EP0379506A1 - Recombinant plasmids for producing human adrenocorticotropic hormone (acth) - Google Patents

Abstract

ACTH and biologically active ACTH derivatives are produced by genetic engineering. The corresponding DNA sequences are incorporated into vector plasmids, which in turn are introduced into the E. coli bacteria in which they are expressed. ACTH and its derivatives thus obtained are useful as medicaments.

Description

 Re orabinante plasmids for the production of human adrenocorticotropic hormone (ACTH).

The adrenocorticotropic hormone, abbreviated ACTH, is a peptide hormone that is formed in the basophilic cells of the anterior lobe of the pituitary gland. It is also known in the literature as corticotrophin or corticotrophin.

ACTH regulates the secretion of glucocorticoids, in particular hydrocortisone and cortisone, in the zona fasciculata of the adrenal cortex and thus influences the carbohydrate, protein and fat metabolism in a complex manner. Likewise, ACTH has a regulating effect on the production of sex hormones in the zona reticularis, which directly surrounds the adrenal center as the inner layer of the bark.

In human medicine, ACTH is used therapeutically to treat inflammatory and allergic processes such as rheumatic fever or bronchial asthma. It is also important in the clinic for the diagnosis of adrenal cortex function. There are currently two types of preparations available:

1) ACTH from animal sources, especially enriched extracts from porcine pituitary glands.

2) Preparations prepared by means of peptide synthesis of an ACTH derivative which is shortened compared to the natural substance.

The amino acid sequence of the enscl 'lend ACTH was determined by the work of Teh H. Lee et al. [J. Biol. Chem. 236, 2970-2974 (1961)] and B. Riniker et al. [Nature New Biol. 235, 114-115 (1972)] clarified: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Prσ-Val-Gly-Lys-

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-

31 32 33 34 35 36 37 38 39 Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe

Human ACTH differs from porcine ACTH only in one amino acid (see Teh H. Lee et al., I.e.). The latter contains a leucyl residue in position 31 instead of the seryl residue in the human sequence.

It is known from the literature that different biological effects can be assigned to individual areas of the natural ACTH molecule (Review: H.J.M. Goverde, Euro Diagnostics Newsletter No. 4, January 1985). According to this, the N-terminal region of the peptide chain, as is contained in the synthetic commercial preparations already mentioned for essential peptides with the 1-24 part sequence of natural ACTH (tetracosactide)], is for the interactions with the various functional areas of the adrenal cortex. On the other hand, the C-terminal portion of the complete peptide has a protective function against biological inactivation processes during ACTH transport in plasma, in particular against the tryptical cleavage of the basic portion of peptides in the region of amino acids 15, which is important for receptor binding. 18th

The availability of larger amounts of pure ACTH with the amino acid sequence of the human hormone is accordingly of considerable medical and scientific interest. A method suitable for the biotechnical production of human ACTH and peptide derivatives derived therefrom is accordingly the subject of the present invention. It is already part of the general state of knowledge to introduce structural genes that encode medically insertable proteins or peptides for cellular synthesis in targeted industrial fermentable prokaryotic or eukaryotic cells and to establish them there in a stable manner. This is known as transformation. Using suitable selection methods, genetically identical cell populations (clones) can be obtained, all of which have the artificially introduced gene. By supplementing such structural genes which have been introduced into the cell by certain control elements which can correspond to the protein synthesis apparatus of the respective host cell, the expression of the foreign genetic material already mentioned, ie its translation into the desired gene product, can be achieved in many cases chen. The most important experimental possibilities for the production of such expression clones are the general standard of molecular biological knowledge and are described in numerous publications [for example W. Gilbert and L. Villa-Komaroff, Scientific American 242, 68-82 (April 1980); A. Aharo Novitz and G. Cohen, Scientific American 245, 106-118 (Sept. 1981); E.-L. Winnacker in Gene and Clones, pp. 191-260 (VC Verl. Weinheim, 1985)].

In this context, genetic engineering has the primary task of developing the range of processes for in-vitro processing of genetic material, i.e. to develop and expand DNA fragments with very different physico-chemical and biological properties.

The construction of a cell clone that is able to translate the genetic information from artificially introduced foreign DNA into the synthesis of corresponding foreign proteins always follows the same procedure in principle:

1. A DNA fragment is provided which contains the desired genetic information. There are several possibilities for that. In the simplest case, it can be Cut out the table from the DNA of a suitable donor species, a frequently used method if the donor is a prokaryote. On the other hand, a gene can be synthesized enzymatically by reverse transcription from messenger RNA (cDNA method). Another possibility is the chemical synthesis of oligonucleotides and their enzymatic linkage to the desired gene fragment.

2. The desired DNA fragment, optionally supplemented with control units for the expression, is enzymatically integrated into a so-called vector DNA (for details, see the literature references mentioned above).

3. The resulting gene construction has the necessary functions for introducing the foreign gene into suitable host cells and for its stable establishment in an autonomously replicating expression unit or built into the host chromosome, as well as marker functions for the selection of successfully transformed in vitro recombined clones.

Prokaryotic host organisms are particularly attractive for the development of biotechnological processes with genetically engineered expression clones. They can be fermented relatively inexpensively using established process technologies and in some cases have been genetically well studied. The latter applies especially to the intestinal bacteria Escherichi coli and derived apathogenic laboratory strains such as E.col K12.

Numerous examples have been described in recent years for the biotechnical synthesis of foreign proteins in E.col, including many that relate to the production of gene products of eukaryotic origin [Review, for example, FAO Marston, Biochem. J. 240, 1-12 (1986); further references s there]. There are three types of expression clones: 1. Clones in which the foreign structural gene is inserted solely between E. coli transcription, translation and termination signals and is directly expressed in the cytoplasm in native form [for example DV Goeddel et al., Natu 281, 544-548 (1979)].

2. Clones in which the foreign structural gene additionally contains codo for a signal peptide sequence which can be cleaved by the E. coli system in front of the amino terminus in the correct reading frame. Such clones are able to process the primary gene product correctly and the foreign protein in native form in the periplasmic To secrete space [e.g. G.W. Becker and H.M. Hsiung, FEBS Left. 204, 145-1 (1986)].

3. Clones in which the foreign structural gene has N-terminal extensions through host-specific control and structural genes in the reading frame. They synthesize a E. coli protein and the fusion protein composed of the target protein, which generally remains in the cytoplasm [e.g. D.V. Goeddel et al. , Proc. Nat. Acad. Be USA 76. / 106-110 (1979)].

The fusion with host-specific genes and the resulting expression as fusion proteins permits the biotechnological synthesis of short-chain target peptides in E. coli [for example, J. Shine et al., Nature 2Z5_ _r 456-461 (1980)]. Such small peptides cannot be directly synthesized in E. coli cells because they do not have a fixed spatial structure and are therefore preferentially degraded by host proteases present in cytoplasm [cf. K. Itakura et al., Science 198, 1056-1063 (1977)]. On the other hand, as parts of fusion proteins, they can be obtained in good yields under suitable conditions. The specific cleavage of such primary products with the release of the native target peptide represents a general problem, for the solutions of which a number of individual examples have been described [cf. FAO Marston, Biochem. J. 240, 1-12 (1986). All of these methods are characteristic Properties of the corresponding fusion proteins depend and are therefore limited in their general applicability [cf. H. Scheidecker et al. , German patent application, file number P 3731875.6].

This also applies to a method already used in application technology, in which the possibilities for cleaving -Met-X bonds (X = N-terminal amino acid of the target peptide) on the carboxyl group of methionine with brocyan are used. This method can only be used if the desired peptide, as in the case of the A and B chains of human insulin, does not contain any further intramolecular methionyl residues [cf. DV Goeddel et al., Proc. Natl. Acad. Be. (USA) 76, 106-110 (1979)]. For many other examples, and this also includes the human ACTH, the aforementioned requirement is not fulfilled. So a cyanogen bromide Spaltun would _(of. .ACTH see. Structure S. 3) perform suitably designed ACTH fusion proteins due to the terminal N-to sätzlichen methionyl residue in the area of the target peptide to a Fragmentierun the desired product. Experimental alternative for examples of this type is offered by a new, specific and highly efficient splitting method with a very wide range of applications, which is described in the German patent application filed in parallel (file number P3731875.6 ⁾ .

Fusion proteins that are to be specifically split by this method must be constructed in such a way that between the amino end of the target peptide and the carboxyl end of the bacterial portion there is an at least repeated tetrapeptide recognition sequence for collagenases of the clo-type. stridiopeptidase A is located. Such constructions have the general structure:

H ₂ N bacterial sequence portion-Gly- (Pro-X-Gly) _n ~ per-target peptide-COOH (n »2).

(Depending on the peptide sequence, n = 1 applies, but for most examples n> 1.)

[Details see parallel patent application G. Siewert et al. (1987)]. After the collagenase cleavage, the target cleavage product obtained is the target peptide extended by the dipeptide Gly-Pro at the N-terminal, which can be quantitatively converted into the native form by cleaving off the glycylproline by means of postproline dipeptidyl aminopeptidase (PPDA) (see FIG EP 20290).

The use of these genetic engineering methods for the construction of in vitro recombined Escherichia coli strains for the bio-technical production of small and medium-sized peptides is also the subject of the present invention. In particular, it describes a new biotechnical production method for the adrenocorticotropic hormone (ACTH) or of derivatives of this active ingredient such as ACTH 1-24 (tetracosactide) or ACTH extended by one or more ^" amino acids. This process comprises the following individual steps:

1. The in vitro construction of Escherichia K12 clones, the ACTH or ACTH derivatives (e.g. ACTH 1-24, one or more amino acids extended ACTH or an ACTH in which one or more amino acids are replaced by others Amino acids were substituted) as the C-terminus of specifically cleavable fusion proteins with N-terminal protein portions of the E. coli-specific alkaline phosphatase, anthranilate synthetase, β-lactamase, β-galactosidase, chloramphenicol acetyltransferase etc. express. For this purpose, synthetically produced gene sequences for ACTH or for the ACTH derivatives have been genetically integrated into special expression vectors, in which a DNA sequence is created by the incorporation, which additionally provides a recognition interface for collagenases of the type clostridipeptidease A between the N-terminal Portions of the pre-protein of the alkaline phasphatase and the respective ACTH portion coded. Expression clones of this type allow high protein yields to be achieved by using the phoA regulation system, ie by optimally dosing the phosphate content in the nutrient media. At the same time, the existing recognition sequence for collagenase offers the possibility for the enzymatic release of Gly- Pro-ACTH, Gly-Pro-ACTH 1-24 or other ACTH derivatives, as already mentioned above, which can be split further with PPDA to the native proteohormones (cf. EP 20290).

2. The in vitro integration of the gene sequences for ACTH, AQT 1-24 or other ACTH derivatives, as they have already been mentioned above, into improved espression vectors which have codons N terminal to the potential installation position for additional collagenase recognition sequences. Expression vectors of this type are described in detail in a German patent application filed in parallel (file number p 3731875. £). The resulting technically improved recombination clones enable the same high synthesis rates for the corresponding ones

Fusion proteins, such as the ACTH clones described in step 1. In contrast to this, however, they permit a decisive release for biotechnology, which is accelerated by orders of magnitude (cf. Tab. 2) and quantitative release of z. B. Gly-Pro-ACTH or Gly-Pro-ACTH 1-24.

