Expression vectors for the production of polypeptides.

0001The invention relates to proteins and polypeptides, pharmaceutical preparations, promoters, operators, genes, vectors, host organisms and methods for their production.

0002The invention relates to proteins and polypeptides, in particular met-aprotinin and met-aprotinin homologs, which are obtained via recombinant DNA technology, expression genes of a new organism, new expression vectors including promoters, operators, amplified genes and a process for the construction of oligomers Genes.

0003The chemically synthesized DNA molecules as disclosed here are characterized by their DNA sequence.

0004In the case of genes for met-aprotinin and met-aprotinin homologs, the DNA sequence codes for new polypeptides or polypeptides which essentially match in their amino acid sequence and composition with that of aprotinin or aprotinin homologues and the biological activity of aprotinin or aprotinin -Homeologists own.

0005Aprotinin is a well-known 58 amino acid peptide that inhibits trypsin, chymotrypsin, plasmin and kallikrein. It is a basic proteinase inhibitor from bovine organs; Aprotinin, referred to as Trasylol®, is a valuable medicine for the treatment of various diseases, such as B. hyperfibrinolytic hemorrhage and traumatic hemorrhagic shock (see H. Fritz and G. Wunderer, 1983, Drug Res. 33, 479-494).

0006It has recently been shown that homologues of aprotinin with other amino acids in position 15 instead of lysine are valuable proteinase inhibitors with modified effects and efficacies compared to aprotinin (DE-OS 33 39 693; HR Wenzel et al 1985 in Chemistry of Peptides and Proteins, Volume 3). These aprotinin homologs have a strong inhibitory effect on pancreatic and leukocyte elastases.

0007Such aprotinin homologues can be used therapeutically for diseases in connection with excessive release of pancreatic elastase (pancreatitis), serum elastase (arteriosclerosis), leukocyte elastase for acute and chronic inflammation with damage to the connective tissue, damage to vascular walls, necrotic diseases and the degeneration of the lung tissue. The role that lysosomal enzymes, especially leukocyte elastase, play in inflammatory reactions due to immunological processes, e.g. B. rheumatoid arthritis, play; in addition, the homologs mentioned can be used as myocardial suppressing factors and in shock syndromes.

0008Although aprotinin and aprotinin homologs can be obtained from bovine organs and by semisynthetic conversion of bovine trypsin inhibitor (Tschesche, H., Wenzel, H., Schmuck R., Schnabel E. DE-OS 33 39 693 from May 15, 1985) the yields are relatively low.

0009It was recognized that the use of recombinant DNA and associated technologies would be the most successful way to provide the large amounts of high quality aprotinin homologs required. The goal was to produce met-aprotinin and met-aprotinin homologues, which are biologically active, as unfused products using recombinant DNA technology in a host organism.

0010DNA encoding polypeptides of known amino acid sequence can be produced using the genomic DNA sequence, the cDNA sequence which is complementary to the mRNA, or by choosing codons according to the genetic code and producing a synthetic gene which contains corresponding codons

0011A partial DNA sequence of a bovine genome clone of bovine pancreatic trypsin inhibitor gene was described by S. Anderson and IB Kingston, 1983, Proc. Natl. Acad. Sci. USA, 80, 6838-6842, to characterize a genomic clone for aprotinin. Recently, approximately 4 kbp segments of the bovine genome, which contains a coding section for bovine pancreatic trypsin inhibitor gene (BPTI e aprotinin) and for bovine spleen inhibitor II, were described by Kingston, IB and Anderson, p. 1986, Biochem. J. 233, 443-450.

0012It has recently been shown that aprotinin homologues can be produced by recombinant DNA technology using a construct in which the gene was linked to β-galactase.

0013It is usually much easier to express a mammalian protein in a bacterial host using the gene fusion method, using the information for an endogenous (homologous) polypeptide that naturally exists in a particular genus or species of host cells Information of a heterologous polypeptide combined to express an unfused heterologous polypeptide in a bacterial host cell. This is due to a number of complex phenomena that are not fully known. Some of the factors that are believed to hinder expression or accumulation of a heterologous polypeptide in a host cell are as follows:<ul id="ul0001" list-style="none"><li>1. The heterologous polypeptide can be degraded by the host cell.</li><li>2nd The heterologous polypeptide can have a toxic effect on the cell.</li></ul>

0014The degradation problem is believed to be more severe with small heterologous polypeptides than with large heterologous polypeptides.

0015Although a large number of heterologous polypeptides with more than approximately 100 amino acid residues have been expressed in E. coli, relatively few small heterologous polypeptides have been expressed in E. coli, despite the numerous attempts.

0016Fusion polypeptides have been used in numerous attempts to overcome the problems of expressing heterologous polypeptides in bacterial cells.

0017The disadvantages of these procedures are as follows:<ul id="ul0002" list-style="none"><li>1. The inserted structural sequence must be in the correct reading frame with respect to the AUG start codon of the E. coli gene.</li><li>2nd The heterologous polypeptide must be cleaved out of the fusion polypeptide with chemicals which modify and / or destroy certain amino acid residues (e.g. methionine by cyanogen bromide; tryptophan by succinimide); therefore, the heterologous polypeptide is required to be free of such amino acid residues.</li></ul>

0018The copy number of the gene to be expressed is normally 1 for each expression vector. Theoretically, it is possible to regulate the expression of several genes using a gene expression control system (N. Lee et al 1984, Nucl. Acids Res. 12, 6797-6812).

0019An increase in the level of expression of heterologous polypeptides or a stabilization of an expressed polypeptide can be achieved with the aid of domain amplifications, as described by SH Shen. 1984, Proc. Nat. Acad. Sci. USA 81, 4627-4631. He demonstrated that multiple copies of the proinsulin coding sequence could increase the stability of expressed products with fused and unfused expression systems. His product was a multiplied polypeptide that has to be cleaved with cyanogen bromide. The best level of expression was achieved with three copies.

