DE3919852A1 - Mini-proinsulin, its preparation and use - Google Patents

Abstract

A mini-proinsulin in which the C chain is replaced by linkage of the A chain and the B chain by the amino acid Arg shows insulin activity and is suitable for the production of pharmaceutical compositions for the treatment of diabetes mellitus. It can furthermore be converted simply with trypsin into an insulin derivative whose B chain is extended by Arg. The latter can be converted with carboxypeptidase B into insulin. However, the mini-proinsulin can also advantageously be converted into insulin directly in a one-pot process.

Description

The invention relates to a new "mini-proinsulin", where the un-shortened B chain only one Arginine residue is attached to the A chain. For this mini proinsulin is human insulin without elaborate chemical Implementation accessible.

Mini-Proinsulins with a shortened C chain are known. Thus, R. Wetzel et al., Gene 16 (1981), 63-71 Proinsulin with a truncated to six amino acids C-chain described. From the European patent application with the publication number (EP-A) 00 55 945 are corresponding Proinsulins are known whose C chain down to two amino acids is shortened.

From EP-A 01 63 529 insulin precursors with a truncated B chain are known in which the C chain is either completely eliminated or - except for an amino acid - is shortened. These precursors are converted to mature human insulin by trypsin-catalyzed transpeptidation with an a- threonine ester.

In contrast, the invention relates to human des- (32-65) -proinsulin or mini-proinsulin of the formula I.

B (1-30) -Arg-A (1-21) (I)

in B (1-30) and A (1-21) represent the B and A chains of human insulin, respectively. This compound serves not only as an intermediate for the production of human insulin Arg ^B31 -OH, hereinafter referred to as "Mono Arg insulin", which is known from European patents (EP-B) 01 32 769 and 01 32 770, as well as human insulin, but it also shows some insulin activity itself.

The invention therefore also relates to the compound of Formula I for use as a remedy, in particular for Treatment of diabetes mellitus, and continue Medicaments containing the compound of the formula I, as well as drugs from a pharmacologically acceptable Carrier and the compound of formula I.

Furthermore, the invention relates to the use of A compound of the formula I for the preparation of the mono-arg-insulin of formula II

in the A (1-21) and B (1-30) are the above Have meanings and the -S-S bridges as in insulin are arranged, and of human insulin, by enzymatic Cleavage. Particularly advantageous is the immediate Transfer of the compound of formula I into insulin in one "One-pot reaction".

The invention further relates to a method for Preparation of the compound of the formula I which is characterized is characterized in that one for this connection coding gene structure in a host cell, preferably in a bacterium such as E. coli, or in a yeast, in particular Saccharomyces cerevisiae, and when expressed encoded the gene structure for a fusion protein, the Releases the compound of formula I from this fusion protein. The invention also relates to DNA sequences useful for the Compound of formula I encode gene structures or Plasmids containing this DNA, as well as host cells, especially bacteria such as E. coli or yeast cells, before  all yeasts of the species Saccharomyces cerevisiae, those Gene structures or plasmids included. The invention further refers to fusion proteins that have a Contain compound of formula I, preferably Fusion proteins in which the compound of the formula I via the bridge link

-Met-Ile-Glu-Gly-Arg-

is bound to the "ballast portion" of the fusion protein.

Further preferred embodiments of the invention are in explained in more detail below.

The figures serve to illustrate the examples, wherein Fig. 1 (and its continuation in Fig. 1a and 1b), the construction of E. coli expression vectors pIK10 and pSW3 and Fig. 2 (and their continuation in Fig. 2a and 2b) of the yeast expression vector p α fB102 and p α fB104, respectively. These vectors encode mini-proinsulin.

It has now been found that the mini-proinsulin the correct Has folding, so that after cleavage with trypsin almost quantitative mono-arg-insulin is produced. So it turns out a surprisingly simple process for the production of Mono-Arg-insulin. For this can be known per se How to make human insulin. Continues to serve Mono Arg insulin as active ingredient in medicines (EP-B 01 32 769).

In EP-A 02 29 998, the expression vector pK50 described. Mini proinsulin may take the form of a Fusion protein according to this construction in one Bacterium such as E. coli.