3. The development of processes that allow biotechnical fermentation and processing of ACTH, ACTH 1-24 and other ACTH derivatives. This methodology is characterized in particular by the fact that the product losses which are characteristic of relatively low molecular weight foreign proteins as a result of unspecific degradation by Escherichia coli proteases could be minimized.

The present invention relates to plasmid vectors which contain a DNA sequence for ACTH or ACTH derivatives and with which bacteria can be transformed into cells which contain these vectors. The invention further relates to methods for producing these plasmid vectors, which are characterized in that DNA insertion fragments, which contain the codons for ACTH or ACTH derivatives and which have Pstl recognition sequences at both ends, are linearized into the Pstl interface and dephosphorylated Inserts vector plasmids with the aid of suitable ligases and E. coli strains containing plasmids with a Ge for ACTH or ACTH derivatives. In addition, the present invention relates to processes for the production of new E. coli strains, which are characterized in that cells of the strain in question are incubated competently with the plasmid DNA for AC or ACTH derivatives under transformation conditions and ACTH clones from the cell population isolated with antibiotic resistance and the use of these new E. coli strains for the production of ACTH or ACTH derivatives

The invention also encompasses a process for the production of new E. coli strains by processes known per se, which is characterized in that competent cells from E. coli E 15 are extracted with a high excess of plasmid DNA for ACTH or ACTH derivatives E. coli SB transformed, and the use of these transformed E. coli E 15 strains for the production of ACTH and ACTH derivatives. In particular, processes for the production of ACTH may be mentioned which are characterized in that the new strains (see claims 13-26) are obtained from a preculture with an aqueous solution of L-proline, L-leucine, casatino acids, vitamin B 1 in concentrated low phosphate medium combined in a fermenter, stirred at 37 ° C. and positive pressure and the ACTH formed is isolated by known methods. The resulting ACTH-containing fusion proteins are subjected to a collagenase and post-proline dipeptidyl aminopeptidase cleavage. Finally, the invention relates to medicaments containing ACTH or ACTH derivatives, which can be prepared by the processes described above, and customary auxiliaries and carriers.

On July 25th, 1987 the following strains were deposited with the DSM, Göttingen:

Escherichia coli E 15 (DSM 4190)

Escherichia coli SB 44 (pSA 125) (DSM 4191) Escherichia coli HB 101 (pSA 504) (DSM 4192) Escherichia coli HB 101 (pSA 271) (DSM 4193)

On July 16, 1979 the following were deposited at the American Type Culture Collection (ATCC) in Rockeville, Md., USA:

Plasmid pBR 322 (ATCC 40015)

Plas id pSB 53 (ATCC 40020)

Escherichia coli SB 44 (pSB 53) (ATCC 31542)

_A m 16.07.1979 were at the DSM, Göttingen deposited: Escherichia coli SB__44 ^{(DSM 1606)}

Escherichia coli HB 101 ⁽ DSM 1607 ⁾

Since November 26, 1973 the DSM has deposited:

-Escherichia coli K 12 wild type ⁽ DSM 498 ⁾

Materials and methods insofar as they are not specified separately in the experiment descriptions

Enzymes: Description of the source:

Restriction Boehringer / Mannheim endonucleases: Pharmacia / P-L, 7800 Freiburg i. Br.

New England Bio Labs GmbH,

6231 Schwalbach

Gibco / BRL, 75H Eggenstein

International Biotechnologies Inc.,

New Haven, CT, U.S.A.

T4 ligase: Boehringer / Mannheim

New England Bio Labs GmbH,

6231 Schwalbach

Pharmacia / P-L, 7800 Freiburg i. Br

DNA polymer gases Boehringer / Mannheim

(Klenow fragment): Pharmacia / P-L, 7800 Freiburg i. Br.

Universal primer for Gibco / BRL, 7514 Eggehstein sequencing using M13mp18 and M13mp19 subclones (see standard procedure A 2.)

alkaline phosphatase from calf intestine: Boehringer / Mannheim

T4 polynucleotide kinase: Pharmacia / PL, 7800 Freiburg i. Br. Host strains for in vitro recombined DNA constructions:

E. coli SB 44 W. Boidol, dissertation Freie Universität Berlin

Genotype: (1984); proA leuB lacZ thi G. Siewert et al. , recA hsdR hscM SupE US Pat. No. 4375514 (1983) str phoA

E. coli E 15 S.J. Hayashi et al., 3rd Biol. Chem. 239 phoAδ relA1 tonA22 3098-3106 (1964)

T E.coli Genetic Stock Center,

2

New Haven, Connecticut, USA

Radioactive chemicals: Amersham Buchler, 3300 Braunschweig

New England Nuclear, 6072 Dreieich

Buffer substances: Merck, Darmstadt

A) Standard procedure

A 1. Plasmid isolation

For the production of purified plasmid DNA, the method described by T. Maniatis et al. [in: 'Molecular Cloning', Cold Spring Harbo Lab., New York, (1982)].

A 2. DNA sequencing

For this, the enzymatic dideoxynucleotide chain termination method according to Sanger et al. [Proc.Nat. Acad. Be J. 5463-5467, (1977) 3. The required single-strand DNA matrices were generally commercially available after recloning the DNA sections to be analyzed into the M13 pages mp18 and mp19; 3rd Mes¬ sing, R. Crea and PH Seeburg, Nucl. Ac. Res. 9_, 309-321, (1981); C. Yanisch-Per _* ron, J. Vieira and J. Messing, Gene -3-3,

103-119, (1985)]. For the radioactive labeling of the resulting polymerase products, Biggin et al.

[Proc. Nat. Acad. Be. 8_0, 3963- 3965, (1983)] Deoxyadeno sin-5 '- [oc-35S] thio-triphosphate used in the substrate mixture.

The resulting radioactive DNA fragment was separated by means of gel electrophoresis in 0.1 mm thin, thermostated (60 ° C.) polyacrylamide / urea gels. To sorge and L. de Maeyer [J. Chromate 202, 45-53, (1980)] using the Macrophor separation system from LKB Instructions.

A 3. Oligonucleotide synthesis

Unless commercially available [e.g. Boehringer / Mannheim or Pharmacia / P-L] available from stock, using the solid phase / phosphotriester method, partially manufactured using a synthesis machine from Applied Bio systems GmbH, 6108 Weiterstadt. The type 381 A was used, the processing and cleaning was carried out according to the information in the corresponding user manual.

E A 4. Oligonucleotide-directed mutagenesis

This procedure was carried out according to the method used by M.J. Zoller and M. Smith [DNA 3_, 479-488, (1984)] carried out modification. The mutants were identified by selective

Hybridization of the Ml3 mutant plaques with the [32P] -labeled mutagen primer nucleotide using the so-called 'plaque-lift' method according to W.D. Benton and R.W. Davis [Science 196, 180-182, (1977)] and the * dot blot * method in the method described by M.J. Zoller and M. Smith [Nucl. Ac. Res. 10, 6487-6500, (1982)].

A 5. Characterization of fusion proteins by 'immunoblotting'

This identification method for expression products which contain parts of the prepeptide of E. coli-specific alkaline phosphatase was described according to H. Towbin, T. Staehelin J. Gordon [Proc. Natl. Acad. Be. 1__, 4350-4354 (1979). A primary antiserum was found after immunization of rabbits under standard conditions [cf. D.M Weir, Handbook of Experimental Immunolgy, Vol. III, Blackwel Scientific Publ., Oxford (1978)] with highly purified alkaline phosphatase from Escherichia coli.

Goat anti-rabbit IgG conjugated with alkaline phosphatase was used as the second antibody [Fa. Cappel represented by Cooper Biomedical, 6000 Frankfur / Main 1] The color reaction was carried out by adding 5-bromo-4-chloro-3 indolyl phosphate.

REPLACEMENT LEAF - 15 -

A 6.5. Isolation of the insoluble proteins:

The insoluble cell disruption products are sedated by means of centrifugation at 0 ° C. and 48,000 × g for 20 minutes. By resuspending twice in 20 ml of 50 mM Tris / HCl pH 8 160 mM NaCl and 10 min. Recentrifugation under the above conditions, the sediments can be obtained free of soluble cellulose substances. They are resuspended in 3 ml of 8 M guanidine ^* HC1 using a dounce homogenizer, heated to 95 ° C. for 1 hour and then cooled to ice temperature. ACTH fusion proteins dissolve stably under these conditions and can be obtained as supernatant after 30 min centrifugation under the above conditions. After 16 hours of dialysis at 2 ° C. against 4 1 50 mM Tris HC1 pH 8, 160 mM NaCl, the proteins are obtained in 4 ml dia lysate as a finely dispersed suspension, which in this form can be used directly for collagenase digestion or can be stored at -20 ° C.

A 6.6. Collagenase digestion

The specific cleavage of the ACTH fusion proteins is expediently carried out using a highly purified collagenase preparation which can be determined by FPLC ion exchange chromatography [cf. Deu patent application file number P 37 31 875.6] of the commercially available Clostridiopeptidase A (EC 3.4.24.3) from Clostri dium histolyticum [Type VII; Sigma GmbH].

For standard analyzes, an amount of protein equivalent to 100 | i main culture, that is 4 μl protein suspension (dialysate), in 10 μl 50 M Tris / HCl pH 8, 5 mM CaCl- with desired units [cf. E. Wünsch et al., Hoppe Seyler's Z Physiol. Chem. 333, 149-151 (1963)] Clostridiopeptidase A incubated for 1 h at 37 ° C. Thereafter, the cleavage product can be separated directly on SDS-polyacrylamide gels under standard conditions [UK Laemmli, Nature 227, 680-685 (1970)], stained in 0.2% Coomassie Blue G-250, and by means of the immunoblotting method described above (cf. A 5). B) Standard solutions:

B 1. Reaction buffer for enzymatic "reactions

B 1.1. Restriction endonucleases

Unless otherwise specified in the individual examples, 3 basic buffers according to T. Maniatis et al. [T. Maniatis, E.F. Fritsch and J. Sambrook in: 'Molecular Cloning', pp. 100-101 and 104-106, Cold Spring Harbor Lab., New York (1982)]:

Low buffer: 10 mM Tris / HCl, 10 mM MgCl ₂ , 1 mM dithiothreit pH 7.5

Medium buffer: 50 mM NaCl, 10 mM Tris / HCl, 10 M MgCl ₂ , 1 mM

Dithiothreitol, pH 7.5

High buffer: 100 mM NaCl, 50 mM Tris / HCl, -10 mM mgCl ₂ ,

1 mM dithiothreitol, pH 7.5

B 1.2. Calf intestine alkaline phosphatase

For this, the methods described by T. Maniatis et al. [in: 'Molecular Cloning ¹ , pp. 133-134, Cold Spring Harbor Lab., New York (1982)].