0020The mechanisms by which abnormal proteins, including heterologous polypeptides, are broken down in E. coli are not fully known. By selecting certain host strains, polypeptides can be stably produced that are otherwise unstable (Gottesmann, S. and Zipser, D., 1970 J. Bacteriol. 133, 844-851). However, these mutants do not stabilize every heterologous polypeptide expressed in E. coli.

0021An object of the invention is to provide met-aprotinin, met-aprotinin homologues, nucleic acids coding for them, vectors in which the nucleic acids are incorporated, new modified cells which are transformed therewith and methods for obtaining Met -Aprotinin and Met-Aprotinin homologues. The term met-aprotinin refers to an aprotinin in which the amino acid in the zero (0) position represents a methionine.

0022For the gene construction for the production of synthetic genes, it was particularly advantageous to select a suitable blueprint with those codons which made possible a wide application. This is particularly the case in the construction of a synthetic master gene which comprises DNA blocks or cassettes which are terminated by unique recognition enzyme recognition sites. Such a construction of the gene allows easy modification or mutation of all DNA sequences within such DNA blocks, and is included in the present invention.

0023Homologs of aprotinin, which are provided with an additional methionine at the amino end, were produced with the aid of recombinant DNA technology. It has been found that such aprotinin homologs, e.g. B. Val-15, Ile-15, Leu-15, Phe-15, Ala-15, Arg-15, Gly-15, Ser-15, Thr-15, Trp-15 -, Met-15 and Tyr-15 aprotinin are equivalent to the known aprotinin and its homologues.

0024A pharmaceutical preparation in unit dose or liquid form is described.

0025The present invention also encompasses a new expression vector that can regulate non-fused heterologous polypeptides, regulatory sequences such as promoters, operators, ribosome binding sites, sequences for ribosome pooling, transcription terminators, as well as typical plasmid functions and genes or oligomers of genes that induce the bacterial host cell express, includes.

0026In a further embodiment, the invention provides a method for producing gene products which comprise oligomers of the gene which are linked to the DNA sequences of the ribosome binding site and which code for individual (unfused) polypeptides by causing a bacterial host to do so to express the oligomerized genes.

0027According to this embodiment, a method for oligomerizing individual genes with a particular construction vector is disclosed, and a bacterial host that has been genetically modified is described.

0028The present invention relates to microbially produced met-aprotinin and met-aprotinin homologs.

0029The microbially produced Met-aprotinin can be substituted in position 15 by any naturally occurring amino acid, in particular with Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr and Ala.

0030The microbially produced met-aprotinin can also be substituted in addition or alone in position 52 by Glu, Leu, Val, Thr or Ser. In this way, one can produce microbially produced Glu-52-Met-Aprotinin or Val-15-Glu-52-Met-Aprotinin or Ile-15-Glu-52-Met-Aprotinin or Leu-15-Glu-52-Met-Aprotinin receive.

0031The DNA encoding met-aprotinin can be substituted on codon 15 and / or on codon 52 by a codon which codes for any naturally occurring amino acid. The DNA may be substituted at codon 15 by a codon which codes for an amino acid selected from the group Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Trp, Tyr, Met and Ala. At codon 15, the DNA can be substituted by a codon which codes for an amino acid which is selected from the group Glu, Leu, Val, Thr and Ser.

0032The invention further relates to a DNA which codes for a protein or a polypeptide and which in the 5 'direction is the DNA of the following sequence<chemistry id="chem0001" num="0001"><img file="EP0244627A2_D0001.tif" /></chemistry> or has functional equivalents thereof and also a DNA which codes for a protein or a polypeptide, and which in the 3 ′ direction with a DNA of the following sequence<chemistry id="chem0002" num="0002"><img file="EP0244627A2_D0002.tif" /></chemistry> or functional equivalents thereof.

0033The invention relates in particular to a DNA which codes for a protein or polypeptide which in the 5 'direction is the DNA of the following sequence<img file="EP0244627A2_D0003.tif" /> and in the 3 'direction the DNA of the following sequence<chemistry id="chem0003" num="0003"><img file="EP0244627A2_D0004.tif" /></chemistry> or has functional equivalents thereof.

0034The coding part of the DNA can e.g. B. encode for the following polypeptides: insulin, proinsulin, vasopressin, oxytocin, ribonuclease, growth hormone, met-aprotinin and homologues of met-aprotinin.

0035The present invention further relates to an expression system for a protein or for a polypeptide, which comprises one or more copies of a DNA which codes for a protein or a polypeptide, each copy of the coding DNA in the 5 'direction being a DNA of the following sequence<chemistry id="chem0004" num="0004"><img file="EP0244627A2_D0005.tif" /></chemistry> and in 3 ′ direction a DNA of the following sequence<chemistry id="chem0005" num="0005"><img file="EP0244627A2_D0006.tif" /></chemistry> and has functional equivalents of these sequences.

0036A DNA of the sequence<chemistry id="chem0006" num="0006"><img file="EP0244627A2_D0007.tif" /></chemistry> is described. This DNA is a DNA sequence that functions as an adapter.

0037In addition, a DNA of the sequence<chemistry id="chem0007" num="0007"><img file="EP0244627A2_D0008.tif" /></chemistry> described. This DNA represents a DNA sequence with a function as a linker sequence.

0038The invention further comprises a DNA with the following sequence:<chemistry id="chem0008" num="0008"><img file="EP0244627A2_D0009.tif" /></chemistry>

0039This DNA codes for a short peptide and functions as an expression enhancer.