The sparingly soluble fusion protein can be washed by washing with neutral buffer solutions are enriched. By  Cyanogen halide cleavage (E. Gross and B. Wittkop, J. Am. Chem. Soc. 82 [1961], 1510-1517) becomes the mini-proinsulin released. This is not yet in the biological active form, but consists of a nonuniform Mixture with different intermolecular and intramolecular ones Disulfide bridges, possibly with others Protein fragments. As chemically uniform, relative stable derivative is the S-sulfonate form of Molecule (P.G. Katsoyannis et al., Biochemistry 6 [1967], 2635-2641). This derivative works very well purified by ion exchange chromatography and is a Proven source material for folding into the native Spatial structure with formation of the correct disulfide bridges (Y.C. Du et al., Sci. Sin. 15 [1965], 229-236; Gattner et al., Hoppe-Seylers, Z. Physiol. Chem. 362 [1981], 1943-1949; B.H. Frank et al. in: "Peptides: Synthesis Structure Function ", D.H. Rich and E. Gross, Eds., [1981], 1043-1049). The success of this convolution will be by HPLC analysis of after cleavage with S. aureus protease V8 arising fragments secured (U. Gray, Diabetes 34 [1985], 1174-1180).

The release of Mono Arg insulin or insulin by Influence of trypsin or carboxypeptidase B or by equivalent enzymes (W. Kemmler et al., J. Biol. Chem. 246 [1971], 2780-2795) is extremely uncomplicated, because it proves to be particularly advantageous here that the Number of possible cleavage sites compared to the normal one Proinsulin is reduced. As a result, the split is significant easier (with regard to the formation of by-products as in the preparation of Des-B30 insulin or Desocta B23 B30 insulin) to control. Both mono-arg insulin Insulin can be in a known manner by Isolate ion exchange chromatography in a highly pure form. The formation of insulin and the mono-Arg derivative, the Course of cleaning and the quality of the final product are prepared by conventional RP-HPLC methods (G. Seipke et al. Angew. Chem. 98 [1986], 530-548).  

Surprisingly, the cleavage of mini-proinsulin, unlike natural proinsulin, is unlikely to produce insulin-Des-B30. Since the latter is very difficult to separate from insulin, in insulin recovery from natural proinsulin, the "second-pot reaction" is preferred, ie the main products of tryptic cleavage, insulin-Arg ^B31 -Arg ^B32 and mono-Arg insulin, are first on ion exchanger in separated from insulin des-B30 and then cleaved by means of carboxypeptidase B to human insulin (EP-B 01 95 691). In contrast, the mini-proinsulin can ideally be converted into human insulin in a "one-pot reaction" with simultaneous use of trypsin and carboxypeptidase B or by means of enzymes having the same effect.

The expression of the compound of formula I in yeast with subsequent secretion is particularly advantageous since the to correctly isolate correctly folded proinsulin derivative is. As host systems, yeasts are chosen, as they are For example, in EP-A 02 48 227 are listed, such. B. Pichia pastoris, Hansenula polymorphis, Schizosaccharomyces pombe or preferably Saccharomyces cerevisiae.

Vectors for expression in yeasts are known in large numbers. The preparation of the insulin derivative according to the invention is described below on the basis of the yeast α- factor system, which is only to be understood as an example since other expression systems can also be used in a manner known per se.

The structure of the yeast pheromone gene MF α is known from the publication Kurjan and Herskovitz, Cell 30 (1982), 933-943, where the possibility of expression of other genes and the secretion of gene products is discussed. In this regard, Brake et al., Proc. Natl. Acad. Sci. USA 81 (1984), 4642-4646.

Alternatively, a yeast "killer toxin" system may be used be, or the secretion on the system of acid Phosphatase or the invertase are exploited.

As Hefevektoren be advantageous so-called "shuttle" vectors used a bacterial Plasmid and a yeast plasmid replication origin as well a gene or genes for selection in both host systems respectively. Furthermore, such vectors contain the Expression of foreign genes necessary promoter sequences and optionally to improve the yield Terminator sequence, so that the heterologous gene - appropriate fused to secretory signals - between promoter and Terminator is arranged. Such vectors are For example, in US-A 47 66 073 described.

The genetic code is known to be "degenerate," d. H. that only for two amino acids a single nucleotide sequence coded while the remaining 18 codable amino acids two to six triplets are assigned. For the synthesis the gene for the mini-proinsulin is thus a great one Variety of codon combinations to choose from. It became now found that the mini-proinsulin coding DNA sequence I (attached in the form of the two gene fragments IK I [Table 1] and IK II [Table 2] is reproduced) is particularly advantageous because it is based on the codon usage is optimized by both yeast and E. coli.