B 1.3. T4 DNA ligase

50 mM Tris / HCl, 10 mM MgCl ₂ , 20 mM dithioerythritol, 1 mM ATP, 50 μg / l bovine serum albumin, pH 7.8

B 1.4. Polynucleotide kinase

(from T4-transfected E.coli B)

100 mM Tris / HCl, 10 mM MgCl ₂ , 10 mM dithiothreitol, pH 8.3 B) Standard solutions:

B 1. Reaction buffer for enzymatic reactions

B 1.1. Restriction endonucleases

Unless otherwise specified in the individual examples, 3 basic buffers according to T. Maniatis et al. [T Maniatis, E.F. Fritsch and J. Sambrook in: 'Molecular Cloning', pp. 100-101 and 104-106, Cold Spring Harbor Lab., Ne York (1982)]:

Low buffer: 10 mM Tris / HCl, 10 mM MgCl ₂ , 1 mM dithiothrei pH 7.5

Medium buffer: 50 M NaCl, 10 M Tris / HCl, 10 mM MgCl ₂ , 1 mM

Dithiothreitol, pH 7.5

High buffer: 100 mM NaCl, 50 mM Tris / HCl, 10 mM gCl ₂ ,

1 mM dithiothreitol, pH 7.5

B 1.2. Calf intestine alkaline phosphatase

For this, the methods described by T. Maniatis et al. [in: 'Molecula Cloning', pp. 133-134, Cold Spring Harbor Lab., New Yor (1982)].

B 1.3. T4 DNA ligase

50 M Tris / HCl, 10 mM MgCl ₂ , 20 mM dithioerythritol, 1 M ATP 50 μg / ml bovine serum albumin, pH 7.8

B 1.4. Polynucleotide kinase

(from T4-transfected E.coli B)

100 mM Tris / HCl, 10 mM MgCl ₂ , 10 mM dithiothreitol, pH 8.3

REPLACEMENT LEAF B 2. Storage buffer for DNS

'TE buffer ^! : 45 M Tris / HCl, 0.1 mM EDTA, pH 7.9

B 3. Electrophoresis buffer

B 3.1. 'TBE buffer': 90 mM Tris / HCl, 80 mM H ₃ B0 ₃ ,

2.5 mM EDTA, pH 8.3 B 3.2. 'TAE buffer': 40 mM Tris acetate, 2 mM EDTA, pH 8.

B 3.3. Working buffer for gel electrophoretic protein separation according to V.K. Laemmli [Nature 227, 680-685 (1970)]

0.25 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3

B 4. Buffer for the extraction of DNA from polyacrylamide gels

0.5 M ammonium acetate, 1 mM EDTA, pH 8.0

B 5. Buffer for the electroelution of DNA from agarose gels

45 mM Tris / HCl, 40 MH ₃ B0 ₃ , μ.25 mM EDTA, pH 8.3

B 6. Buffer for selection of bacterial mutants according to the colony filter hybridization method

All necessary buffer solutions were used, as with R.B. Wallace et al. described [Nucl. Ac. Res. 9_, 879-89 (1981)].

B 7. Buffer for the 'DOT BLO' identification of M13 phage plaques

All required buffer solutions were used as described by MJ Zoller and M. Smith [Nucl. Ac. Res. 10 6487-6500 (1982) 1. C) Execution examples

C 1. Production of the E. coli SB 44 expression clone (pSA 172)

This clone synthesizes a fusion protein consisting of the N-terminal amino acids 1-446 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1983)], followed by the collagenase recognition sequence -Pro-Ala-Gly-Pro and the ACTH-24 sequence (SA 172):

Alk. Phosphatase (1-446) -Pro-Ala-Gly-Pro-ACTH 1-24 ι i

Collagenase recognition SA 172 sequence

C 1.1. Integration of an 86 bp-Pstl restriction fragment with codons for ACTH 1-24 into the PstI interface in position 1624 of the phoA plasmid pSA 125 [as E.coli SB44 (pSA 125) = DSM 4191J.

[see. Fig. 1}

C 1.1.1. Production of the linear vector plas ids

1 μg of pSA 125 DNA was incubated in 50 μl of medium buffer (solution B 1.1. With 0.43 units of PstI [Boehringer / Mannheim] for 5 min at 37 ° C. Partial digestion of the plasmid takes place under these conditions The reaction mixture was made 15 mM of EDTA and the enzyme was thereby inactivated.The two linear 6458 bp fragments could be separated by gel electrophoresis in 0.7% agarose (buffer B 3.2.) And were identified using an ethidium bromide-stained marker DNA the DNA was done by electroelutio in 1 ml elution buffer (solution B 5.) according to Maniatis ^• et al. [i 'Molecular Cloning', pp. 164-165, Cold Spring Harbor Lab., N York (1982)]. The solution was adsorbed by means of a 2 ml Einwe syringe in adsorption buffer (TE buffer, solution B 2. + 0.2 NaCl) on a finished column [type Elutip-d from Schleich & Schüll] and treated according to the manufacturer's instructions. The desorption was carried out accordingly with TE buffer (solution B 2.) 1 M NaCl and gave 0.5 ml of eluate. By adding 1 ml of ethanol, heating for 3 hours at -25 ° C and following centrifugation (10 min., 12,000 xg), the 6458 B DNA mixture could be precipitated and sedimented in pure form.

C 1.1.2. Dephosphorylation of the linear vector plasmid

The DNA was dissolved in 5 μl TE buffer (solution B 2.) and 10 μl reaction volume with alkaline phosphatase from the small intestine according to Maniatis et al. (Solutions according to B 1.2.) Depho-phorylated. After extraction with 300 μl TE-saturated pheno and twice extraction with 600 μl ether each, 150 were made of NaCl and precipitated with 0.5 ml ethanol (as described in C 1.1.1. B) and sedimented.

C 1.1.3. Preparation of the ACTH 1-24 DNA insertion fragment

A 86 bp DNA fragment with PstI recognition sequences at the ends, which contains the codons for ACTH 1-24, was deposited from plasmid pSA 271 (see FIG. 2) [as E. coli HB 101 (pSA 271) = DSM 4193].

50 μg pSA 271-DNA were incubated in 200 μl medium buffer (solution 1.1.) With 198 units PstI [Boehringer / Mannheim] for 2 S at 37 ° C. After extractions with 200 μl TE-Puff saturated phenol and 2x 400 μl ether, 0.3 mM of acetate was made and temperature-controlled with 500 μl ethanol at -80 for 20 min. The DNA mixture was sedimented by centrifugation at 12,000 × g for 20 min. And was poured into 10 μl Buffer (solution B 2.) and 2 μl charge buffer type II [T. Manitis, EF Fritsch and J. Sambrook in: 'Molecular Cloning', 160, Cold Spring Harbor Lab., New York (1982)]. After gel electrophoretic separation, the Bp fragment could be cut out from an 8% acrylic gel and according to Maniatis al. [in: 'Molecular Cloning', p. 178, Cold Spring Harb Lab., New York (1982)].

C 1.1.4. Linking vector and insertion fragment with T4 DNA ligase

0.5 μg of desphosphorylated pSA 125 vector DNA were m 0.5 μg of the isolated 86 bp fragment in 100 μl ligase powder (solution B 1.3.) With 4.5 Weiss units [B. Weiss et al. J. Biol. Chem. 243, 4543-4555 (1968)] T4 DNA ligase incubated for hours at 23 ° C. The reaction mixture was used for the transformation of competent recipient cells.

C 1.2. Transformation of Escherichia coli SB 44

C 1.2.1.

50 ul T4 ligase reaction products were used to transform competent E. coli SB 44 cells among those described by M. Dage and S.D. Ehrlich [Gene 6_, 23-28 (1979)] conditions used.

C 1.2.2. Selection of transformants

The ampicillin resistance is retained when it is incorporated into the desired Pstl site in positi 1624 of the vector. The transformation mixture was therefore placed on LB agar [T. M niatis, E.F. Fritsch and J. Sambrook in: 'Molecular Cloning' p. 440, Cold Spring Harbor Lab., New York (1982)], which contained 100 μg / ml ampicillin. Twelve clones were selected from 220 transformant colonies for the isolation of plasma mid-DNA.

REPLACEMENT LEAF C 1.3. Characterization of transformants by size determination of DNA fragments after implementation with special restriction endonucleases

The plasmid isolation from 12 randomly selected clones was carried out according to standard conditions [cf. A 1.]. By digesting back with PstI, the incorporation of the 86 bp fragment could be ensured in all cases.

C 1.3.1.

5 μg DNA were incubated in 100 μl medium buffer (solution 1.1.) With units PstI [Boehringer / Mannheim] for 2 hours at 37 ° C. After extraction with 100 μl of TE-saturated phenol and 300 μl of ether, 0 ^' . 3 M of Na acetate was made and the DN was precipitated with 3 volumes of ethanol for 20 min at -80 ° C. Nac

Sedimentation according to 1.1.1. the fragment sought was found in a 4% NuSieve ™ agarose gel [Marine Colloids, Rockland

ME, USA] in TBE buffer (solution B 3.1.) Gel electrophoresis and identify them with ethidium bromide after staining.

C 1.3.2.

By double digestion of the plasmid DNA with EcoRI and Ncol and subsequent size determination of the resulting fragment, clones could be identified which contain the ACTH 1-24 in the desired orientation:

5 μg DNA were in 100 μl high buffer (solution B 1.1.) With j 10 units EcoRI and Ncol [BioLabs, cf. P. 9] for 2 hours at 37 ° C and, as in C 1.3.1. described, extracted and ethanol-fallen. The product mixture was separated by electrophoresis in a 5% polyacrylamide gel in TBE buffer (solution B 3.1.) And the separation result after staining with ethidium bromide was evaluated visually. Clones which contain the ACTH 1-24 insertion fragment inserted in the desired pho-A transcription direction have fragments of

96, 232, 235 and 5981 base pairs.

Once the counter strand has been integrated in a corresponding manner, the fragment sizes can be found

96, 235, 282 and 5931 base pairs.

5 test clones showed the typical band pattern for correct installation.

C 1.3.3. Confirmation of full integration of the

86 bp ACTH 1-24 gene fragment by DNA sequencing

3 test clones each with an integrated 86 bp-Pstl-DNA frag were found in both possible orientations, as in C 1.1.3. b wrote, implemented with PstI and isolated the Pstl insertion fragments. After recloning in the M13 phage mpl8, sequencing was carried out under standard conditions [cf. A 2.].

One of the transformant clones, which according to C 1.3. containing the ACTH 1-24 gene fragment completely and incorporated in the phoA transcription direction was further cultivated under the name SB 44 (p 172) for the subsequent protein analysis.

[see. Fig 3, pSA 172]

C 2. Production of the E. coli SB 44 expression clone (pSA 186)

This clone synthesizes a fusion protein consisting of d N-terminal amino acids 1-446 of the prepeptide of the alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1983)], followed by the Collagenaseseque -Pro-Ala-Gly-Pro and the ACTH sequence (SA 186) consists of:

Alk. Phosphatase (1-446) -Pro-Ala-Gly-Pro-ACTH i 1

Collagenase recognition SA 186 sequence

C 2.1. Integration of a 131 bp Pstl restriction fragment with codons for ACTH into the Pst / interface i position 1624 of the phoA plasmid pSA 125 (see Fig. 1)

C 2.1.1. Production of the linear vector plasmid

(as described in C 1.1.1.)

C 2.1.2. Dehosphorylation of the linear vector plasmid

(as described in C 1.1.2.)