0040A DNA is described with a function as an operator with the following sequence<chemistry id="chem0009" num="0009"><img file="EP0244627A2_D0010.tif" /></chemistry> and a DNA with a function as a promoter with the following sequence:<chemistry id="chem0010" num="0010"><img file="EP0244627A2_D0011.tif" /></chemistry>

0041It is known to the person skilled in the relevant art that the substitution of one or more bases in the sequence described does not necessarily lead to a loss or a change in the function. The invention therefore also includes functional equivalents of the sequences mentioned.

0042Both DNA sequences can be combined to form a DNA which functions as a promoter and operator and has the following sequence<chemistry id="chem0011" num="0011"><img file="EP0244627A2_D0012.tif" /></chemistry> or which has functional equivalents of said sequence.

0043The expression system described above can be provided in the 5′-direction with the DNA sequences which are composed of the DNA with a function as a promoter and operator and are then incorporated into a plasmid.

0044The term "expression system" refers to a DNA sequence which comprises a structural sequence which codes for a protein or polypeptide and regulatory sequences such as promoter and operator.

0045This plasmid is suitable for transforming a microorganism, in particular an E. coli microorganism.

0046Most preferred is an E. coli BR17 microorganism which is transformed with the plasmid and has the DSM designation DSM 3685 and DSM 3686.

0047The invention relates to plasmids which have the restriction map of PiWiT 10 wL1 and PiWiT 11 and also the plasmids piWiT10 wi7 and piWi T11.

0048The plasmids piWiT 10 wL1 and piWiT 11 are suitable in a process for the production of proteins and polypeptides, in particular a process for the production of a polypeptide selected from the group consisting of insulin, proinsulin, vasopressin, oxytocin, ribonuclease, growth hormone, met-aprotinin and homologues of Met -Protinin.

0049The microorganisms used in accordance with the invention were deposited with the German Collection of Microorganisms, Griesbachstrasse 8, D-3400 Göttingen. The DSM deposit number of the host microorganism E. coli BR 17 is DSM 3684.

0050The DSM deposit number of the host microorganism E. Coli BR 17, which is transformed with the plasmids piWiT10 wi11 and piWiT10 wi7, is DSM 3685 and DSM 3686.

0051Brief description of the drawings<ul id="ul0003" list-style="none"><li>Fig. 1a. Structure of the synthetic gene</li><li>Fig. 1b. DNA sequence of DNA fragments</li><li>Fig. 2a. Construction vector piWi T9</li><li>Fig. 2b. Partial DNA sequences of piWi T9</li><li>Fig. 3. Amplification scheme for oligomerization of the synthetic gene</li><li>Fig. 4a. Expression vector piWi T 11</li><li>Fig. 4b. Partial DNA sequence of piWi T11</li><li>Fig. 5a. Plasmid PiWi T10 wL1</li><li>Fig. 5b. Partial DNA sequences of piWi T10 wL1</li><li>Figure 6. SDS protein gel electrophoresis</li><li>Figure 7. SDS protein gel electrophoresis</li><li>Fig. 8. Renaturation of Met aprotinin</li><li>Fig. 9. Western blot</li><li>Fig. 10. Chromatography of Met aprotinin</li></ul>

0052The following are the strategy for the construction and selection, the production of DNA fragments which code for met-aprotinin, met-aprotinin homologs, construction vectors, expression vectors, expression plasmids, microorganisms which are transformed with them, and the microbial production of Met-aprotinin and met-aprotinin homologues, as well as pharmaceutical preparations are disclosed.

0053Standard recombinant DNA technology methods have been used, as described by Maniatis et al, 1982, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, sometimes using modifications as described below.

Strategy for the construction and selection of DNA fragments encoding a synthetic inhibitor gene

0054The known protein sequence and the genetic code of aprotinin and aprotinin homologues were used to determine a DNA sequence which codes for such polypeptides.

0055The degeneracy of the genetic code allows a certain freedom in the choice of codons for a given amino acid sequence.

0056All possible base substitutions among the codons that determine the amino acid sequence of this protein have been determined. Accordingly, all potential restriction sites located within the possible DNA sequences were determined.

0057The codon selection for master genes was accompanied by the following considerations:<ul id="ul0004" list-style="none"><li>1. Codons and fragments were selected and the fragment composition planned in such a way that undesired complementarity of the fragments was avoided.</li><li>2nd The area above the initiation codon contains a ribosomal binding site; as codons around the region of the initiation codon, those were chosen which enhance ribosome binding (Lit GFE Scherer et al, 1980, Nucl. Acids Res. 8, 3895-3907).</li><li>3rd Restriction sites were selected that are necessary to facilitate the determination of transformants or to replace bases by replacing suitable fragments with other fragments, so that modifications of aprotinin can be easily produced, and to create a broad spectrum of possibilities for cloning and expression.</li><li>4th The majority of the codons chosen are those which are preferred in the expression of microbial genes (see H. Grosjean and W. Fiers, Gene, 18 (1982) 192-209; M. Gouy and C. Gautier, Nucleic Acids Research, 10 ( 1982) 7055-7074).</li></ul>

0058The basic blueprint for synthetic aprotinin genes and their homologues is shown in Fig. 1a. reproduced.

Construction of synthetic genes

0059The synthetic genes for Met aprotinin homologues were constructed via a master gene by assembling 14 purified oligonucleotides. This construction was carried out as in Fig. 1a. shown. The DNA sequences of the fragments for the synthetic inhibitor are shown in Fig. 1b. forth.

0060The master gene comprises a ribosome 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 III, additionally the internal restriction sites for Xho I, Apa I, Stu I, Acc I, Pst I, Sst II and Sph I. These restriction sites, particularly 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 a different codon usage. The entire spectrum of protein engineering is possible with such a construct.