At the 5 'end of the coding strand of the DNA sequence I there is an "overhanging" DNA sequence, accordingly the restriction endonuclease KpnI. At the 3'-end of the coding strand, however, is the single-stranded sequence according to the restriction enzyme HindIII overhanging. These two different recognition sequences ensure the insertion of the DNA sequence I into plasmids in the desired orientation. Follow the coding sequence on the triplet No. 65 for asparagine two  Translation termination codons (stop codons). An internal one unique site for the restriction enzyme PstI (Codon 41/42) allows the subcloning of two Gene fragments expressed in well-studied plasmids such as pUC18 or derivatives of these plasmids can be incorporated.

In addition, within the structural gene, a number of further singular recognition sequences for Restriction enzymes incorporated, on the one hand an access to create partial sequences of the proinsulin and on the other hand allow the execution of mutations:

restriction enzyme Cut to nucleotide no. (coding strand) Acc 201 DraIII 46 FnuDII 107 HgaI 105 Hind 213 HinfI 17 Hpa 22 HphI 76 Mae I 155 Maelll 191 Mbo 89 MluI 106 NcoI 207 NlaIII 208 PvuII 175 Sall 201 Spel 154 Styl 207 TaqI 202

The DNA sequence I has been distinguished in essential points from the modified natural sequence. This was the introduction  the numerous singular interfaces for Restriction enzymes possible.

The DNA sequence I can be composed of a total of 6 oligonucleotides with a chain length of 47 to 96 nucleotide units build up. The procedure is as described below.

The gene fragment IK I (Table 1) can be built up from 4 oligonucleotides with a chain length of 47 to 74 units by first synthesizing them chemically and then enzymatically linking them via "sticky ends" of 3 nucleotides. The sticky ends correspond to those of the restriction enzyme DraIII, which is advantageous for later modifications.
The gene fragment IK II (Table 2) is obtained from two chemically synthesized oligonucleotides with a length of 88 and 96 nucleotide units.

Example 1 a) Chemical synthesis of a single-stranded oligonucleotide

The example of the oligonucleotide no. 4 (Table 1) is the Synthesis of the DNA building blocks explained. For the Solid phase synthesis becomes the 3'-end nucleoside, In the present case, therefore, adenine (nucleotide No. 125), about the 3'-hydroxy function covalently bonded to a support used. Carrier material is long-chain Aminoalkyl radicals functionalized CPG ("Controlled Pore Glass ").

In the following synthesis steps, the base component is used as 5'-O-dimethoxytrityl nucleoside 3'-phosphorous acid β- cyanoethyl ester dialkylamide, wherein the adenine as N⁶-benzoyl compound, the cytosine as N⁴-benzoyl compound, the guanine as N² Isobutyryl compound and the thymine without protective group.

25 mg of the polymeric carrier containing 0.2 μmol of 5'-O-dimethoxy trityl-N⁴-benzoyl-2'-deoxyadenosine bound treated successively with the following agents:

• A) acetonitrile
• B) 3% trichloroacetic acid in dichloromethane
• C) acetonitrile
• D) 5 μmol of the corresponding nucleoside 3'-O-phosphite and 25 μmol tetrazole in 0.15 ml anhydrous acetonitrile
• E) acetonitrile
• F) 20% acetic anhydride in tetrahydrofuran with 40% lutidine and 10% dimethylaminopyridine
• G) Acetonitrile
• H) 3% iodine in lutidine / water / tetrahydrofuran in Volume ratio 5: 4: 1

By "phosphite" is meant here the 2'-deoxyribose-3'-mono-phosphorous acid mono- β- cyanoethyl ester, the third valence being saturated by a diisopropylamino radical. The yields of the individual synthesis steps can each be determined spectrophotometrically by measuring the absorption of the dimethoxytrityl cation at the wavelength of 496 nm after the detritylation reaction B).

After completion of the synthesis, the cleavage of the dimethoxytrityl group is carried out as described in A) to C). By treatment with ammonia, the oligonucleotide is cleaved by the carrier and at the same time the β- cyanoethyl groups are eliminated. A 16-hour treatment of the oligomers with concentrated ammonia at 50 ° C quantitatively cleaves the amino protecting groups of the bases. The crude product thus obtained is purified by polyacrylamide gel electrophoresis.