C 2.1.3. Preparation of the ACTH-DNA insertion fragment

A 131 bp DNA fragment with PstI recognition sequences at the ends, which contains the codons for ACTH [cf. Fig. 4a ACTH sequence] was from plasmid pSA 504 [deposited as E. coli HB 101 (pSA 504) - DSM 4192].

[see. Fig. 4, pSA 504]

50 μg pSA 504-DNA were among the in C 1.1.3. described conditions implemented with PstI. According to C 1.1.3. the required 131 bp-Pstl fragment could also be obtained. C 2.1.4. Linking vector and insertion fragment with T4 DNA ligase

0.5 μg of the isolated 131 bp fragment was obtained according to the methods described in 1.1.4. link conditions described with the vector DNA

C 2.2. Transformation of Escherichia coli SB 44

50 μl of the ligase approach were carried out as described in C 1.2. described, used.

C 2.2.1. Selection of transformants

The under C 1.2.1. described selection on A picillin-LB agar plates could also be used here. From 180 transformant colonies, 12 clones were selected for the isolation of plasmid DNA.

C 2.3. Characterization of transformants by size determination after reaction with special restriction endonucleases

C 2.3.1.

The detection of the 131 bp fragment incorporation by digestion with PstI and gel electrophoretic analysis was carried out in accordance with. C 1.3.1. with 5 μg plasmid DNA from each of the 12 test clones.

C 2.3.2.

The determination of the installation direction was carried out under the same conditions as in C 1.3.2. described, each with 5 μg of DNA ^' .

Clones which contain the ACTH insertion fragment inserted in the desired phoA transcription direction have fragments of

96, 232, 235 and 6026 base pairs. If the counter strand is integrated in a corresponding manner, the fragment sizes can be found

96, 235, 327 and 5931 base pairs.

4 test clones showed the typical band pattern for correct installation.

C 2.3.3. Confirmation of full integration of the 131 bp ACTH gene fragment by DNA sequencing

The 4 in C 2.3.2. Test clones described and 2 clones with opposite installation orientation were gem. C 1.3.3. i Ml3mpl8 subcloned and sequenced.

One of the transformant clones, which according to C 2.3. containing the ACTH gene fragment completely and built into the phoA transcription direction was further cultivated under the name SB 44 (pSA 186 for the subsequent protein analysis.

[see. Fig. 5, pSA 186]

C 3. Production of the expression clone SB44 (pSA 341)

This clone synthesizes a fusion protein consisting of the N-terminal amino acids 1-444 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1983)], followed by glycine and 5 collagenase identification sequences before the ACTH sequence (SA 341):

Alk.phosphatase (1-444) -Gly-Pro-His-Gly- (Pro-Ala-Gly), -Pro- A

5 collagenase recognition sequences SA 341

REPLACEMENT LEAF C 3.1. Single or multiple integration of 131 bp-Pstl restriction fragments with codons for ACTH into the Pstl interface in position 1657 of the vector plas mids pSA 506

^{(Vg l.} H. Scheidecker et al., German Patent Application No. P 37 31 875.6 and A _b b.. ₆₎

C 3.1.1. Production of the linear vector plasmid

1 μg pSA 506-DNA (produced according to P 37 31 875.6) was as in C 1.1.1. described with 0.2

Units PstI [Boehringer / Mannheim! partially digested at 37 ° C for 5 min and the mixture of linear 6499 bp fragments after electrophoretic separation in a 0.8% agarose gel according to C 1.1.1. isolated.

C 3.1.2. Preparation of the ACTH-DNA insertion fragment

(as described in C 2.1.3.)

C 3.1.3. Linking vector and insertion question ent with T4 DNA ligase

0.1 μg of Pstl-linearized pSA506 vector DNA were mixed with 0.1 μ of the isolated 131 bp ACTH fragment in 50 μl of ligase puff (B solution 1.3.) With 1 Weiss unit [B. Weiss et al., J. Biol Chem. _24_3_ _/ 4543-4555 (1968)] T4 DNA ligase [Boehringer / Mann heim] incubated overnight at 9 ° C.

C 3.2. Transformation of Escherichia coli SB 44

C 3.2.1.

The entire ligase reaction mixture was used to transform competent E. coli SB 44 cells as described under 1.2.1. refereed, used.

REPLACEMENT LEAF 3.2.2. Selection of transformations

On LB agar [T. Maniatis, E.F. Fritsch and J. Sambrook in 'Molecular Cloning', p. 440ff, Cold Spring Harbor Lab., Ne York (1982)] with 100 μg / ml A picillin content, approximately 800 resistant clones were obtained.

C 3.3. Characterization of transformants

Because of the symmetrical Pstl ends of the vector and the insertion fragment, different installation orientations were assumed. In addition, clones with repeatedly integrated 131 bp fragments or partially deleted multiple collagen recognition sequence were to be expected, and moreover, the vector mixture with the sequence positions 1630 and 1657 bp [cf. Fig. 6] two competing integrators are available. The characterization of the correspondingly complex complex transformant spectrum had to be done in several steps.

C 3.3.1. Identification of clones containing ACTH gene sequences

For this purpose, 700 clones were placed on LB agar [T. Maniatis, E.F. Frits and J. Sambrook in: 'Molecular Cloning', p. 440, Cold Spri Harbor Lab., New York (1982)] inoculated with 50 μg / ml ampicillin and transferred to Whatman 540 filters. The identification was made according to one of R.B. Wallace et al. [Nucl. A Res. 9_, 879-894 (1982)] and J.P. Gergen et al. [Nucl. A Res. _8_, 2115-2136 (1979)] drive hybridization described. A 15 bp oligonucleotide which had been used to prepare the ACTH plasmid pSA 504 could be used as a specific hybridization sample [commercial preparation from Applied Biosystems, 6108 Weiterstadt]. It has the sequence:

CTTCTTGCCCACCGG-3

REPLACEMENT - 29 -

0.6 αg of the nucleotide were according to MD Edge et al. [Nature 292, 756-762 (1981)] with 20 μCi [^ - ³² P] ATP [Fa. Amersham

Buchler1 and 1 unit of T4 polynucleotide kinase phosphorylated and thus radioactively labeled. The 32P-phosphorylated product was separated off according to K.-M. Lo et al. [Proc.

Natl. Acad. Be jTl, 2285-2289 (1984)] using a SEP-Pak

TM C 18 single-use column [Fa. Waters GmbH, 6240 Koenigstein / Ts. ].

280 clones showed a positive hybridization signal with the

P sample.

C 3.3.2. Identification of clones containing codons for 5 collagenase recognition sequences

This test was also carried out according to the in 3.3.1. described colony hybridization with a radioactively marked 45 base oligonucleotide sample carried out for the production of the vector plasmid pSA 506 [Fig. 6 and German patent application, file number P3731875.6 ^~ ~ under standard conditions [cf. A 3.3 had been synthesized.

5 '-CGGACCTGCAGGGCCTGCTGGACCCGCCGGGCCTGCAGGACCATG-3' to pSA 341 complementary area

The 3 'end of this oligonucleotide is complementary over a region of 41 bases (90%) to the gene segment in the information strand of the plas ids pSA 341 [Fig. 7], which for the 5 consecutive collagenase recognition sequences in the fusion protein [cf. C 3.] coded. do

Of 100 randomly selected clones, which according to C 3.3.1. Containing ACTH inserts showed 35 positive hybridization signals with the oligonucleotide specific for the multiple collagenase recognition sequence (see above). C 3.3.3. Characterization of transformants by size determination of DNA fragments after implementation with special restriction endonucleases

The plasmid isolation from 16 randomly selected clones according to the result of 3.3.2. takes place according to standard conditions [cf. A I.].

C 3.3.3.1. Determination of the number of 131 bp ACTH installed

Fragments

5 μg of DNA were mixed with 20 units of EcoRI [BioLabs, cf. Pp. 10 and 10 units of SphI [Boehringer / Mannheim] as described in C 1.3.2, implemented, the products separated by gel electrophoresis and the result evaluated visually.

C 3.3.3.2. Determination of the installation orientation for the

131 bp ACTH fragment

5 μg of DNA were mixed with 7 units of Ncol [BioLabs, cf. S. 1 and 3 units SphI [Boehringer / Mannheim], as in C 1.3. described, implemented, the products separated by gel electrophoresis and the result evaluated visually.

C 3.3.3.3. Identification of clones whose insertion at the Pstl interface position 1657, i.e. 'downstream' to the quintuple collagenase Ge fragment

10 μg of DNA were mixed with 18 units of Hinfl [BioLabs, cf. P. 1 digested in 90 μl 'Medium buffer' (solution B 1.1.) For 2 hours at 37. Then 10 μl 0.41 M Tris / HCl pH 7.5 + 0.55 NaCl + 18 units Ncol [BioLabs, cf. P. 10] added and incubated for a further 2 hours at 37 ° C. The test was again processed and evaluated in accordance with C 1.3.2. By comparative analysis of the according to C 3.3.3.1, C 3.3.3.2 and C 3.3.3.3. The results obtained revealed a recombinant clone whose gel band pattern corresponds to the structure of the plasmid pSA 341 [Fig. 7] corresponded.

C 3.3.4. Structure confirmation through DNA sequencing

From the DNS implementation according to C 3.3.3.1. a 519 bp EcoRI / Sphl restriction fragment as described in C 1.1.3. described, isolate and subclone aliquots of it into phages M13mpl8 and Ml3mpl9. Using suitable Polymeras I primers, standard sequencing conditions [cf. A 2.] the identity of the clone SB 44 (pSA 341) with de tandem-shaped integration of two 131 bp ACTH-DNA fragments into the Pstl interface at position 1657 of the vector pSA 506 are confirmed.

[see. Fig. 7, pSA 341]

C 3.3.5. Other expression clones from the cloning procedure given for plasmid pSA 341

Among the remaining 15 plasmid isolates from C 3.3.3. Using the various analysis steps described there, 2 further clones could be identified which express fusion proteins suitable for the production of ACTH.

C .4. Characteristics of the expression clone 'SB 44 (pSA 343)

This clone synthesizes a fusion protein which consists of the N-terminal amino acids 1-444 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1983)], followed by glycine and 2 collagenase recognition sequences before the ACTH sequence (SA 343), consists of:

Alk. Phosphatase (1-444) -Gly-Pro-His-Gly-Pro-Ala-Gly-Pro-ACT

2 collagenase detection SA 343 sequences From the analytical data [cf. C 3.] it was found that ei

131 bp-Pstl restriction fragment with the codons for ACTH i the Pstl site in position 1630 of the vector plasmid pSA 506 [cf. Fig. 6] was integrated.

[see. Fig. 8, pSA 343]

C 5. Properties of the expression clone SB 44 (pSA 345)

This clone synthesizes a fusion protein that is identical to the product expressed by v SB 44 (pSA 341) [vg C 3.].

From the analytical data it follows that a 131 bp-Pst restriction fragment with the codons for ACTH into the Pst interface in position 1657 of the vector plasmid pSA 5 [cf. Fig. 6] 'was integrated.