0061To construct genes for met-aprotinin homologues, all that is required is to exchange a restriction fragment with a suitable DNA sequence. Sequences for such fragments encoding amino acid changes in position 15, e.g. B. Apa I - Stu I fragments are shown in Fig. 1b. reproduced.

Construction vector piWi T9 and amplification of the genes

0062The plasmid chosen for experimental Met-aprotinin cloning was piWi T9 (see also FIG. 2a.).

0063The construction vector is composed of two fragments of pBR 322, each bearing the origin of replication and the tetracycin resistance gene, a fragment of the bacteriophage fd 11 DNA which carries a transcription termination signal, and a fragment from the lac operon by E. coli, which extends from the C-terminus of the lac I gene to just behind the lac Z gene, the entire lac promoter operator region and the first five codons of the lac Z gene being deleted and by a short piece of a synthetic DNA were replaced. The synthetic DNA has constitutive promoter activity which is directed to the lac-Z gene and contains the cloning sites Xba I, Xma I and Hind III 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 is also translated depends on the sequence. The lac-Z gene of piWi T9 is not translated, active β-galactosidase is only produced if additional DNA is inserted into any of the cloning sites which an initiation codon brings in the reading frame for the lac-Z gene. The DNA sequences of important regions of the construction vector piWi T9 are shown in Fig. 2b.

0064For the oligomerization of genes, the synthetic gene (see FIG. 1a) was cloned between the restriction sites Xba I and Hind III of the construction vector piWi T9. For a tandem duplication of the synthetic gene, a Bam HI - Hind III fragment, which contains a copy of the gene located above the Hind III site, was attached to an Xba I - Bam HI fragment in the presence of a Hind III-Xba I adapter, which contains a copy of the gene located below the Xba I site (see Fig. 3) ligated. The Hind III-Xba I adapter fits into the protruding ends as formed by Hind III and Xba I, but does not regenerate these sites. The plasmid piWi T9 TL2 obtained has a single Xba I site above the first gene and a single Hind III site below the second gene. Thus, the duplication can be repeated using the same series of reactions to obtain additional plasmids containing 4, 8, 16 and 32 genes repeated in tandem, each preceded 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 vector piWi T11 and expression plasmid piWi T10 wL1

0065For the expression of Met-aprotinin and Met-aprotinin homologues, a plasmid was constructed in which the suitable genes could be expressed under the control of a strong promoter operator, and which further comprises DNA sequences which are very suitable for the expression of heterologous proteins and polypeptides are suitable. The physical map of the expression vector piWi T11 is shown in Fig. 4a.

0066The expression vector piWi T11 is very similar to the construction vector piWi T9; they have a common 3.3 Kb long Xho I - Eco RI fragment which contains 36 bp lac I DNA, 883 bp pBR 322 DNA (origin of replication), 2067 (+ 8) bp pBR 322 DNA (tetracycline resistance gene), 9 bp polylinker, 332 bp fd 11 DNA transcription termination signal, 8 bp Bam HI octalinker and 68 bp lac-Z-DNA. To enhance expression, a synthetic DNA fragment was constructed and ligated between the Eco RI - Xho I sites. The sequence of this synthetic fragment is shown in Fig. 4b. The fragment comprises sequences for a strong promoter, an RNA start, a lac repressor binding site (operator), a ribosomal binding site, a protein start codon, a short coding section which contains five unique restriction sites for cloning (Hind III, Xba I, Pst I, Bgl II and Xma I).

0067For expression purposes, the small Xho I - Xba I fragment of a construction vector with oligomers of the synthetic gene as described above was replaced with a small Xho I - Xba I fragment from piWi T11, the latter being a promoter, operator, a ribosome binding site and one Contained coding sequence for a hexapeptide. A recombinant plasmid obtained is e.g. B. the expression plasmid piWi T10 wL1 with oligomers of Met-Aprotinens.

0068DNA sequences of important sections of the expression plasmid are shown in FIG. 5b.

The microorganism

0069A large number of microorganisms which are suitable for transformation are known in the prior art. These are unicellular organisms that are suitable for cultivation in cultures or for fermentations. Preferred organisms for transformation include bacteria, yeast and fungi.

0070The organism chosen for the present work was E. coli.

0071A particularly suitable strain is E. coli B lamda R⁻, mal⁻, which was noted for recA using the method described by JH Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory.

Manufacture of mead aprotinin

0072Many proteins that are synthesized in large quantities in bacteria accumulate in insoluble form (DC Williams, RM van Frank, JB Burnett, WL Muth, 1982, Science 215, 687).

0073These insoluble proteins are called inclusion bodies. You can usually only with polar substances such. B. urea or guanidinium hydrochloride.

0074The E. coli strain BR17 was transformed with the plasmid piWi T10 wL1, which codes for the aprotin gene fourteen times below an E. coli promoter, operator and a ribosome binding site. An overnight culture of E. coli strain BR17 piWi T10 wL1 was centrifuged, the pellet was resuspended in buffer for disruption, and the cells were analyzed using a "french press". lysed.

0075The cell lysate was centrifuged to obtain the inclusion bodies. The pellet was washed twice with buffer (breaking buffer).

0076The cleaning steps were checked using SDS-polyacrylamide gel electrophoresis according to Laemmli (UK Laemmli 1970, Nature 277, 680-685); Fig. 7.

0077The inactive inhibitor was renatured using a Creighton method (TE Creighton, Proceedings of Genex-UCLA Symposium 1985 Kingstones); Fig. 8. The active inhibitor could be detected by trypsin determination and Western blot analysis; Fig. 9.

