spread on a minimal glucose plate supplemented with thymine to a final concentration of 0.05% and trimethoprim to a final concentration of 0.001% in order to select for thyA mutants. One of the colonies that had grown on this plate after 30 h incubation at 37was purified on another minimal glucose agar plate with thymine and trimethoprim and called B13.From this plate a single colony was chosen and mated with E. coli RZ423 a recA deficient Hfr strain which was also lac-. The mating mixture was plated on minimal lactose plates. The female B1 3 could not grow on these plates because of its thymine auxotrophy. The male RZ423 was not able to use lactose as carbon source. Thus only those B13 cells that - after mating with RZ423 - had stably incorporated that part of the RZ423 chromosome that coded for a functional thyA gene could grow. 24 such colonies were purified and individually tested for being mal- (on EMBmal plates) and for recA~ (by comparing growth in the presence of methyl methane sulfonate.All of the tested colonies were mal-, thus it was ensured that they were not accidental lac+ revertants of RZ423, three of them showed reduced growth in the presence of rriethyl methane sulfonate which indicated that these bacteria had incorporated the recA~ allele from RZ423 simultaneously with the closely linked wildtype thyA gene. They had not acquired the Hfr genotype as they proved to be unable to plate the male specific bacteriophage M13.

One of the three clones was used as host strain for the expression plasmids carrying the inhibitor genes. It was called E. coli BR17 and deposited at the "Deutsche Sammlung Mikroorganismen" Göttingen, DSM No.

3684 EXAMPLE 7 Preparation of Met-aprotinin solutions Buffer A, pH 8.2 50 mmol Tris-HCI 1 mmol EDTA Adjust to pH 8.2 Buffer B, pH 8.2 8 M Urea 1 mmol Bis-(2-hydroxyethyl)-disulfide 1 mmol 2-Mercaptoethanol in Buffer A; Buffer C, pH 8.2 1 mmol Bis-(2-hydroxyethyl-disulfide) 1 mmol 2-Mercaptoethanol in Buffer A; BufferD,pH8.2 0.6 mol Sodiumchloride in Buffer A.

For preparation purposes 5 1 of an E. coli overnight culture of the strain BR17 piWI T10 wL1 were centrifuged for 15 min. at 8.000 rpm. The cell pellet, about lOg, in weight, was resuspended in 25 ml 0,1 M Tris. HCI, 0.001 M EDTA and 0.1 mg Lysozyme/ml. The cells were lysed by a French press.

The cell lysate was centrifuged for 20 min. at 20.000 rpm. The supernatant was discarded. The pellet was washed two times with 20 mi of breaking buffer without lysozyme.

The pellet was dissolved in 10 ml of Buffer B. The solution was reduced for 1 hat 50"C under nitrogen atmosphere. The solution was then applied to an Econocolumn (25+10 mm) filled with about 10 ml CM-Sepharose Fast FlowR. The column was equilibrated with buffer B. The column was washed with buffer B until the baseline was stable. In a first linear gradient elution the column was developed with 100 ml buffer B and 100 ml of buffer C. Before the second linear elution gradient was applied the column was washed with buffer A until the baseline was stable.

The second gradient was formed with 100 ml buffer A and 100 ml buffer D. The peal fractions were tested for trypsin inhibitory activity, ELISA and Western blot.

In a series of experiments the yield estimated by the different tests was in the range of 5-20 mg.

Purification or Met-aprotinin by chromatography on a trypsin sepharose column: 5 mg of renatured Met-aprotinin was dissolved in 1 ml O. 1 M Tris-buffer pH 6.5 and applied to an Econo-column filled with 30 ml trypsin sepharose.

The column had been previously equilibrated with 0.1 M Tris-buffer pH 6.5. The column was then washed with the 0.1 M Tris-buffer pH 6.5 until the baseline was stable.

The column was washed with five volumes of 0.2 M Na-acetate buffer pH 4 and then with five volumes of 0.2 M acetic acid/HCI pH 1.8.