In an analogous manner, the oligonucleotides 1-3 (Table 1), 5 and 6 (Table 2).

b) Enzymatic linkage of single-stranded oligonucleotides

For the enzymatic phosphorylation of the oligonucleotides on 5'-terminus are each 1 .mu.mol of the oligonucleotides 1 and 4 with 5 μmol adenosine triphosphate with four units of T4 Polynucleotide kinase in 20 μl of 50 mM Tris-HCl buffer (pH 7.6), 10 mM magnesium chloride and 10 mM dithiothreitol (DTT) treated at 37 ° C for 30 minutes. The enzyme is going through 5 minutes heating to 95 ° C deactivated. The Oligonucleotides 2 and 3, which are the "overhanging" do not form single-stranded sequences phosphorylated. This prevents in the following Ligation the formation of larger gene fragments.

The oligonucleotides 1 to 4 are ligated as follows: 1 μmol each oligonucleotides 1 and 2, and 3 and 4, respectively hybridized in pairs by adding 50 μM each in 20 μl Tris-HCl buffer (pH 7.6), 10 mM magnesium chloride and 10 mM DTT, this solution heated to 95 ° C for 5 minutes and cooled to room temperature within 2 hours. In this case, the oligonucleotides 1 and 4 in the form of their 5'-phosphates used. To further link the formed bihelical DNA fragments become the solutions the same, heated to 60 ° C for 15 minutes and on Room temperature cooled. Then you set 2 ul 0.1 M DTT, 16 ul 2.5 mM adenosine triphosphate (pH 7) and 1 μl T4 DNA ligase (400 units) and incubated for 16 hours at 22 ° C.

The purification of the gene fragments obtained in this way (Tables 1 and 2) by gel electrophoresis on a 10% Polyacrylamide gel (without added urea, 40 × 20 × 0.1 cm), wherein as marker substance ØX174 DNA (BRL), cut with HinfI, or pBR322 cut with HaeIII.  

example 2 a) Cloning of the synthesized DNA fragments

The commercially available plasmid pUC19 is mixed with the Restriction enzymes KpnI and PstI opened and the big one Fragment (1) separated via a 0.8% "Seaplaque" gel. This fragment is labeled with the synthetic DNA (2) according to Table 1 reacted with T4 DNA ligase and the Ligation mixture with competent E. coli 79/22 cells incubated. The transformation mixture is applied to IPTG / Xgal plates, containing 20 mg / l ampicillin, plated. Out the white colonies, the plasmid DNA is isolated and by Restriction and DNA sequence analysis characterized. The desired plasmids receive the name pIK1.

Accordingly, the DNA (5) according to Table 2 in pUC19 ligated, which was opened with PstI and HindIII (4). you receives the plasmid pIK2 (6).

b) Construction of the mini-proinsulin gene

Plasmids pIK1 (3) and pIK2 (6) become the DNA sequences (2) and (5) according to Table 1 and 2 reisolated and ligated with pUC19 opened with KpnI and HindIII (7). This gives the plasmid pIK3 (8), which is for a modified human insulin sequence encoded.

The plasmid pIK3 (8) is opened with MluI and SpeI and the big fragment (9) isolated. This is done with the DNA sequence (10)

ligate the last codon of the B chain (B30) by one Arginine codon supplements and the excised codons replaced for the first 7 amino acids of the A chain and the Codons for amino acids 8 and 9 of this chain added.

This gives the plasmid pIK4 (11), which is responsible for the encoded according to the invention human mini-proinsulin.

c) expression vectors for mini-proinsulin PIK I

The plasmid pK50 (12) known from EP-A 02 29 998 (there Example 3, Figure 3 [33]) is cleaved with EcoRI and HindIII. Both fragments (13) and (14) are isolated. The small fragment (14) with the IL-2 partial sequence is cleaved with MluI and the IL-2 partial sequence (15) is isolated.

The plasmid pIK4 (11) is digested with EcoRI and HpaI and the big fragment (16) isolated. This will now be with the IL-2 partial sequence (15) and the synthetic DNA (17)

ligated to obtain the plasmid pIK8 (18). This encodes a fusion protein in which the first 38 Amino acids of IL-2 a bridge member Met-Ile-Glu-Gly-Arg and then the amino acid sequence of the mini-proinsulin consequences.