0078The active inhibitor was furthermore determined by chromatography on a trypsin-Sepharose column according to Fritz (H. Fritz, M. Gebhardt, R. Meister, K. Illchmann, K. Hochstraßer, 1970, Hoppe-Seyler's Z. Physiol. Chem. 351 , 571-574); Fig. 10.

0079The inhibitor was then characterized by microsequencing according to Hewick (RM Hewick, MW Hunkapillar, LE Hood, WI Dreger 1981, J. Biol. Chem. 256, 7970-7997).

0080The first 20 residues at the N-terminus were determined. Except for the methionine in the zero position, the amino acid sequence was completely identical to 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 means of the trypsin-inhibiting effect shows comparable values ​​(Table 3).

0081All of these experiments show that it is possible to produce met-aprotinin in E. coli and to renature it to the active inhibitor.

Production of Met-Ile-15-Aprotinin

0082Met-Ile-15-aprotinin can be prepared in E. coli in a similar manner to that described for Met-aprotinin (see also Figure 6).

0083The inhibitory effect was determined by means of an elastase inhibition assay (K. Nakajima, M. Zimmermann, JC Powers, MJ Castillo, BM Ashe 1979, J. Biol. Chem. 254, 4027).

0084The inhibitor was characterized by amino acid analysis and N-terminal sequencing (Table 1, 2).

0085All other derivatives of aprotinin could be prepared in a manner similar to that described for Met-Aprotinin and Met-Ile-15-Aprotinin.

Pharmaceutical preparations

0086The present invention encompasses pharmaceutical preparations which, in addition to non-toxic, inert, pharmaceutically suitable excipients, comprise 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 preparation of these preparations.

0087The present invention also encompasses pharmaceutical preparations in unit doses. This means that the preparations are in the form of individual parts, e.g. B. as tablets, coated tablets, capsules, pills, suppositories and ampoules, the content of active compound corresponding to a part or a multiple of an individual dose. The dose units can e.g. B. contain one, two, three or four individual doses or a half, a third or a 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 the whole, a half, a third or a quarter of a daily dose.

0088Non-toxic, inert, pharmaceutically suitable excipients are understood to mean solid, semi-solid or liquid diluents, fillers and formulation auxiliaries of all kinds.

0089Preferred pharmaceutical preparations include tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays.

0090Tablets, coated tablets, capsules, pills and granules can contain the active compound or compounds together with conventional excipients, such as. B. (a) fillers and extenders, e.g. B. starches, lactose, sucrose, glucose, mannitol and silicon dioxide, (b) binders, e.g. B. carboxymethyl cellulose, alginates, gelatin and polyvinyl pyrrolidone, (c) moisturizing agents, e.g. B. glycerin, (d) disintegrant, e.g. B. Agar-agar, calcium carbonate and sodium carbonate, (e) solution retarders, e.g. B. quaternary ammonium compounds, (g) wetting agents, e.g. B. cetyl alcohol and glycerol monostearate, (h) adsorbents, e.g. B. kaolin and bentonite, and (i) lubricants, e.g. B. talc, calcium and magnesium stearate and solid polyethylene glycols, or mixtures of the substances listed under (a) to (i).

0091The tablets, coated tablets, capsules, pills and granules can be provided with customary coatings and coatings, and optionally contain opacifying agents; they can also be a preparation which releases the active compound or compounds only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of suitable embedding materials are polymeric substances and waxes.

0092The active compound or compounds, optionally together with one or more of the above-mentioned excipients, can also be in a microencapsulated form.

0093Suppositories can have the usual water-soluble or water-insoluble excipients in addition to the active compound or compounds, e.g. B. polyethylene glycols, fats, e.g. B. cocoa fat and higher esters (z. B. C₁₄ alcohol with C₁₆ fatty acid) or mixtures of these substances.

0094Ointments, pastes, creams and gels can contain conventional excipients in addition to the active compound or compounds, e.g. B. animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicon dioxide, talc and zinc oxide, or mixtures of these substances.

0095Powders and sprays can contain conventional excipients in addition to the active compound or compounds, e.g. B. lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can also contain conventional propellants, e.g. B. chlorofluorocarbons.

0096Solutions and emulsions can contain conventional excipients in addition to the active compound or compounds, such as solvents, solubilizers and emulsifiers, e.g. B. Water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerin, glycerol formaldehyde, tetrahydrofuran fatty acid, tetrahydrofuryl fatty acid, tetrahydrofuran fatty acid, tetrahydrofuran fatty acid, tetrahydrofuran , or mixtures of these substances.

0097For parenteral administration, the solutions and emulsions can also be in a sterile form that is isotonic with blood.

0098The suspensions may contain conventional excipients in addition to the active compound or compounds, such as liquid diluents, e.g. B. water, ethyl alcohol or propylene glycol, suspending agent, for. B. ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

0099The formulation forms mentioned can also contain dyes, preservatives and additives which improve the smell and taste, e.g. Peppermint oil and eucalyptus oil, and sweeteners, e.g. B. saccharin.

0100The therapeutically active compounds should be present in the above-mentioned pharmaceutical preparations preferably in a concentration of approximately 0.1 to 99.5, preferably 0.5 to 95% by weight, based on the total mixture.

0101The above-mentioned pharmaceutical preparations can also contain other pharmaceutically active compounds in addition to the compounds according to the invention.

0102The pharmaceutical preparations mentioned above are prepared in a conventional manner by known processes, for. B. by mixing the active compound or compounds with the excipient or excipients.

0103The 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.

0104In general, it has proven advantageous in both human and veterinary medicine to use the active compound or compounds according to the invention in total amounts of from about 0.5 to about 500, preferably from 5 to 100 mg / kg of body weight every 24 h, if necessary in the form of several individual administrations, to achieve the desired results. An individual administration contains the active compound or the active compounds according to the invention preferably in amounts of approx. 1 to approx. 250, in particular 3 to 60 mg / kg body weight. However, it may be necessary to deviate from this dose, in particular depending on the nature and body weight of the patient to be treated, the type and severity of the disease, the type of preparation and administration of the medicine, and the time or interval, through which the administration takes place.