[see. Fig. 9, pSA 345]

C 6. Production of Expression Clone SB 44 (pSA 353)

This clone synthesizes a fusion protein consisting of d amino acids 1-444 of the prepeptide of alkaline phosphates [M. Simonis, dissertation Freie Universität Berlin, Berl (1983)], followed by glycine and 5 collagenase recognition sequences before the ACTH 1-24 sequence (SA 353), consists of:

Alk.phosphatase (1-444) -Gly-Pro-His-Gly- (Pro-Ala-Gly) -Pro-ACTHl-24

4 l I

5 collagenase detection sequences SA 353 C 6.1. Integration of an 86 bp-Pstl restriction fragment with codons for ACTH 1-24 into the Pstl interface in position 1657 of the vector plasmid pSA 506

C 6.1.1. Production of the linear vector plasmid

(as described in C 3.1.1.)

C 6.1.2. Preparation of the ACTH 1-24 DNA insertion fragment

(as described in C 1.1.3.)

C 6.1.3. Linking vector and insertion question ent with T4 DNA ligase

0.1 μg Pstl-linearized pSA 506 vector DNA were mixed with 0.1 μg of the isolated 86 bp ACTH 1-24 fragment under the i C 3.1.3. described conditions implemented.

C 6.2. Transformation of Escherichia coli SB 44

C 6.2.1.

(as described in C 3.2.1.)

C 6.2.2. Selection of transformants

Implementation as in C 3.2.2. described. About 600 ampicillin-resistant clones were obtained.

C 6.3. Characterization of transformants

(see comment on C 3.3) C 6.3.1. Identification of clones containing ACTH gene sequences

As in C 3.3.1. described. 315 clones showed a positive hybridization signal with the [32P] sample.

C 6.3.2. Identification of clones containing codons for 5 collagen recognition sequences

As in C 3.3.2. described. 41 clones were obtained, that is with the oligonucleotide specific for the multiple collagenase recognition sequence [cf. C 3.3.2.] Hybridized under stringent conditions (15 minutes, 50 ° C, 50% formamide).

C 6.3.3. Characterization of transformants by size determination of DNA fragments after implementation with special restriction endonucleases

As in C 3.3.3. described. By comparative analysis de as in C 3.3.3.1. - C 3.3.3.3. The results obtained showed a recombination clone whose gel band pattern corresponded to the structure of the plasmid pSA 353 (Fig. 10)

C 6.3.4. Structure confirmation through DNA sequencing

As in C 3.3.4. described. The incorporation of the 86 bp ACTH 1-24 DNA fragment into the PstI site at position 1657 d vector pSA 506 was confirmed.

[see. Fig. 10, pSA 353]

C 7. Production of Expression Clone SB 44 (pSA 509)

This clone synthesizes a fusion protein which consists of d N-terminal amino acids 1-444 of the prepeptide of the alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1982)], followed by glycine and 5 collagenase-E

REPLACEMENT identification sequences before the ACTH sequence (SA 509), there is:

Alk..Phosphaεase ^(' 1-444) -Gly-Pro-His-Gly- (Pro-Ala-Gly) -Pro-lyr-Gly-Pro-ACTH ul

5 ollagenase recognition sequence

SA 509

C 7.1. Integration of a 158 bp Pstl fragment with codons for 3 1/2 collagenase interfaces before ACTH into the Pstl interface in position 1630 of the vector plasmid pHS 4133

[see. German patent application file number P 37 31 875.63

C 7.1.1. Production of the linear vector plasmid 6.7 μg pHS 133-DNA (produced according to P 37 31 875.6) was carried out in 60 μl 'medium buffer' (solution B 1.1.) With 2.4 units PstI (Boehringer / Mannheim) for 1 hour at 37 ° C partially digested. The preparation of the batch and the gel electrophoretic isolation of the linear 6473 bp-DNA mixture were carried out in accordance with C 1.1.1.

C 7.1.2. Dephosphorylation of the linear vector plasmid

The DNA was dissolved in 26 μl TE buffer (solution B 2.) and in 30 μl reaction volume, as in C 1.1.2. described, dephosphorylated and processed.

C 7.1.3. Preparation of the ACTH-DNA insertion fragment

This fragment could be ligase-linked to a sub-fragment from pSA 186 [cf. Fig. 5] with 2 synthetically manufactured oligonucleotides. C 7.1.3.1. Preparation of a 113 bp DNA fragment from pSA 186 with partial sequences for ACTH

90 μg pSA 186 were digested in 180 μl 'medium buffer' (solution B 1.1.) With 300 units PstI [Boehringer / Mannheim] for 2 1/2 hours at 37 ° C. Then 120 μl 0.11 M Tris / HCl pH 7.5 + 0.175 M NaCl + 60 units Ncol [BioLabs, cf. P. 10] added and incubated for a further 2 1/2 hours at 37 ° C. After extraction with 300 TE-saturated phenol and zweima¬ liger ul extraction with 600 .mu.l ether was 0.2 M Na-Äcetat ^"made, the DNA overnight at -25 ° C precipitated and gem. C 1.1.1. Sedimented. After Dissolving in 16 μl TE buffer, the product mixture was separated by gel electrophoresis in 6% polyacrylamide in TBE buffer (solution B 3.1.) And the 113 bp-Pstl / Ncol restriction fragment was isolated according to C 1.1.1.

C 7.1.3.2. Production of two oligonucleotides to supplement the one described in 7.1.3.1. produced ACTH partial fragment

The two oligonucleotides are complementary to one another and, under suitable conditions, form a 45 bp-Pstl / Ncol hybrid. Synthesized and worked up under standard conditions [cf. A 3.] became the 41 base nucleotide

5 '-GGCCCGGCGGGTCCAGCAGGCCCTTATGGACCTAGCTACTC-3'

and the 49 base nucleotide

5 '-CATGGAGTAGCTAGGTCCATAAGGGCCTGCTGGACCCGCCGGGCCTGCA-3'.

Both oligonucleotides were phosphorylated at the 5 'end:

2.7 μg each of the 41-base and 3.3. μg of the 49-base nucleotide were each in 20 μl reaction volume in kinase buffer (solution B 1.4.) with 10 units each of nucleotide kinase [Phar macia / PL, cf. P. 10] for 1 hour at 37 ° C. Tue Reactions were stopped by inactivating the enzyme at 65 ° C. for 10 minutes each.

C 7.1.3.3. Linking the according to C 7.1.3.1. and C 7.1.3.2 products obtained using T4 DNA ligase

The inactivated kinase product mixtures were combined with 10 μl 10 × concentrated ligase buffer (solution B 1.3. 10 × concentrated), 1 μg 113 bp DNA fragment from C 7.1.3.1 and H ^ O to 92 μl and supplemented to 50 ° C tempered. After 30 min. Cooling to room temperature, the reaction volume was added by adding 48 Weiss units [B. Weiss et al., J Biol. Chem. 2 A3_, 4543-4555, (1968)] to 100 μl and incubated overnight at 13 ° C. Finally, 100 μl of TE-saturated phenol and two 200 μl of ether were extracted and as in C 7.1.3.1. ethanol fallen and worked up

C 7.1.3.4. Processing of oligomer products

Post-digestion with PstI

The ligase products were incubated in 100 μl of medium buffer (solution 1.1.) With 9 units of PstI [Boehringer / Mannheim] for 3 hours at 37 ° C. and again according to C 7.1.3.3. worked up

C 7.1.3.5. Isolation of the 158 bp-Pstl DNA fragment with

Codons for 3 1/2 collagenase recognition sequences before ACTH

The mixture of the Pstl-digested ligase products was dissolved in 16 μl _, TE buffer and separated by gel electrophoresis in 10 polyacrylamide in TBE buffer (solution B 3.1.). The 158 bp fragment sought was isolated in accordance with C 1.1.1. C 7.1.4. Linking vector and insertion fragment with T4 DNA ligase

2 μg desphosphorylated pHS 4133 vector DNA 'from C 7.1.2. were obtained with 14 ng 158 bp fragment in 20 μl ligase buffer (solution B 1.3.) with 9 white units [B. Weiss et al., J. Biol. Chem. 243, 4543-4555 (1968)] T4 DNA ligase incubated overnight at 13 ° C. Finally, according to 7.1.3.1. extracted with phenol and ether, ethanol precipitated and worked up.

C 7.2. Transformation of Escherichia coli SB 44

1/3 of the ligase products from C 7.1.4. was dissolved in 50 μl TE buffer and used to transform competent E. coli SB 44 cells, as in C 1.2.1. lectured, used. 203 ampicillin-resistant clones were found.

C 7.3. Characterization of transformants by determining the size of DNA fragments after reaction with PstI and Ncol

The plasmid isolation from 16 clones selected at random was carried out under standard conditions [cf. A I.].

C 7.3.1. Digestion with PstI

As in C 1.3.1. described. 10 clones were found which had the 158 bp-Pstl insert.

C 7.3.2. Determination of the installation orientation for the 158 bp ACTH fragment

5 μg DNA each were in 20 μl 'high buffer' (solution B 1.1.) With 7 units Ncol [BioLabs, cf. P. 10] for 3 hours at 37 ° C. The size of the restriction fragments was then determined according to gel electrophoresis. C 1.3.2. in counter were from DNA size markers. Clones which contain the insertion fragment in the desired phoA transcription direction have fragments of

500 and 6130 base pairs.

If the counter strand is integrated in a corresponding manner, fragment sizes of

568 and 6062 base pairs.

A test clone (pSA 509) showed the typical Ncol band pattern for correct installation.

C 7.3.3. Structure confirmation through DNA sequencing

15 μg pSA.509 DNA were in 30 μl high buffer (solution B 1.1.-) with 25 units SphI [Boehringer / Mannheim] and 40 units EcoRI [BioLabs, cf. P. 10] for 5 hours at 37 ° C. The mixture of fragments was then analyzed by gel electrophoresis. C 1.1.3. separated and a 388 bp fragment isolated, which contained the entire coding region for the collagenase interfaces and the ACTH. This fragment was subcloned into the phages Ml3mpl8 and Ml3mpl9 and under standard conditions [cf. A 2.] sequenced.

The structure of the ACTH expression clone pSA 509 was confirmed from the sequence data:

[see. Fig 11, pSA 509]

C 8. Production of Expression Clone SB 44 (pHS 6134)

This clone synthesizes a _^ fusion protein which consists of the N-terminal amino acids 1-297 of the prepeptide of the alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1983)], followed by proline, tryptophan and 4 collagenase recognition sequences before the ACTH sequences (HS 6134), consists of:

ALk.phosphatase (l-297) -Pro-Trp- (Pro-Ala-Gly) -Pro-Iyr-Gly-Pro-ACTH

4 cell-detection sequences.

HS 6134

C 8.1. Integration of a 158 bp-Pstl restriction fragment with codons for 3 1/2 collagenase cleavage site before ACTH 1-39 into the Pstl cleavage site in position 1183 of the vector plasmid pSA 302

^[ cf. German patent application file number P 37 31 875.6]

C 8.1.1. Production of the linear vector plasmid ₁ μg p _{SA 3} 0 ₂ (produced according to P 37 31 875.6) was carried out in 50 μl reaction volume, as in C 1.1.1. described, partially digested with PstI [Boehringer / Mannheim]. The mixture of the two possible linear 6472 bp fragments could be, likewise according to. C 1.1.1., Gel electrophoresis.