The plasmid pIK8 (18) becomes the EcoRI-HindIII fragment (19) cut out for the said fusion protein coded. This fragment becomes with the big fragment (13) ligated, which was obtained in the cleavage of pK50. you  thus receives the expression vector pIK10 (20), which is responsible for the encoded above characterized fusion protein.

pSW3

If one removes the NdeI-BstEII segment from the vector pIK10 (20), that includes the "bom site", you get one Vector, which is present in higher copy number in the cell and because of the lack of "bom site" by conjugative plasmids is no longer mobilizable.

For this, the vector pIK10 (20) with BstEII and NdeI cut, the DNA precipitated with ethanol, in DNA polymerase buffer transferred and subjected to a Klenow polymerase reaction. The resulting blunt-ended DNA fragments are separated gelelektrophoretisch and the larger Fragment (21) isolated. By ligation one obtains the Vector pSW3 (22). After transformation of competent E. coli Mc1061 cells and amplification becomes the plasmid pSW3 (22) isolated and characterized.

example 3 Expression in strain E. coli W3110

An overnight culture of E. coli cells containing the plasmid pIK10 (20) or pSW3 (22) is extracted with LB medium (J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 50 μg / ml ampicillin, diluted in the ratio of about 1: 100 and growth over Followed by OD measurement. At OD = 0.5, the culture is reduced to 1 mM IPTG adjusted and the bacteria after 150 to 180 minutes centrifuged. The bacteria will be in one for 5 minutes Buffer mixture (7 M urea, 0.1% SDS, 0.1 M Sodium phosphate, pH 7.0) and samples taken to one SDS gel electrophoresis plate applied. To gel electrophoretic analysis is in the range of about 10 Kd observed an additional band, the expected Corresponds to fusion protein. This gang responds in the "Western Blot" experiment with antibodies directed against insulin.  Close the cells under pressure and centrifuge then the digestion, then the fusion protein in the sediment along with other insoluble cell constituents found.

The specified induction conditions apply to shake; for larger fermentations is the choice other media and conditions, eg. B. to achieve modified O.D. values, appropriate.

example 4 a) Preparation of Mono Arg Insulin

40 g of the by centrifugation and washing with Phosphate buffer (pH 7) or water-enriched Fusion protein (dry matter content about 25%) are in Dissolved 75 ml 98-100% phosphoric acid and with 5 g BrCN added. After 6 hours of reaction at room temperature the mixture is mixed with 2 liters of water and freeze-dried.

The fragment mixture (10 g) is dissolved in 1 l of buffer solution (8 M Urea, 0.2 M Tris-HCl [pH 8.5]), heated to 30 ° C. and with 10 g of sodium sulfite and 2.5 g of sodium tetrathionate added. After 90 minutes at 30 ° C, 3 liters of cold Add water and adjust the pH to 7.0. The resulting flocculation is centrifuged off. The Hexa-S-sulfonate of the mini-proinsulin is removed from the supernatant precipitated to 3.5 by pH adjustment. After 15 hours Incubation at + 4 ° C is centrifuged. The rainfall is washed with 200 ml of water and freeze-dried. you receives 4.8 g of a mixture of substances in which by RP-HPLC a mini-proinsulin content of 900 mg is determined. The Enrichment of the S-sulfonate takes place in 2 stages:

• 1. Anion exchange chromatography over a 5 x 60 cm column with ®Fractogel TSK DEAE-650 M in 3 M urea;  0.05 M Tris-HCl (pH 8.3). The elution comes with a Gradients of 0.05-0.5 M NaCl (6 L each) made. After analysis of the eluate by isoelectric Focusing one drops the product from the united ones Fractions by dilution to 1 M urea and pH setting to 3.5 off.
• 2. Removal of high and low molecular weight Impurities by gel filtration over ®Sephacryl S200 in 3M urea; 0.05M Tris-HCl; 0.05 M NaCl (pH 8.3). Analysis of the fractions and isolation of the Product will be as in the previous step carried out. The precipitate is mixed with 20 ml of water washed and freeze-dried. This gives 1.10 g of the on 69% purity enriched product.