0105Thus, in some cases it may suffice to make do with less than the above-mentioned amount of active compound, while in other cases the above-mentioned amount of active compound has to be exceeded. The particular optimal dose required and the mode of administration of the active compound can be readily determined by the person skilled in the art on the basis of his specialist knowledge.

Examples

example 1

Synthesis and purification of DNA fragments coding for Met-Aprotinin and Met-Ile-15-Aprotinin

0106The oligonucleotides comprising the gene were made using solid phase synthesis methods. The synthesis scheme for the oligomers corresponded to that stated and used proton-activated, protected 2′-deoxyribonucleotide-phosphoramidites. All sequence steps were carried out in an automated manner on an Applied Biosystems model 380 DNA synthesizer using protected nucleotides, solvents, chemicals and reagents, which were obtained from the manufacturer. The solid phase support, which is from the same manufacturer, was c "controlled pore glass" to which the starting 3'-nucleotide was already applied. Certain modifications have been introduced in the automated response cycle in accordance with the "Manufacturers Operating Instructions" and "Users Bulletins". After completion of the synthesis, the oligomers were unblocked and cleaved from the solid phase support within the DNA synthesizer as specified by the manufacturer.

0107Removal of the blocking groups was completed by heating the aqueous solution containing the oligomer with concentrated ammonium hydroxide for 4 to 24 hours at 55 ° C in a sealed ampoule. The resulting solution was evaporated, the residue dissolved in 0.01 M triethylammonium bicarbonate buffer, pH 7.0 (TEAB buffer). This solution was chromatographed on Sephadex-G 50 ® gel filtration resin.

0108The column was prepared in the same TEAB buffer and eluted with it. The material that eluted with the void volume was combined and the solution evaporated.

0109A part of the residue (10 to 40% of the absorbance units at 260 nm), dissolved in loading buffer (composition: 0.1% bromophenol blue, 0.1% xylene cyanol, 10 mM disodium EDTA, in formamide) was electrophoresed in polyacrylamide gels further cleaned. The gel dimension was 10 × 32 cm with a thickness of 1.5 mm. The gel pocket for each oligomer purified in this way was 2 to 5 cm wide; up to five oligomers were purified using a gel. The acrylamide concentration of the respective gel varied from 14 to 20%, depending on the chain length of the products to be cleaned. The 14% acrylamide gel was preferred for longer oligomers, 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 also consisted of the tris-borate-EDTA mixture. Electrophoresis was carried out at 20 to 60 watts, constant current, for 6 to 18 hours. Such standardized methods can be found in various "User Information Bulletins" from Applied Biosystems.

0110After the electrophoresis had ended, the gel was placed on a plastic film and the oligomers were made visible by "UV shadowing". This image is achieved by placing the gel with foil on a fluorescence thin-layer chromatography plate and observing the gel under a short-wave UV light source. The desired product appears as the slowest migrating blue DNA main fragment using this imaging method. The desired 8ande is cut out of the gel. The DNA oligomer is eluted from the gel slice on powdered diethylaminoethyl (DEAE) cellulose using an EpiGene D-Gel® electrophoresis apparatus. The oligomer is obtained from the cellulose by elution with 1 M TEAB buffer. The buffer solution containing the oligomer is evaporated, the residue dissolved in 0.01 M TEAB buffer and then desalted by chromatography on a Sephadex-G 50® column as described above. The material eluting in the empty volume is combined and lyophilized to obtain the end product.

0111Using the methods described above, about 0.5 to 5.0 A₂₆₀ units of each of the purified oligomers were obtained.

Example 2

Construction of synthetic genes

0112The synthetic oligonucleotides, which had been purified as in Example 1, were treated with polynucleotide kinase and adenosine triphosphate (ATP) under standard conditions (Maniatis et al. (1982)). One-tenth of each sample was incubated with δ [32P] ATP instead of ATP. Large samples were then mixed and, as described in Example 1, subjected to electrophoresis. The labeled bands were eluted from the gel and purified as previously described.

0113The 14 oligonucleotides (FIG. 1a, b) were then mixed with 30 μl of double-concentrated ligase buffer (Maniatis et al. (1982 and subjected to a ligation reaction for 20 min at 40 ° C. and 30 min at room temperature. Then 2 μl of 100 mM ATP, pH 7.5, 6 ul T4DNA ligase and 22 ul water added and the ligation continued at about 10 ° C overnight.

0114The products of the ligation reaction were then cut with Xba I and HindIII to obtain individual synthetic gene segments with a total length of 204 bp, which included an open XbaI site, a ribosomal binding site, an initiation codon ATG, the aprotinin coding sequence, a termination codon TAG and one open HindIII position.

Example 3

Construction of recombinant plasmids

0115The construction vector DNA (Fig. 2a, b) was digested with XbaI and HindIII (Maniatis), mixed with synthetic gene segments, which had been prepared as described in Example 2, mixed and ligated overnight. The sample was then used to transform E. coli Su3 [lac-pro]_Δmet⁻ arg_{at the} supF thi⁻) according to the method of Hahnahan (D. Hahnahan, J. Mol. Biol., 1983, 166, 557-580). The cells were plated on full medium plates containing 10 µg tetracycline / ml and 0.005% x-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside; J. Miler, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) contained and were incubated overnight at 37 ° C. The plasmid DNA was extracted from blue colonies and examined for the presence of restriction enzyme recognition sites located in the synthetic gene segment. Finally, the DNA sequence was determined by the Maxam and Gilbert method (A. Maxam &amp; W. Gilbert, in Meth. Enzymol., 1983, 65, 499-560).