C 8.1.2. Dephosphorylation of the linear vector plasmid

(as described in C 1.1.2.)

C 8.1.3. Preparation of the 158 bp Pstl insertion fragment

50 μg pSA 509-DNA are made according to C 1.1.3. digested with PstI and worked up after gel electrophoretic isolation.

C 8.1.4. Linking vector and insertion fragment with T4 DNA ligase

0.1 μg dephosphorylated pSA 302 vector DNA was 0.25 μg of the isolated 158 bp fragment in 50 μl Ligase-P fer (solution B 1.3.) with 1 white unit [B. Weiss et al., J. Biol. Chem. 243, 4543-4555 (1968)] T4 DNA ligase [Boehringer / Mannheim] incubated overnight at 16 ° C.

C 8.2. Transformation of Escherichia coli SB 44

C 8.2.1.

The entire ligase reaction mixture from C 8.1.4. was acc. C 1.2.1. used to transform competent E.coli SB 44 cells.

C 8.2.2. Selection of transformants

The ampicillin resistance is retained when incorporated into the desired Pstl interface in position 1183 of the vector. The transformer selection could therefore according to C 1.2.2. 15 ampicillin-resistant clones were selected for further investigations.

C 8.3. Characterization of transformants by determining the size of DNA fragments after reaction with PstI and Rsal

C 8.3.1. Digestion with PstI

As in C 1.3.1. described. The 158 bp insert was clearly detected for 10 transformant clones.

C 8.3.2. Determination of the insertion orientation of the 158 bp-Pstl insertion fragment

4 μg of plasmid DNA were mixed with 7.5 units of Rsal [BioLabs, cf. P. 10] in 100 μl 'Medium buffer' (solution B 1.1.) Incubated overnight at 37 ° C. The product mixture was separated and visually evaluated in accordance with C 1.3.2. Of the total of 9 possible Rsa restriction fragments, two are lengths

97 and 114 base pairs

characteristic for installation in the desired phoA transcription direction.

With appropriate integration of the counter strand, ma finds the fragment sizes instead

^'68 and 143 base pairs.

2 test clones showed the typical band pattern for correct installation. They received the designations pHS 6134 and pHS 613

[see. Fig 12, pHS 6134]

C 9. Production of Expression Clone SB 44 (pSA 351)

This clone synthesizes a fusion protein which is identical to the product expressed by SB 44 (pHS 6134) [cf. C 8.1.

The expression plasmid pSA 351 contains, compared to the plasmid pHS 6134, a deletion of 461 base pairs in the non-translated DNA region between the ocher codon of the ACTH and the terminator region [cf. Chung Nan Chang et al. , Gene 44 121-125 (1986)] of the phoA gene.

C 9.1. Linearization of the expression plasmid pHS 6134 (Fig. 12)

10 μg pHS 6134-DNA were incubated in 50 μl 'high buffer * (Ls B 1.1.) With 10 units SphI [Boehringer / Mannheim] for 2 hours at 37 ° C, according to. C 1.1.3. extracted with phenol and ether and ethanol precipitated. C 9.1.1. Production of a linear 6173 Bp-Pstl / Sphl restriction fragment by deletion of the Bas 1345-1795 in pHS 6134

10 0.5 μg aliquots of the Sphl-linearized DNA from C 9. were dissolved in 10 × 10 μl 'medium buffer' (solution 1.1.) And partially with 0.4 units PstI [Boehringer / Mannheim] for 5 Mi at 37 ° C digested. After separation of the product mixtures according to C 1.1.1. the fragment sought, contaminated with the DNA band shortened by 158 bp, could be obtained as ethano sediment.

C 9.1.2. Removal of unpaired 3 'bases using exonuclease III activity in T4 DNA polymerase

0.75 μg of that according to C 9.1.1. isolated DNA fragments were 50 ul reaction volume with 11 units of T4 DNA polymera [Pharmacia / P-L, 7800 Freiburg] according to Maniatis et al. [i 'Molecular Cloning', p. 395, Cold Spring Harbor Lab., N York (1982)] incubated for 10 min at 37 ° C. After inactivating the enzyme by heating it at 70 ° C for 5 minutes, phenol extraction, ether extraction and ethanol precipitation take place according to C 1.1.1.

C 9.1.3. Recircularization of the reaction products from C 9.1.2. with T4 DNA ligase

The sedimented reaction products after cleavage of the paired 3 '-based bases were dissolved in 50 μl ligase puff (solution B 1.3.) And with 5 Weiss units (B. Weiss al., J. Biol. Chem. 2Λ 2_, 4543 -4555 (1968)] T4-DNA-Liga [Boehringer / Mannheim] incubated overnight at 9 ° C. C 9.2. Transformation of Escherichia coli SB 44

C 9.2.1.

The ligase products according to C 9.1.3. were completely transformed into competent cells from E. coli SB 44 under de by M. Dagert and S.D. Ehrlich [Gene 6_, 23-28 (1979)] described conditions used.

C 9.2.2. Selection of transformants

The DNA-treated cell suspension was divided into 5 aliquots to inoculate 20 ml of LB medium [T. Maniatis E.F. Fritsch and J. Sambrook in: 'Molecular Cloning', p. 440 Cold Spring Harbor Lab., New York (1982)] with 50 μg / ml Amp cillin. After 16 hours of shaking incubation at 37 °, diluted aliquots were plated on LB agar and after 24 hours of incubation at 37 ° C. 6 plates with 70-80 ampicin-resistant colonies each were selected for the characterization experiments.

C 9.3. Characterization of transformants

C 9.3.1. Identification of transformants with codons for ACTH using 'colony hybridization'

In order to be able to test a larger number of ampicillin-resistant clones, the colony hybridization method was used again, as described in C 3.3.1. described, used. The 158 bp-PstI fragment from pSA 5 [cf. C 8.3.1.], Which contains codons for 3 1/2 collagenase sections in front of the ACTH sequence, in a standard nick translation reaction according to T. Maniatis et al. [i 'Molecular Cloning', pp. 109-112, Cold Spring Harbor Lab., N York (1982)] radioactively labeled. For this purpose, 0.2 μg of the purified DNA fragment were placed in 100 μl reaction volume

32 addition of 50 μCi [α-P] dATP [6000 Ci / mmol; Amersham Buc ler, cf. P. 10] with 5 units of DNA polymerase I [Boehr ger / Mannheim] for 1 hour at 15 ° C. After stopping the reaction in 10 M EDTA, the [32 labeled fragment was removed after adsorption on an Elutip column, as in C 1.1.1. described.

After performing the hybridization procedure according to C 3.3.1. showed 50% of the approx. 450 transformants distributed on 6 test agar plates C 9.2.2.) under stringent conditions (55 ° C, in some cases of 50% formamide) positive hybridization signals after autoradiographic evaluation.

C 9.3.2. Characterization of transformants by size determination of DNA fragments after implementation of special restriction endonucleases

The plasmid isolation from 6 randomly selected clones according to the result of C 9.3.1. in accordance with standard conditions [cf. A I.]. PHS 6134 [cf. C 8. and Fig. 12] under the same conditions.

C 9.3.2.1. Implementation with the restriction endonuclease

Rsal (negative control)

5 μg of each of the 7 test or control plasmids were placed in 100 μl 'medium buffer' (solution B 1.1.) For 2 hours 10 units Rsal [BioLabs., Cf. P. 10] at 37 ° C incubate with 100 μl TE-saturated phenol and 2x 200 μl ether and ext. C 1.3.2. ethanol-precipitated and analyzed by gel electrophotography.

None of the 6 test clones showed the gene fragments of 97 bp and 159 bp characteristic of pHS 6134.

REPLACEMENT C 9.3.2.2. Implementation with the restriction endonuclease

Sau3AI (positive control)

The reaction was carried out analogously to C 9.3.2.1. carried out. The enzyme activity used was 15 units Sau3AI [Boehringer / Mannheim].

All 6 clones showed the 458 bp fragment typical for the deletion clone sought, while the 598 bp fragment characteristic of the control pH 6134 was missing.

C 9.3.2.3. Double digestion with the restriction endonucleic acids Sau3AI and EcoRI

5 μg of DNA were initially gem. C 9.3.2.2. with Sau3AI i 90 μl reaction volume for 2 hours at 37 ° C. After the addition of 10 μl of 0.4 M Tris / HCl pH 7.5 + 0.55 NaCl + 10 units of EcoRI [Boehringer / Mannheim], Danac was incubated for a further 2 S and mixed in accordance with. C 9.3.2.1. worked up.

All 6 test clones showed the typical 294 bp fragment for the deletion clone, while the 434 bp fragment indicated for the control pHS 6134 was missing.

C 9.3.3 Structure confirmation by DNA sequencing

That from the double digestion of DNA according to C 9.3.2.3. result 294 bp fragment could be according to C 1.1.3. isolate and subclone into the EcoRI / BamHI gap of phage M13mpl8. Under standard sequencing conditions [cf. A 2.] the identity of the pHS 6134 deletion derivative was fully confirmed. The new clone was named pSA 351.

[see. Fig 13, pSA 351] C 10. Production of the expression clone SB 44 (pSA 360)

This clone synthesizes a fusion protein consisting of the N-terminal amino acids 1-444 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie Universität Berlin, Berlin (1983)], followed by glycine and 8 collagenase recognition sequences before the ACTH sequence (SA 360), consists of:

Alk.phosphatase (1-444) -Gly-Pro-His-Gly- (Pro-Ala-Gly), - Pro-Iyτ-Gly-Pro-ACTH

I t i

8 collagenase recognition sequences

SA 360

C 10.1. Preparation of the vector plasmid pSA 510

This plasmid differs from pSA 506 [cf. Fig. Only by a T / A—> C / G base exchange in Positi 1629, whereby the corresponding Pstl recognition sequence a pSA 506 is missing.

[see. Fig. 14, pSA 510]

PSA 510 was produced in the same way as described for pSA 506 [see. German file number P 37 31 875.6]. For insertion into the Sp I interface of the base vector pSB 94 (produced according to P 37 31 875.6), two 5-base nucleotides with a correspondingly modified sequence were accordingly synthesized under standard conditions [cf. A 3]:

5 '-GTCCGGCAGGCCCGGCGGGTCCAGCAGGCCCTGCAGGTCCGCATü-J' and

5 '-CGGACCTGCAGGGCCTGCTGGACCCGCCGGGCCTGCCGGACCATG-3'. C 10.2. Integration of a 158 bp Pstl fragment with codons for 3 1/2 collagenase sites before ACTH into the Pstl site in position 1657 of the vector plasmid pSA 510

C 10.2.1. Production of the linear vector plasmid

0.7 μg of pSA 510 DNA were determined as in C 1.1.1. described, partially digested with 0.6 units of PstI [Boehringer / Mannheim] at 37 ° C. for 5 minutes and the reaction products, including the two 6499 bp-PstI fragments, according to C 1.1.1. worked up.

C 10.2.2. Production of the 158 bp-Pstl integration fragment

The required fragment was isolated from 50 μg pHS 6134 according to C 1.1.3 by Pstl digestion and subsequent processing.

C 10.2.3. Linking vector and insertion fragment with T4 DNA ligase

I μg Pstl-linearized pSA 510 fragment mixture from C 10.2. was treated with 0.1 μg 158 bp fragment according to C 3.1.3. incubated with T4 DNA ligase at 9 ° C.