For folding and disulfide bridge formation, the S-sulfonate in 50 ml of 8 M urea; 0.02 M Tris-HCl dissolved at pH 8.6. After adding a few drops of octanol for 15 minutes purified nitrogen introduced. By adding 1.1 ml (16 mmol) of 2-mercaptoethanol takes place within 1 hour at room temperature the complete reduction. The solution is applied to a ®Sephadex G25 column (5 x 60 cm) and eluted with 0.05 M glycine / NaOH (pH 10.6). The Protein fraction in 300 ml of the glycine buffer is after Control and possible correction of the pH value (10,6) Kept for 2 days at 4 ° C. Thereafter, the pH becomes 6.8 adjusted and the solution with 1 mg (3.5 U) trypsin (Merck, with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone [TPCK] treated) at room temperature for 4 hours. Subsequently, the pH is adjusted to 8.5, the Solution with 1 mg soybean trypsin inhibitor (Sigma) and 3 ml of 10% ZnCl₂ added and returned to pH 6.8 reset. The resulting precipitate is through Centrifugation separated. It contains predominantly mono-arg insulin, that by ion exchange chromatography on S-Sepharose® (2.5 x 40 cm) in a 50 mM buffer  Lactic acid, 30% isopropanol (pH 3.5) is purified. The Elution is carried out by means of a gradient of 0.05-0.50 M NaCl (1 l each). The eluate is analyzed by HPLC; from the product-containing fractions fall the mono-Arg insulin after 1: 1 dilution with H₂O by addition of 10 ml of 10% ZnCl₂ per 1 l and pH adjustment to 6.8. By Centrifugation separated precipitate is from a buffer 1 g / l phenol, 10.5 g / l citric acid and 200 mg / l ZnCl₂ at pH 6 crystallized. Obtained after freeze-drying the washed with a little water crystals 390 mg Mono Arg insulin in over 90% purity.

example 5 Production of insulin

200 mg of mono-arg-insulin (see Example 4) are dissolved in 100 ml 0.05 M Tris-HCl (pH 8.5) dissolved. Then 1 U (about 4 μg) Carboxypeptidase B added and the solution at Room temperature stirred gently. After 3 hours that will be Human insulin by acidification to pH 3.5 and addition of 1 ml 10% ZnCl₂ crystallized at pH 5.5. 200 mg are obtained crystalline insulin with a purity of more than 85%. This material will undergo a cleaning Ion exchange chromatography on a column with Fractogel TSK-DEAE-650 M (2.5 × 40 cm) in 0.1% ® Lutensol ON 100 (BASF AG, Oxethylate of a linear, saturated Fatty alcohol of essentially 12 C atoms); 0.05M Tris-HCl (pH 8.3), eluting with a Gradients of 0-0.4 M NaCl (1 l each) takes place. From the by HPLC identified product containing fractions the insulin after addition of 10 ml of 10% ZnCl₂ and 1 ml 10% Citric acid crystallized at pH 5.5. After slow Stirring overnight is centrifuged and the resulting Sediment from 20 ml of a buffer of 5 g / l citric acid, 125 ml / l acetone and 200 mg / l ZnCl₂ at pH 5.5 recrystallized. 160 mg of insulin are obtained with one Purity of more than 95%.  

example 6 Production of Insulin from Mono Arg Insulin by combined use of trypsin and carboxypeptidase B

5 mg Mono Arg insulin are dissolved in 20 ml 0.1 M Tris-HCl (pH 8.0). dissolved and heated to 30 ° C. There will be 2.5 μl at the same time Trypsin solution (1 U / ml) and 150 μl of carboxypeptidase B solution (1 U / ml) was added. After 3 hours the solution becomes adjusted to pH 3.5 and 2.5 μl trypsin inhibitor solution (1 U / ml) and 200 ul of 10% ZnCl₂ solution was added. The Human insulin is precipitated by adjustment to pH 6.8, centrifuged and crystallized as in Example 5. The crystallized insulin has a purity <95%.

example 7 Construction of a yeast expression vector

First, the DNA sequence (23) (Table 3) is synthesized by the phosphite method. This DNA sequence (23) codes for the amino acids 49 to 80 of the MF α -Vorläuferproteins and corresponds essentially to the natural DNA sequence.

The DNA sequence (23) is first used as a probe to isolate the gene for the α- factor and this marked with ³²P. This probe is isolated from a λ gt11 genomic yeast library (as now commercially available and available, for example, from Clontech Laboratories Inc., 4055 Fabian Way, Palo Alto, CA 94 303). For this, λ gt11 phages carrying the α- factor gene are identified in a plaque hybridization experiment. Phages from positively identified plaques are isolated, propagated and the DNA recovered. This is digested with EcoRI and analyzed on a 0.8% agarose gel. After a "Southern transfer" experiment, the membrane is hybridized against the 32 P-labeled DNA sequence (23). Phage DNA having an approximately 1.75 kb fragment (24) that hybridizes to the DNA sequence (23) is cleaved again with the enzyme and the corresponding fragment (24) isolated. The vector pUC 19 is opened with EcoRI (25) and reacted with the 1.75 kb fragment (24) with T4 ligase. The cloning vector (26) is obtained.