Example 4

Construction of multimers of the gene

0116piWiT9L1-DNA, which had been prepared as described in Example 3 above, was used for the preparation of multimers of the aprotinin gene. The plasmid DNA was digested with HindIII and BamHI and the smaller fragment containing the 3'-end of the tetracycline resistance gene, the origin of replication, the promoter and the met-aprotinin gene was purified (Maniatis et al. (1982 )). A second sample of the plasmid DNA was digested with SbaI and BamHI and the larger fragment, which in turn contains the met aprotinin gene, the lac Z gene, the transcription termination signal and the 5'-end of the tetracycline resistance gene, was purified as above . Both fragments are ligated (see Example 2) in the presence of the purified adapter molecules,<chemistry id="chem0012" num="0012"><img file="EP0244627A2_D0013.tif" /></chemistry> and with this is transformed into E. coli BR17 (see example 5). Since the adapter molecule has protruding ends that each fit into a HindIII site and an XbaI site without regenerating one of these sites, the resulting new plasmid with two tandemly recurring aprotinin genes also has only a single XbaI site above the ribosomal binding site of the first aprotinining and also only a single Hind III site below the termination codon of the second met atrining. Thus, the same reactions can be performed using this new piWiT9L2 DNA to give another plasmid that carries four tandemly recurring Met-Aprotinin genes, each preceded by its own ribosomal binding site.

0117Before a third duplication of the now four aprotinin genes, the 3Kb EcoRI fragment was deleted from the plasmid in order to reduce its overall size (FIG. 3). After the third doubling, we happened to receive a plasmid with seven aprotinin genes instead of eight. Plasmids with odd number of genes are usually found among the products of such a low frequency ligation reaction. We believe that their formation is due to partial denaturation of the DNA fragments during the melting of the "Seaplaque® agarose (Maniatis et al. (I982)) in the course of the purification process. We could not deviate from a given number of genes after the determine further growth of these plasmids in E. coli BR17.

0118Thus, the fourth duplication, performed as described above, gave a plasmid with fourteen aprotinin genes assembled in an artificial operon that could be transcribed into a polycistronic mRNA, which was then translated into 14 separate identical aprotinin peptides.

Example 5

Construction of expression plasmids

0119The importance of the 5'-untranslated section of the mRNA, the so-called leader sequence of the mRNA, on the translation rate of the gene or of the genes which code on this mRNA cannot yet be fully explained. We examined several of these leader sequences for their translation efficiency and obtained different results with different genes. A leader sequence with which the best results were obtained with our synthetic aprotinin gene is the sequence which is present in the expression vector piWiT11 (FIG. 4a, b).

0120A fragment comprising 14 identical aprotinin genes, each containing its own ribosome binding site, was excised from the construction plasmid with XbaI and EcoRI and inserted between the XbaI and EcoRI sites of the polylinker of the expression vector piWiT11. Figure 5b shows that a hexapeptide encoded by the mRNA is translated in addition to the 14 aprotinin peptides. Such small peptides have been reported to translate the following gene or of the following genes (BE Schoner et al, Proc. Natl. Acad. Sci. USA, 1984, 81, 5403-5407).

Example 6

Construction of the E. coli host strain BR17

0121E. coli B1.8 was isolated in Berkeley. His phenotype, given by mal⁻ and λ^R was confirmed. This strain was subjected to the following procedures: 1 ml of saturated culture was centrifuged for 1 min in an Eppendorf centrifuge, the peletted cells were resuspended in 1 ml of 0.9% NaCl solution. 0.2 ml of this suspension was plated on a minimal glucose plate containing thymine to a final concentration of 0.05% and trimethoprim to a final concentration of 0.001% to select the thyA mutants. One of these colonies, which had grown on this plate after 30 h of incubation at 37 ° C., was grown on a further minimal glucose agar plate with thymine and trimethoprim and designated B13. A single colony was selected from this plate and E. coli RZ423, a recA-deficient Hfr strain, which is also lac-paired. These sexually merging E. colis were streaked on minimal lactose plates. The female B13 strains could not grow on these plates due to their thymine auxotrophy. The male RZ423 strains could not use lactose as a carbon source. Thus, only those B13 cells could grow that - after pairing with RZ423 - had stably incorporated the part of the RZ423 chromosome that codes for a functional thyA gene. 24th such colonies were cleaned and individually examined for the characteristics mal⁻ (on EMBmal plates) and recA⁻ (by comparing the growth in the presence of methyl methanesulfonate). All of the colonies tested were mal⁻; in this way it was ensured that they did not happen to be lac von revertands of RZ423; three of them showed reduced growth in the presence of methyl methanesulfonate, indicating that these bacteria had incorporated the recA⁻ allele from RZ423 at the same time as the closely related wild-type thyA gene. They did not have the Hfr genotype because they did not have to be used to plate out the male-specific bacteriophage M13.

0122One of the three clones was used as a host strain for the expression plasmids that have the inhibitor genes. It was designated E. coli BR17 and deposited with the German Collection for Microorganisms, Göttigen, under the number DMS 3684.

Example 7

Manufacture of mead aprotinin

solutions

0123Buffer A, pH 8.2 50 mmol Tris-HCl 1 mmol EDTA adjusted to pH 8.2 Buffer B, pH 8.2 8 M urea 1 mmole of bis (2-hydroxyethyl) disulfide 1 mmole of 2-mercaptoethanol in buffer A; Buffer C, pH 8.2 1 mmole of bis (2-hydroxyethyl disulfide) 1 mmole of 2-mercaptoethanol in buffer A; Buffer D, pH 8.2 0.6 mol sodium chloride in buffer A.