C 10.3. Transformation of Escherichia coli SB 44

C 10.3.1.

1/5 of the ligase reaction mixture was used to transform competent E. coli SB 44 cells, as described in C 1.2.1. refereed, used.

C 10.3.2. Selection of transformants

On LB agar [T. Maniatis, E.F. Fritsch and J. Sambrook in 'Molecular Cloning', pp. 440ff, Cold Spring Harbor Lab., Ne

* York (1982)] with 100 μg / ml ampicillin content, 70 ampicillin-resistant transformant clones were isolated.

C 10.4. Characterization of transformants

C 10.4.1. Identification of transformants with codons for ACTH using 'colony hybridization *

The procedure is described in C 3.3.1. described. As specific

Again, 0.75 μg of the 158 bp-Pstl fragment from pHS 6134 was used in the sample. The [32P] phosphorylation and workup was carried out according to the method already described in C 9.3.1. described method with 100 μCi [- 32P] dATP. Colonies of the clones SB 44 (pSA 510), SB 44 (pHS 6134) and HB 101 (pSB 94) [cf. German patent application, file number P 3731875.6 _ψ Fig. 2] included in the test.

Under stringent washing conditions (60 ° C, 50% formamide), 21 clones showed positive hybridization signals after 16 hours of autoradiography at -70 ° C.

C 10.4.2. Characterization of transformants by size determination of DNA fragments with PstI and Ncol

The plasmid isolation from 16 clones selected at random according to the result of C 10.4.1. was carried out under standard conditions [cf. A I.].

C 10.4.2.1. Digestion with the restriction endonαclease

PstI

This experiment is under C 1.2.1. described. 11 clones showed the expected band pattern of 158, 2441 and 4058 bp fragment lengths.

HE / 88/00536

- 50 -

C 10.4.2.2. Determination of the installation orientation for the

Integration fragment by reaction with the restriction endonuclease Ncol

This experiment was carried out under C 7.3.2. described. Clones which contain the 158 bp-PstI fragment inserted in the desired phoA transcription direction have fragments of

527 and 6130 base pairs.

If the opposite strand is integrated in a corresponding manner, fragment sizes of

595 and 6062 base pairs.

3 test clones showed the typical Ncol band pattern for correct installation. One of them was further cultivated under the name pSA 360 for the subsequent protein analysis.

[see. Fig. 15, pSA 360]

D) Expression products

D 1. Cloning of the ACTH expression plasmids into E. coli E 15

As an alternative phoA-negative host strain for the isolation and characterization of phoA fusion proteins, E. c li E 15 (see p. 10) has significant advantages over SB 44.One time it lacks the amber suppressor mutation supE, i.e. The TAG triplets used for the ACTH 1-24 hybrid genes pSA 172 and pSA 353 are, unlike in SB 44, strictly read as stop codons; on the other hand, the protein yields b E 15 clones are significantly higher under comparable culture conditions than with homologous SB 44 cloning.

For these reasons, 5-10 μg aliquots of the ACTH plaids described in the preceding sections C 1. to C 10. were used under standard conditions [cf. M. Dagert and SD Eh lich, Gene _6 _T 23-28 (1979)] in E 15. The relatively high DNA excess required for a successful transformation is necessary because E.coli E 15, unlike SB 44, has an active restriction and modification system.

D 2. Analytical determination of fusion proteins and the clostridiopeptidase A cleavage products

The production, cleavage and characterization of ACTH fusion proteins for analytical purposes was carried out according to d under A 5. to A 6.6. described standard conditions. After fermentation of the corresponding phoA-ACTH clones in low phosphate medium, the fusion proteins were isolated and their molecular weight was confirmed by means of SDS gel electrophoresis and co-electrophoresis of calibration proteins. In addition to some other characteristic data, they are compiled in Table 1 for the cloning examples previously described Tab. 1: Summary of some data for the described phoA-ACTH clones including the molecular weights of their expression products

All clones produce the expected fusion proteins in good yields as insoluble cytoplasm components. Some individual examples are documented in Figures 16-20. After cleavage with collagenase, the expected Gly Pro-ACTH levels (Fig. 18 and 19) are found next to the corresponding residual levels of alkaline phosphatase (Fig. 18-20). The identity of the protein bands could be confirmed in each case by standard 'immunoblotting' tests.

D 3. Determination of product formation per unit time ^"By J clostridiopeptidase A cleavage of 4 ACTH clones mi different numbers of collagenase interfaces

These experiments allow a quantitative estimate of the amounts of ACTH fusion protein with clostridiopeptidase A that can be cleaved per unit of time and enzyme, depending on the number (s) of repetitive collagenase recognition sequences According to the procedures described by H. Scheidecker et al. , German file number P 37318 ⁽ 1987)] given the conditions specified, the fusion proteins of clones E 15 (pSA 186), E 15 (pSA 343), E 15 (pSA 341) and E 15 (pSA 360) were used with limited amounts of purified collagenase [cf. . above German patent application in ^~~

100 ul reaction volumes implemented. The determination of the clone-specific release of cleavage products after time-dependent removal of 10 μl aliquots by their densitometric determination after gel electrophoretic separation and staining with Coomassie blue was also carried out according to the description cited above. The mean values from each of 3 test series carried out in parallel with different enzyme concentrations are summarized in Table 2.

Tab. 2

Origin of the number of repetitive product formation rate of the fusion protein collagenase (pmol / min / enzyme interface unit) n

E 15 (pSA 186) 1 <0.01

E 15 (pSA 343) 2 40

E 15 (pSA 341) 5 350

E 15 (pSA 360) 8 540

As the comparison of the product formation rates shows, the expected increase in the reaction rates could be fully confirmed by increasing the number of repetitive collagenase recognition sequences. D 4. Production and isolation of ACTH from a 10 l fermenter culture of clone E 15 (pSA 341)

D 4.1. Pre-cultures

30 ml LB medium [T. Maniatis, E.F. Fritsch and J. Sambrook in: 'Molecular Cloning', p. 440, Cold Spring Harbor Lab., New York (1982)] with 100 μg / ml ampicillin are inoculated with a single colony of E 15 (pSA 341) and used for incubated for approximately 16 hours at 37 ° C. The fully grown cell culture is used to inoculate a second preculture with 800 ml of the same medium, which is incubated by shaking under identical conditions.

D 4.2. Main culture

For this purpose, 7 1 H_0 are expediently mixed with 2.2 1 of a sterile-filtered solution of 0.2 g L-proline, 0.2 g 'leucine, 10 g Casamino acids [Difco Lab. Detroit, USA] and 10 mg vitamin B 1 in 545-fold concentrated low-phosphate medium [cf. LP medium according to K. Kreuzer et al. , Genetics 8_1, 459-468 (1975)] and the 0.8 1 preculture combined in a closed glass fermenter with 15 1 capacity and for 6 hours at 37 ° C. with 10 1 air / min (0.7 atm pressure) and a stirrer speed stirred for 220 min. The subsequent sedimentation of the bacterial cells by means of a "continuous centrifuge of the type Sharples T-1A [Deutsche Sharples GmbH, 4100 Duisburg-Meiderich] results in a cell yield of approx. 30 g wet weight.

D 4.3 Cell disruption

The 30 g of low-phosphate cells are removed using an Ultra-Turrax T 25 [Fa. Jahnke & Kunke GmbH & Co., Kg, 7813 Staufen] in 300 ml 50 mM Tris / HC pH 7.5, 160 mM NaCl, 20 mM MgCl_, with 200 μ DNase I [Boehringer / Mannheim], 300 μg pepstatin A un 10 mg phenyl-methyl-sulfonyl chloride (PMSF) [Sigma Chemie cf. A 6.3.] And temperature-controlled in the cold room to 2-4 ° C. A complete cell disruption under the mentioned temperature conditions can be achieved by using the high pressure homogenizer 15M-8TA [Fa. Manton Gaulin SA, Hilversum / NL] after 3 cycles at 9000-12000 psi. The temperature is then adjusted to DNase digestion at 0 ° C for 1 hour.

D 4.4. Protease inactivation

After centrifugation of the homogenate at 0-2 ° C. and 24,000 × g for 1 hour, the sediment obtained is resuspended in 400 ml of 50 M Tris HC1 pH 8.0, 160 mM NaCl and sedimented as above. This washing process is repeated. Then the sediment free of soluble cell components is resuspended using Ultra-Turrax T 25 [Fa. Jahnke & Kunkel GmbH & Co. KG 7813 Staufen] in 100 ml 8 M guanidine hydrochloride in 50 m Tris / HCl pH 8.0 and heated to 95 ° C for 1 hour. Insoluble parts can be sedimented by a subsequent centrifugation of 20 minutes at 40,000 x g and are discarded

D 4.5. Collagenase cleavage

The clear 8 M guanidine chloride extract is first dialyzed against 2 1 H 0 for 30 min, then twice for 4 hours against 2 50 mM Tris / HCl pH8.0, 160 mM NaCl. This gives 70-80% pure fusion protein in insoluble form as finely dispersed suspension. The dialysate suspension is mixed with 10 units of purified clostridiopeptidase A [see A 6.6.] And 15 ml of 0.5 M Tris / HCl pH 8.0, 50 mM CaCl ₂ and with 50 mM Tris / HCl pH 8.0, 160 mM NaCl to 150 ml of reac volume added. This is followed by 3 hours of shaking incubation in a 37 ° C water bath. D 4.6. Processing of the reaction products

D 4.6.1. Adsorption on silica gel

Insoluble residues of the collagenase digestion are sedimented by centrifugation at 45,000 x g for 30 minutes. Using a 'Roto-Torque' mixer rotor [Fa. Cole Parmer Ins r. Co., Chicago, Illinois, USA], the clear centrifugation supernatant is mixed with 30 g of Mallinckrodt 100 esh silica gel [Serva Feinbiochemica GmbH & Co., 6900 Heidelberg 1 in a sealed bottle for 30 min. Rotation mixture [R.A. Donald, J. Endocrin 3_9_, 451-452 (1967)]. The adsorbent had previously been moistened by slurrying with H_0 and suction using a G3 glass frit. In the same way it also follows the recovery of the silica gel after quantitative adsorption of the collagenase reaction products, predominate Gly-Pro-ACTH. This material can easily be stored at 0 ° C for 10-20 hours. The peptides fixed to the diatomaceous earth can be desorbed by 60 min. Of mixed rotation (see above) with 100 ml of an acetone / water / acetic acid mixture (volume ratios 20: 79: 1) and, after the adsorbent has been suctioned off (see above), can be largely clear Eluate who receive. Using a rotary evaporator, the mixture is concentrated to about 30 ml at a bath temperature of 30-35 ° C. Low residual contamination from silica gel can be sedimented for 15 min. Centrifugation at 30,000 x g.