The different fractions were neutralised and their activities were determined by the trypsin inhibition assay.

The active fractions were pooled and were then desalted by dialysis against dest. water. The resulting inhibitor was characterized by N-terminal sequencing and amino acid analysis.

EXAMPLE 8 Preparation ofMet-lle-15aprotinin The fermentation of E. coli transformed with the plasmid piWi T10 Wi7 and the renaturation of the Met-lle-15-aprotinin was performed according to the same procedures as described in Example 7, with the difference that the activity was tested by an elastase inhibition assay instead of the trypsin inhibitor assay. In a series of experiments the yield estimated by the different tests was in the range of 5-10 mg.

The renaturated Met-lIe-i 5-aprotinin was further purified by chromatography on an anti-aprotinin polyclonal antibody column.

1 mg of Met-lle-15-aprotinin was dissolved in 2 ml of 0.05 M phosphate buffer, 1 M NaCI pH 7.0 and applied to an Econo Column (41+100 mm) filled with 40 ml polyclonal anti-aprotinin antibody sepharose.

The column was washed with three volumes starting buffer. The elastase inhibitor Met-lle-l 5-aprotinin was desorbed by 0.2 M KCI/HCI pH 2.2. The active fractions were pooled and dialysed against dest. water. The inhibitor was obtained by lyophilization (about 600 ig by weight). 1 nmol of the inhibitor was loaded to the gas phase sequenator and characterised by N-terminal sequencing, another nmol was used for amino acid analysis.

TABLE 1 Amino acid sequencing of Met-aprotinin and Met-lle- 15-apro tin in 1. Met-aprotinin; about 1 nmol of the substance was sequenced ver 20 cycles 1 5 10 Met-Arg-Pro-Asp-Phe-Cys-Leu-Giu-Pro-Pro-Tyr-Th r-Gly-Pro 15 19 Cys-Lys-Ala-Arg-lle-lle2.Met-lle-15-aprotinin; about 1 nmol of the substance was sequenced over 20 cycles 1 5 10 Met-Arg-Pro-Asp-Phe-Cys-Leu-G lu-Pro-Pro-Tyr-Th r-Gly-Pro- 15 19 Cys-lle-Ala-Arg-lle-lle TABLE 2 Amino acid analysis ofMet-aprotinin, Met-lle- 15-aprotinin and aprotinin Amino acid Met-aprotinin Met-lle-15-aprotinin a pro tin in Asp 5,06 (5) 5,12 (5) 4,90 (5) Thr 2,71 (3) 2,80 (3) 2,89 (3) Ser 0,98(1) 0,95(1) 0,94(1) Glu 3,15(3) 3,20(3) 3,05(3) Gly 5,97 (6) 5,92 (6) 6,05 (6) Ala 6,00 (6) 6,00 (6) 6,00 (6) Val 0,94(1) 1,02(1) 1,04(1) Met 1,60 (2) 1,76 (2) 0,95 (1) lle 1,36 (2) 2,45 (3) 1,29 (2) Leu 2,02 (2) 2,08(2) 2,10 (2) Thyr 4,01(4) 3,92(4) 3,85(4) Phe 3,98 (4) 3,94 (4) 3,86 (4) Lys 4,19 (4) 3,08 (3) 3,99 (4) Arg 6,09(6) 5,82 (6) 5,90(6) The amino acids were measured after the post column derivatisation with o-phthalaldehyde. Cys and Pro were not determined.

TABLE 3 Comparison of the inhibitory activity of aprotinin and Met-aprotinin. The inhibition oftryptic activity was measured by means of N-Benzoyl-DL-arginin-p-nitroanilide (BANA).

Aprotinin Gene product Amount of inhi- Amount of inhibitor per assay O.D./10 min bitor per assay O.D./10 min.