0124For production purposes, 5 l of an E. coli overnight culture of the strain BR17 piWi T10 wL1.15 min were centrifuged at 8000 rpm. The cell pellet, weighing approximately 10 g, was resuspended in 25 ml 0.1 M Tris.HCl, 0.001 M EDTA and 0.1 mg lysozyme / ml. The cells were lysed with a "french press".

0125The cell lysate was centrifuged at 20,000 rpm for 20 min. The supernatant was discarded. The pellet was washed twice with 20 ml buffer.

0126The pellet was dissolved in 10 ml of buffer B. The solution was reduced for 1 h at 50 ° C under a nitrogen atmosphere. The solution was then placed on an Econo column (25 × 100 mm) containing approx. 10 ml CM-Sepharose Fast Flow^R was filled. The column had been 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 eluted with 100 ml buffer B and 100 ml buffer C. Before applying the second linear elution gradient, the column was washed with buffer A until the baseline was stable.

0127The second gradient was formed from 100 ml of buffer A and 100 ml of buffer D. The peak fractions were tested for their trypsin inhibitory activity; an ELISA and a Western blot were also carried out.

0128In a series of experiments, the yield determined with various tests was 5 to 20 mg.

Purification of Met-Aprotinin by Chromatography on a Trypsin-Sepharose Column:

01295 mg of renatured met-aprotinin were dissolved in 1 ml of 0.1 M Tris buffer, pH 6.5, and on an Econo column. which was filled with 30 ml of trypsin-Sepharose.

0130The column had previously been equilibrated with 0.1 M Tris buffer, pH 6.5. The column was then washed with 0.1 M Tris buffer, pH 6.5, until a stable baseline was obtained.

0131The 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 / HCl, pH 1.8.

0132The various fractions were neutralized and their activities determined using a trypsin inhibition assay.

0133The active fractions were pooled and then desalted by dialysis against distilled water. The inhibitor obtained was characterized by N-terminal sequencing and amino acid analysis.

Example 8

Production of Met-Ile-15-Aprotinin

0134The fermentation of E. coli transformed with plasmid piWi T10 Wi7 and the renaturation of Met-Ile-15-aprotinin were carried out by the same method as described in Example 7, with the exception that the activity was carried out with the aid of an elastase inhibition assay was tested instead of a trypsin inhibition assay. In a series of experiments, the yield determined by various tests was 5-10 mg.

0135The renatured Met-Ile-15-aprotinin was further purified by chromatography on a column filled with polyclonal anti-aprotinin antibody.

01361 mg of Met-Ile-15-aprotinin was dissolved in 2 ml of 0.05 M phosphate buffer, 1 M NaCl, pH 7.0 and placed on an Econo column (41 × 100 mm) which was filled with 40 ml of polyclonal anti-aprotinin antibody. Sepharose was filled, applied.

0137The column was washed with three volumes of starting buffer. The elastase inhibitor Met-Ile-15-aprotinin was desorbed using 0.2 M KCl / HCl pH 2.2. The active fractions were pooled and dialyzed against distilled water. The inhibitor was obtained by lyophilization (approx. 600 µg weight). 1 nmol of the inhibitor was applied to the gas phase sequencer and characterized by N-terminal sequencing; 1 nmol was also used for amino acid analysis.

Table 1: N-terminal sequencing of Met-Aprotinin and Met-Ile-15-Aprotinin

0138<ul id="ul0005" list-style="none"><li>1. Mead aprotinin; approx. 1 nmol of the substance was sequenced over 20 cycles.<chemistry id="chem0013" num="0013"><img file="EP0244627A2_D0014.tif" /></chemistry></li><li>2nd Met-Ile-15 aprotinin; approx. 1 nmol of the substance was sequenced over 20 cycles.<chemistry id="chem0014" num="0014"><img file="EP0244627A2_D0015.tif" /></chemistry></li></ul><tables id="tabl0001" num="0001"><img file="EP0244627A2_D0016.tif" /></tables>

0139The amino acids were determined by "post column" derivatization with o-phthalaldehyde. Cys and Pro were not determined.<tables id="tabl0002" num="0002"><img file="EP0244627A2_D0017.tif" /></tables>

0140Legends too<ul id="ul0006" list-style="none"><li>6: SDS gel electrophoresis (15% polyacrylamide) of E. coli proteins from E. coli BR 17 [piWi T10 wL1], E. coli BR 17 [piWi T10 wi7] and E. coli BR 17 [piWi T10w]<ul id="ul0007" list-style="none"><li>1. piWi T10 wi7,10⁹ cells;</li><li>2nd piWi T10 wL1, 10⁹ cells;</li><li>3rd soluble proteins piWi T10 wi7;</li><li>4th soluble proteins piWi T10 wL1;</li><li>5. soluble proteins piWi T10w;</li><li>6. piWi T10w, 2 x 10⁸ cells;</li><li>7. piWi T10 wi7, 2 × 10⁸ cells;</li><li>8th. piWi T10 wL1, 2 x 10⁸ cells.</li></ul></li><li>Fig. 7: SDS gel electrophoresis (10-20% polyacrylamide) of the proteins from E. coli strain BR 17 piWi T10 wL1.</li><li>Fig. 9: Recombinant aprotinin obtained by renaturation and trypsin-Sepharose chromatography was subjected to electrophoresis on a Na-DodS0₄ gradient (10-20%) polyacrylamide gel and then a Western blot according to Towbin was carried out.<ul id="ul0008" list-style="none"><li>Lane 1: aprotinin;</li><li>Lane 2: recombinant aprotinin after renaturation;</li><li>Lane 3: recombinant aprotinin after trypsin-Sepharose chromatography.</li></ul></li></ul>