D 4.6.2. Gly-Pro-ACTH isolation using reversed phase chromatography

Pre-packed separation column and the Fast Protein Liquid Chromatograph (FPLC) system from Pharmacia [Deutsche Pharmacia GmbH, 7800 Freiburg 1] ei were predominantly used for the column chromatographic purification steps described below. The silica gel eluate is fixed on a PepRPC HR 16/10 column and with a linear 450 ml gradient of 10 mM KH-PO. in 0 (buffer A) - 50% (buffer B) acetonitrile 5 ml volumes at a flow rate of 5 ml / min. fractionally eluted. The Gly-Pro-ACTH peak fractions can be identified photometrically at about 27.5% acetonitrile content (approx. 55% buffer B) after light absorption at 214 nm. They are combined and concentrated to 3-4 ml on a rotary evaporator (see above) . The phosphate portion was removed by standard gel filtration over a 60 x 2.6 cm Sephade G-10 column with water as the eluent. The peak fractions determined photometrically at 280 nm can, as described above, be concentrated to 5 ml.

D 4.6.3. Proof of identity for Gly-Pro-ACTH

The identity of the isolated Gly-Pro-ACTH could be verified in two ways:

a) According to the 'immunoblotting' method [cf. Standards A 5. and A 6.6] using a rabbit ACTH antiserum as primary antibody. b) By peptide sequencing according to B. Wittmann-Liebold and M. Kimura [in Proteins Vol. _1_, ed. J.M. Walther, Human Press, Clifton, New Jersey (1984)]. Thereafter, the sequence of the 5 amino terminal amino acids was left

Detect Gly-Pro-Ser-Tyr-Ser safely.

D 4.7. Enzymatic conversion of Gly-Pro-ACTH with post-proline dipeptidyl aminopeptidase (PPDA)

The specific cleavage of the N-terminal glycylprolyl residue leads to native corticotrophin. For this purpose, the 5 m of the purified Gly-Pro-ACTH fraction (cf. D 4.6.2.] With 360 μg to 50% purified PPDA (systematic designation Dipeptidyl-Peptidase IV (EC 4.4.14.5.), [Purification after Tadash , Yoshimoto, Walter, BBA 485, 391-401 (1977)] for 3 hours at 37 ° C. The reaction mixture is then used directly for purification by column chromatography D 4.8. Working up the reaction product

D 4.8.1. ACTH isolation by means of 'reversed phase' chromatography

The reaction mixture is separated again using a PepRPC HR16 / 10 column under the conditions described in Section D 4.6.2. described technical conditions with the exception of the eluent. In this case, the linear gradient consists of 225 ml 0.1% trifluoroacetic acid in H ₂ 0 (buffer A) and 225 ml 0.1% trifluoroacetic acid in 60% acetonitrile (buffer B). The ACTH peak fractions can be identified photometrically in approximately 29.4% acetonitrile (approximately 49% buffer B) after light absorption at 214 nm (cf. Fig. 21).

D 4.8.2. Proof of identity for ACTH

Experimental data for the identity of the peak material as pure ACTH can be obtained in three ways:

a) According to the 'immunoblotting' method using an rabbit ACTH antiserum as the primary antibody. This is a modified implementation of the method already described [cf. A 5.], in which the sample material in addition to 0.1-10 μg aliquots of authentic human ACTH [Fa. Bachern AG, CH-4416 Bubendorf] directly on BA85 nitrocellulose membranes [Schleicher & Schüll, 3354 Dassel] and further processed according to the standard method (see above). b) By comparative thin layer chromatography on silica gel 60 [Merck, Darmstadt] against authentic Huma ACTH [Fa. Bachern, s. top] Aliquots of the peak material from D 4.8.1. show identical R ^ values in

Isopropanol: conc. NH_ 1: 1 (R value 0.7) Gly-Pro-ACTH has an R value of 0.5 in the same solvent system. c) By peptide sequencing according to B. Wittmann-Liebold and M. Kimura [in: Proteins Vol. JL, ed. JM Walther, Huma¬ na Press, Clifton, New Jersey (1984)]. Then the sequence of 9 amino terminal amino acids

Detect Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp safely. d) By detection of a uniform product peak after analytical 'reversed phase' chromatography on a Pep RPC HR 5/5 column in the isocratic system. The retention time is identical to that of a comparative sample of authentic ACTH (see Fig. 22).

Fig. 15: SDS gel electropherogram of protein extracts

E. coli SB 44 (phoA, supE) and E. coli E 15 (phoA) with the plasmids psB 94 [native phoA gene, cf. Parallel registration Dr. G. Siewert et al., (1987)], psA 172 (phoA / ACTH 1-24) and psA 186 (phoA / ACTH 1-39)

2 3

1. Purified alkaline phosphatase (MW 47 147)

2. Host strain E. coli SB 44 (plasmid-free)

3. E. coli SB 44 (psB 94)

Molecular weight of the alkaline phosphatase prepeptide: 49,383

4. E. coli SB 44 (pSA 186)

Molecular weight of the pre-phoA / ACTH 1-39: 51,566 fusion protein 5. E. coli SB 44 (pSA 172)

Because of the supE mutation, the clone expresses a fusion protein 27 amino acids long; MW 52 849

6. E. coli E 15 (pSA 172)

Molecular weight of the fusion protein Pr-phoA / ACTH 1-24: 49,960

7. Host strain E. coli E 15 (free of plasmids)

8. Molecular weight markers

Fig. 17: Preparation of the pre-phoA / ACTH 1-39 fusion protein from

E. coli E 15 (pSA 341) by fermentation on a 10 1 scale [cf. D4., P. 51ff]

SDS gel electrophoresis (12.5 '/ polyacrylamide)

94,000 67,000

47 147

* 43 ÖόO (AP, 450 AS)

30,000

20 100

1. Fusion protein pre-phoA / ACTH 1-39 according to D 4.4. [see. P. 52].

The amount of protein corresponds to 0.2 ml main culture. Molecular weight 52,435

2. E. coli E 15 (plasmid rei)

3. Molecular weight markers

4. Purified alkaline phosphatase (AP) molecular weight 47,147 Fig. 18: SDS gel electropherogram of soluble and insoluble substrate and product components in clostridiopeptidase A digestion of the fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 341)

1. Reaction product (pre-phoA ant

30,000 2nd reaction product 20 100 (Gly-Pro ACTH 1-39) 14 400

1. Molecular weight markers

2. Total collagenase reaction mixture before enzyme addition. Molecular weight of the fusion protein pre-phoA / ACTH 1-39: 52,453

3. Insoluble fraction of the collagenase reaction mixture before adding the enzyme (centrifugation sediment)

4. Soluble fraction of the collagenase reaction mixture before adding the enzyme (centrifugation supernatant)

5. Total reaction mixture after collagenase digestion Molecular weight of the 1st reaction product: 46,863 Molecular weight of the 2nd reaction product: 4,690 (Gly-Pro-ACTH 1-39) 6. Insoluble fraction of the reaction mixture after collagenase digestion (centrifugation sediment)

7. Soluble portion of the reaction mixture after collagenase digestion (centrifugation supernatant)

Fig. 19: Clostridiopeptidase A digestion of the pre-phoA / ACTH 1-39 fusion proteins from E. coli E 15 with the plasmids pSA 341 and pSA 509, substrates and reaction products after SDS gel electrophoresis (12.5-25 7. polyacrylamide gradient)

94,000 67,000

30,000

20 100 14 400

1. Molecular weight markers

2. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 341); Molecular weight 52,453

3. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 341) reaction product a: pre-phoA portion

Molecular weight 46,863 reaction product b: Gly-Pro-ACTH 1-39

Molecular weight 4,690

4. Marker protein ACTH 1-39, molecular weight 4,536 5. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 509) reaction product a: pre-phoA portion

Molecular weight 46,863 reaction product b: Gly-Pro-ACTH 1-39

Molecular weight 4,690

6. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 509) molecular weight 52,527

7. Molecular weight markers

8. Marker protein ACTH 1-39, molecular weight 4,536

Fig. 20: Clostridiopeptidase A digestion of the pre-phoA / ACTH 1-39 fusion proteins from E. coli E 15 with the plasmids pSA 341 and pSA 351 substrates and reaction products after SDS gel electrophoresis (12.5 7. polyacrylamide )

43,000

30,000

1. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 341); Molecular weight: 52 453

2. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 341),

1. Reaction product (pre-phoA content) after 6 hours of clostridiopeptidase

A digestion

Molecular weight: 46,863

3. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 351); Molecular weight: 36 710

4. Fusion protein pre-phoA / ACTH 1-39 from E. coli E 15 (pSA 351),

1. Reaction product (pre-phoA content) after 6 hours of clostridiopeptidase

digestion

Molecular weight: 31,271

_ /

5. Position of 2 molecular weight marker proteins

Claims

 Claims

1) Plasmid vectors which contain a DNA sequence for ACTH or ACTH derivatives and with which bacteria can be transformed into cells which contain these vectors. Plasmid pSA 172 Plasmid pSA 186 Plasmid pSA 341 Plasmid pSA 343 Plasmid pSA 345 Plasmid pSA 353 Plasmid pSA 509

9 plasmid pHS 6134 10 plasmid pSA 351 11 plasmid pSA 360 12 A method for producing plasmid vectors according to claim 1, characterized in that DNA insertion fragments which contain the codons for ACTH or ACTH derivatives' and which have Pstl- at both ends. Have recognition sequences, insert linearized and dephosphorylated vector plasmids into the PstI interface with the aid of a suitable ligase

13) E. coli strains containing plasmids with a gene for ACTH or ACTH derivatives according to claim 1.

14) E.coli SB 44 (pSA 172) 15) E.coli SB 44 (pSA 186) 16) E.coli SB 44 (pSA 341) 17) E.coli SB 44 (pSA 343) 18) E.coli SB 44 (pSA 345) 19) E.coli SB 44 (pSA 353) 20) E.coli SB 44 (pSA 509) 21) E.coli SB 44 (pHS 6134) 22) E.coli SB 44 (pSA 351) 23 ) E.coli SB 44 (pSA 360) 24) E.coli E 15 (pSA 343) 25) E.coli E 15 (pSA 341) 26) E.coli E 15 (pSA 360) 27) Process for the production of new E. coli strains according to claim 13, characterized in that competent cells of the strain in question are incubated with the plasmid DNA according to claim 1 under transformation conditions and isolated from the cell population by clones containing ACTH their antibiotic resistance can be identified.

28) Use of E. coli strains which contain a plasmid with a gene for ACTH or ACTH derivatives for the production of ACTH or ACTH derivatives.

29) Process for the production of new E. coli strains by methods known per se, characterized in that competent cells from E. coli E 15 with a large excess of plasmid DNA according to claim 1 are transformed from E. coli SB k

30) Use of E. coli E 15 strains containing a plasmid with a gene for ACTH for the production of ACTH.

31) Process for the preparation of ACTH, characterized in that the strains according to claims 13-26 from a preculture with an aqueous solution of L-proline, L-leucine, casamino acids, vitamin B 1 in concentrated low-phosphate medium combined in a fermenter, stirred at 37 ° C. and excess pressure and the ACTH formed is isolated by known processes.

32) Method according to claim 31, characterized in that the resulting ACTH-containing fusion proteins are subjected to a collagenase cleavage and the reaction products are isolated while avoiding proteolytic decomposition and then Gly-Pro-ACTH is cleaved by means of post-proline dipeptidyl aminopeptidase

33) Medicaments containing ACTH or ACTH derivatives, which were prepared by the method according to claim 31, and conventional auxiliaries and carriers.