0 ng 0.444 0 ng 0.466 100 ng 0.391 68 ng 0.398 200 ng 0.261 136 ng 0.355 400 ng 0.073 272 ng 0.268 Slope: -.00096 Slope: -.00071 Intercept with Intercept with abscissa: 479 abscissa: 642 Correlation coeffi- Correlation coefficient: .993 cient: .994 Legends to Figure 6: SDS-gel electrophoresis (15% polyacrylamide) of E.coli proteins from E.coli BR 17 [piWiT10wL1], E.Coli BR 17 (piWi T10 wi7] and E.coli BR 17 [piWiTlOw] 1. piWi T10 wi7,109 cells; 2. piWi T10wL1,199 cells; 3. soluble proteins piWi T10 wi7; 4. soluble proteins piWi T1OwL1; 5. soluble proteins piWi Ti Ow; 6. piWi Trow, 2x108 cells; 8. piWiT10wL1,2x108cells.

Figure 7: SDS-gel electrophoresis (10-20% polyacrylamide) of the proteins from the E.coli strain BR 17 piWi T10 wL1.

Figure 9: Recombinant protinin from renaturation and trypsin sepharose chromatography was subjected to electrophoresis on Na-DodSO4-gradient (10-20%) polyacrylamide gel and then western blotted according to Towbin.

Lane 1 aprotinin; lane 2 recombinant aprotinin after renaturation; lane 3 recombinant aprotinin after trypsin sepharose chromatography.

Claims (44)

1. Microbially produced Met-aprotinin and Met-aprotinin homologues.

2. Microbially produced Met-aprotinin substituted at position 15 by any naturally ocurring amino acid.

3. Microbially produced Met-aprotinin substituted in position 15 by Arg, Val, Thr, lle, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala.

4. Microbially produced Met-aprotinin according to any of claims 1 to 3 substituted in position 52 by Glu, Leu, Val, Thr or Ser.

5. Microbially produced Glu-52-Met-aprotinin.

6. Microbially produced Val-1 5-Glu-52-Met-aprotinin.

7. Microbially produced lle-1 5-Glu-52-Met-aprotinin.

8. Microbially produced Leu-1 5-Glu-52-Met-aprotinin.

9. DNA having the sequence

1 5 10 met arg pro asp phe cys eu glu pro pro tyr thr gly prp ATG AGA CCA GAT TTC TGC qTC GAG CCG CCG TAC ACT GGG CCU TAC TCT GGT CTA AAG ACG GAG CM GGC GGC ATG ATG C C1GGG XhoI ApaI 15 20 25 cys lys ala arg ile ile arg tyr phe tyr asN ala lys TGC AAA GCT CGT ATC ATC COT TAC TTC TAC AAT GCA AAG ACG TTT CGA GGA TAG TAG GCA ATG AAG ATG TTA CGT TTC 30 35 ala g y leu cys glN thr phe V 1 tyr gly gly cys arg GCA G C CTG TGT CAG ACC TTC GiA TAC GGC GGC TGC A A COT CgG GAG ACA GTC TGG AAG CAT TO CCG CCG ACO TCT Stu I AccI PstI 40 45 50 ala lys arg asN asN phe lys ser ala glu asp GCT AAG COT AAC AAC TTC AAA TCC GCI3 GAA GAC CGA TTC GCA TTG TTG AAG TTT AGG1CGC CTT CTG SstII 55 cys met arg thr cys gly gly ala TGC ATG CGT ACT TGC GGT GGT GCT TAG AC TAC GCA TGA ACG CCA CCA COA ATC SphI and functional equivalents thereof.

10. The DNA of claim 9 wherein codon 15 and/or codon 52 is substituted by a codon which is coding for any natural occuring aminoacid.

11. The DNA of any of claims 9 and 10 wherein codon 15 is substituted by a codon which is coding for an aminoacid selected from the group Arg, Val, THr, lle, Leu, Phe, Gly, Met, Ser, Trp, Tyr and Ala.

12. The DNA of any of claims 9 to 11 wherein codon 52 is substituted by a codon which is coding for an aminoacid selected from the group Glu, Leu, Val, Thr and Ser.