With the ligation mixture of the strain E. coli 79/02 transformed. White colonies are isolated, from this the Plasmid DNA was recovered and plasmids (26) containing the 1.75 kb EcoRI fragment included, identified.

The natural DNA sequence of the precursor protein for MF α contains a PstI site in the region of amino acids 8 to 10 and a TaqI site in the region of amino acids 48/49. From the isolated plasmid DNA (26), the fragment (27) which codes for the amino acids 9 to 48 of the MF α precursor sequence is now isolated by reaction with PstI and TaqI. The vector pUC18 is opened with PstI and KpnI (28) and reacted with the PstI-TaqI fragment (27) as well as with the synthetic DNA sequence (23) by means of T4 ligase. The ligation mixture is used to transform E. coli 79/02. The transformation mixture is plated on IPTG-Xgal Ap plates. White colonies are isolated and the plasmid DNA of these clones characterized by restriction analysis. This gives the cloning vector (29) which codes for amino acids 8 to 80 of the MF α precursor sequence.

From the cloning vector (29), said coding sequence (30) is excised by reaction with PstI and KpnI and introduced into the ligation described below. For this purpose, the cloning vector (26) is reacted with EcoRI and partial with PstI and isolated the coding sequence for the first 8 amino acids of the MF α -Vorläufersequenz comprising fragment (31). Furthermore, the vector pUC19 is opened with EcoRI and KpnI (32) and ligated with the two described fragments (30) and (31) to form the cloning vector (33). This encodes the entire precursor sequence of MF α to amino acid 80.

The cloning vector (33) is cloned with KpnI and HindIII opened and the big fragment (34) isolated. This will with the KpnI-HindIII fragment (35) from plasmid (11), the encoded for the mini-proinsulin, ligated. You get that Plasmid pIK20 (36), its structure by restriction analysis is confirmed.

The plasmid Yep13 (Broach et al., Gene 8 [1979], 121) opened with BamHI and the protruding ends with Klenow polymerase filled up (38). The DNA is precipitated with ethanol and treated with alkaline bovine phosphatase.

From the cloning vector (36), the fragment coding for the insulin derivative and the precursor sequence of the MF α is cut out with HindIII and EcoRI and the protruding ends are filled in as described (37).

The two blunt-ended DNA sequences (37) and (38) are ligated together, resulting in the plasmids p α fB102 (39) and p α fB104 (40). These two plasmids differ only in the orientation of the inserted fragment.

As described in EP-A 01 71 024, a terminator can be used behind the inserted sequence ( FIGS. 4 to 6 of EP-A 01 71 024). The NcoI and / or BamHI interfaces are suitable for this purpose.

After amplification of the plasmid DNA in E. coli MM294, the plasmid p a fB102 (39) is transformed into the leucine-requiring yeast strains Y79 ( α , trp1-1, leu2-1) (Cantrell et al., Proc. Acad. Natl. Sci., USA 82 [1985], 6250) and DM6-6 ( α / α leu2-3, 112 :: ura3⁺ / leu2 :: lys2⁺, trp1⁻ / trp1⁻, his3-11, 15 / his3-11 , 15, ura3⁻ / ura3⁻, lys2⁻ / lys2⁻, arg4-17 / arg4⁺, adel⁻ / adel⁺) (Maya Hanna, Dept. Mol. Biol., Massachusetts General Hospital, Boston, USA) after lithium Method of Ito, H. et al., J. Bacteriol., 153 (1983), 163. Colonies that can grow on selective medium without leucine supplement are isolated and separated. Yeast minimal medium is inoculated with the individual colonies and incubated for 24 hours at 28 ° C. The cells are centrifuged off and the supernatant is checked for insulin activity in an RIA assay. From yeast clones whose supernatant shows insulin activity, the plasmid DNA is reisolated and characterized by restriction analysis. The transformed yeast strains are used for the following expression.