13. A DNA coding for a protein or a polypeptide provided upstream with DNA having the sequence

and functional equivalents thereof.

14. A DNA coding for protein or a polypeptide provided downstream with DNA having the sequence

and functional equivalents thereof.

15. A DNA coding for a protein or a polypeptide provided upstream with DNA having the sequence

and downstream with DNA having the sequence

and functional equivalents thereof.

16. The DNA of any of claims 13 to 15 wherein the DNA is coding for a polypeptide selected from the group Met-proinsulin, Met-vasopressin, Met-oxytocin, Met-ribonuclease, Met-groth hormone, Met-interleukin, Metinterferon, Met-aprotinin and homologues of Met-aprotinin.

17. An expression system for a protein or for a polypeptide comprising of one or more copies of a DNA coding for a protein or a polypeptide wherein any copy of the coding DNA is provided upstream with DNA having the sequence

and downstream with DNA having the sequence

or functional equivalents of said sequences.

18. The expression system of claim 17 wherein the coding DNA is coding for a polypeptide selected from the group Met-proinsulin, Met-vasopressin, Met-oxytocin, Met-ribonuclease, Met-groth hormone, Metinterleukin, Met-interferon, Met-aprotinin and homologues of Met-aprotinin.

19. A DNA having the sequence

20. The DNA according to claim 19 having a function as adaptor and functional equivalents thereof.

21. A DNA having the sequence

22. The DNA according to claim 21 having a function as promotor and functional equivalents thereof.

23. A DNA having the sequence

24. The DNA according to claim 23 having a function as operator and functional equivalents thereof.

25. A DNA having the sequence

26. The DNA according to claim 25 having a function as promotor and operator and functional equivalents thereof.

27. A DNA having the sequence

28. The DNA according to claims 27 having a function as linking sequence and functional equivalents thereof.

29. A DNA having the sequence

30. The DNA according to claim 29 having a function as expression enhancer and functional equivalents thereof.

31. An expression system for a protein or for a polypeptide comprising one or more of the DNA sequences according to claims 13,14 and 19 to 29.

32. A plasmid comprising an expression system according to claim 31.

33. A method for producing of a protein or a polypeptide wherein a microorganism is cultivated in an appropriate medium and the protein or the polypeptide is isolated thereafter characterized in that the microorganism is transformed with the plasmid of claim 32.

34. The method of claim 33 whrein the polypeptide is selected from the group Met-proinsulin, Metvasopressin, Met-oxytocin, Met-ribonuclease, Met-groth hormone, Met-interleukin, Met-interferon, Metaprotinin and homologues of Met-aprotinin.

35. A microorganism transformed with the plasmid of claim 32.

36. An E.coli microorganism which is transformed with the plasmid of claim 32.

37. An E.coli BR17 microorganism which is transformed with the plasmid of claim 32.

38. Plasmids having the restriction cleavage map of piWiT 10 wL1, piWi T10 wi7 and piWi T1 1.

39. Plasmids piWi T10 wL1, piWi T10 wi7 and piWi T1 1.

40. An E.coli BR17 microorganism which is transformed with the plasmids of claim 39 having the DSM designation DSM 3685 and DSM 3686.

41. Use of plasmids piWi T10 wL1, piWi T1 0 wi7 and piWiT11 in a process for production of proteins and polypeptides.

42. Use of plasmids piWi T1 0 wL1, piWi T10 wi7 and piWi T1 1 in a process for production of a polypeptide selected from the group Met-proinsulin, Met-vasopressin, Met-oxyticin, Met-ribonuclease, Met-groth hormone, Met-interleukin, Met-interferon, Met-aprotinin and homologues of Met-aprotinin.

43. Pharmaceutical composition containing microbially produced Met-aprotinin and/or Met-aprotinin homologues.

44. Use of microbially produced Met-aprotinin and Met-aprotinin in the production of pharmaceuticals.