example 8 Expression in yeast

10 ml of whole yeast medium is mixed with cells that are fresh Overnight culture of a strain obtained according to Example 7 from Selective medium were inoculated so that a optical density OD₆₀₀ = 0.1 is reached. The culture will Shaken for 8 hours at 28 ° C, whereupon 90 ml of fresh medium be added. Subsequently, the culture for more Shaken for 20 hours. The cells are centrifuged off and the insulin concentration in the supernatant determined. The Conditions change for larger fermentation, e.g. B. Fresh medium can be added continuously.

example 9 Purification of Mono Arg Insulin yeast supernatant

The fermentation supernatant is passed through an adsorption column with a porous adsorbent resin of a copolymer of Styrene and divinylbenzene (®Diaion HP20). The pillar was previously treated with a 20-50 mM acetate buffer (pH 5) equilibrated. After washing with Tris buffer (pH 8) becomes Isopropanol gradient (0-50%) with 10 times Column volume created. Insulin-containing fractions are adjusted to pH 6, with ®MATREX CELLUFINE AM (Fa.  Amicon), stirred and filtered with suction and with 50 mM Acetate buffer (pH 6) washed. The washing fraction and the Main fraction are combined with lactic acid to pH 3.5 adjusted and over an S-Sepharose column, with 50 mM Lactic acid (pH 5) / 30% isopropanol was equilibrated, given.

Elution is carried out by means of a 0-0.6 M NaCl gradient. Mini-proinsulin elutes in the range 0.25-0.3 M.

The proinsulin-containing fractions are added to ¼ of the Concentrated volumes and on a column with Biogel P10 (Bio-Rad) equilibrated in 6% acetic acid (pH 2). The insulin-containing eluate is lyophilized and over a preparative "reversed-phase" HPLC step (RP18 material, 0.1% TFA, acetonitrile gradient 20-40%). To subsequent freeze-drying is carried out in Tris buffer (pH 6.8) dissolved and with 4 units of trypsin per gram of mono-arg-proinsulin incubated at room temperature for 3 to 5 hours. The Reaction process is via "reversed-phase" analysis controlled. It turns out that almost quantitatively Mono-Arg insulin arises. At the end of the reaction becomes a pH adjusted by 3.5 and the reaction by adding a equivalent amount trypsin inhibitor terminated. Subsequently is a zinc chloride concentration of 0.21 g / l and a pH set by 6.8. You get a flaky Precipitate dissolved in lactic acid buffer. The Components are purified by S-Sepharose chromatography separated from each other. Fractions, the mono-arg insulin are combined and combined with water Mixed 1: 1. Then the solution with 10 ml 10% ZnCl₂ added per 1 liter. At pH 6.8 then falls Mono-Arg insulin, which is then known in (for insulin) Recrystallized.

example 10 Production of human insulin

The mono-Arg insulin shown in Example 9 serves as  Starting substance for the carboxypeptidase B cleavage. To the insulin derivative is dissolved in Tris buffer (pH 8.5) and with 5 units of carboxypeptidase B per gram Mono Arg insulin added. With gentle stirring Room temperature, the reaction is over 3 hours carried out. Then as described in Example 9 with ZnCl₂ like. Human insulin is then administered in a known manner cleaned (DE-B 26 29 568).  

Table 1

Gene fragment IK I (2)

Table 2

Gene fragment IK II (5)

Table 3

DNA sequence (23)

Claims (10)

1. Compound of formula I B (1-30) -Arg-A (1-21) (I) in the B (1-30) and A (1-21) the B and A chain of the Human insulin mean. 2. Compound of formula I for use as a remedy, in particular for the treatment of diabetes mellitus. 3. Medicaments containing the compound of the formula I. 4. Medicines from a pharmacologically acceptable Carrier and the compound of formula I. 5. Use of the compound of the formula I for the preparation of the compound of the formula II in which A (1-21) and B (1-30) have the meaning given in claim 1 and the -SS bridges are arranged as in insulin, or of insulin, preferably in a one-pot reaction. 6. A process for the preparation of the compound of the formula I, characterized in that one for this Compound-encoding gene structure in a host cell, preferably in a bacterium or in a yeast, expressed and, if the gene structure for a Fusion protein encoded, from the obtained  Fusion protein releases the compound of formula I. 7. DNA coding for the compounds of the formula I. 8. Gene structure or plasmid containing the DNA according to Claim 7. 9. host cell, preferably a bacterium or a yeast, containing the gene structure or a plasmid Claim 8. 10. fusion protein containing the compound of formula I, preferably via a bridge member -Met-Ile-Glu-Gly-Arg-bound to the "ballast portion" of the fusion protein.