DK172458B1 - Signal peptide, DNA sequence encoding this peptide, gene structure containing the DNA sequence, plasmid containing DNA sequence - Google Patents

Abstract

1. Claims for the contracting states : BE. CH. LI. DE. FR. GB. IT. LU. NL. SE DNA sequence B, obtainable from Streptomyces tendae strains, which produce tendamistat and have preferably been treated with sublethal doses of acriflavine, by isolation of the complete DNA, digestion with Pst l, Southern hybridization with the DNA sequence A, 5'-(**32 P-)CCT TCA GTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst l fragment, cutting with BamHl, Southern hybridization with the sequence A, isolation of the 0.94 kb Pst l-BamHl subfragment, cutting with Sau 3a, Southern hybridization with the sequence A, isolation of the 0.525 kb BamHi-Sau 3a subfragment and sequencing of the DNA, and which has the following features : a) it is located immediately upstream of the tendamistat structural gene, b) it codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) it codes at the carboxyl terminal end for Ala-Ser-Ala and d) it codes in the middle for a hydrophobic region X which comprises 10 to 25, preferably 17 to 20, amino acids. 1. Claims for contracting state AT A process for the preparation of polypeptides composed of genetically codable amino acids, characterized by the expression, in a host cell of the genus Streptomyces, of a gene structure which contains in the reading frame with the structural gene for the desired polypeptide the DNA sequence B of a prepeptide, which sequence is obtainable from Streptomyces tendae strains, which produce tendamistat and have preferably been treated with sublethal doses of acriflavine, by isolation of the complete DNA, digestion with Pst l, Southern hybridization with the DNA sequence A, 5'-(**32 P-)CCT TCA GTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst l fragment, cutting with BamHI, Southern hybridization with the sequence A, isolation of the 0.94 kb Pst l-BamHl subfragment, cutting with Sau 3a, Southern hybriridization with the sequence A, isolation of the 0.525 kb BamHl-Sau 3a subfragment and sequencing of the DNA, and a) is located immediately upstream of the structural gene, b) codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) codes at the carboxyl terminal end for Ala-Ser-Ala and d) codes in the middle for a hydrophobic region X which comprises 10 to 25 amino acids.

Description

in DK PR 172458 B1

The present invention relates to a signal peptide which is a component of a propeptide which, in a streptomycet cell containing a signal peptidase, is cleaved into the signal peptide and a polypeptide, whereby the latter is removed from the cell and secreted into the culture medium. The invention further relates to a DNA sequence encoding this signal peptide, gene structures containing this DNA sequence in read frame with a structural gene, a plasmid containing such DNA sequence or gene structure, and a host organism containing such a plasmid. The invention further relates to a method for producing a polypeptide using such a host organism.

German Patent Application No. P 33 31 860.3 proposes a process for the preparation of tendamistat 15 by fermentation of streptomyces tendae, which is characterized by the use of tendamistat-producing S. tendae strains treated with sublethal doses of acriflavine. From the strains obtained, a DNA fragment is isolated by the tendamistat gene, namely a 2,3-kb-Pst X fragment. By incorporating this fragment into a pBR 322 cut with Pst I, this DNA could be propagated in E. coli and isolated again.

It has now been found that on this 2,3-kb fragment directly in front of the structural gene for tendamist 25, a signal peptide (prepeptide) of the formula is encoded

Met-Arg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala I

wherein S is a hydrophobic region comprising 17-20 amino acids.

The invention thus relates to a peptide which is peculiar to its formula

Met-Arg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala 35 wherein X is a hydrophobic region comprising 10-25, preferably 17-20 amino acids.

The invention also relates to the corresponding DNA sequence 5B which can be obtained from tendamistat-producing Streptomyces tendae strains, which are preferably treated with sublethal doses of acriflavin, by isolating the total DNA, digestion with Pst I, Southern hybridization. with the DNA sequence A

5 '- (32P-) CCT TCA GTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst I fragment, sectioning with BamHI, Southern hybridization with sequence A, isolation of the 0, 94 kb PstI-BamHI subfragment, sectioning with Sau 15 3a, Southern hybridization with sequence A, isolation of the 0.525 kb BamHI-Sau 3a subfragment and sequencing of the DNA, which is characterized by the characteristics: (A) it encodes immediately before the tendamistat structure of the gene, (b) it encodes at the amino-terminus of Met-Arg-Val-Arg-Ala-Leu-Arg, (c) it encodes at the carboxy terminus of Ala- Ser-Ala, and (d) it encodes in the center of a hydrophobic region comprising 10-25, preferably 17-20 amino acids.

The kb-number values obtained by comparison with standard markers have the usual accuracy.

Instead of Sequence A, Southern hybridization may select any sequence that is complementary to the tendamistat gene or the counter strand.

In practice, for the various steps to characterize DNA sequence B, the DNA is introduced into a suitable vector, transformed into a host cell, propagated there, transformants obtained by colony hybridization with sequence A, and DNA isolated again. These steps are known per se.

The DNA sequence C, whose coding strand is listed below in the Appendix to the specification, has the nucleotide sequence of the tendamistat structural gene derived from S. ten-days.

The invention also relates to a gene structure which is unique in that it contains the DNA sequence B, preferably immediately in the reading frame with a structural gene, in particular the structural gene for tendamistat, in particular the DNA sequence C.

The invention further relates to a plasmid which is peculiar to the above DNA sequence B or gene structure, preferably with a streptomyceter replicon and / or an E. coli replicon.

If the plasmid contains an E. coli replicon, it is capable of propagating the DNA of E. coli and optionally also expressing it. If the plasmid contains a 25-streptomycetre replicon, a streptomycet is transformed with such a plasmid capable of expressing the peptide-determined peptide of the formula II, which then cleaves with a signal peptidase within the framework of the reprocessing and secretes it. desired peptide in the culture medium.

Advantageous are the so-called "shuttle" plasmids, which contain a replicon that is active both in E. coli and in streptomycytes. These "shuttle" vectors can be propagated in E. coli and, after re-isolation, transformed into streptomycetes, where production of the desired polypeptide takes place.

4 DK PR 172458 B1

The invention also relates to a host organism of the genus Streptomyces, especially the species S. tendae or S. lividans, which is characterized in that it contains a plasmid according to the invention.

The invention further relates to a method for producing a polypeptide which is characterized in that a host organism according to the invention is used which contains a signal peptidase which separates the prepeptide corresponding to the DNA sequence B.

While there are numerous vectors for gram-negative bacteria, only a few vectors for gram-positive bacteria have been described. Vectors for bacteria of the species S. tendae have so far been unknown. Thus, the invention opens up the possibility of access to the utilization of S. tendae as host organisms.

A particular advantage of the invention is that transformed streptomyces strains, especially S. lividans, have optimal spore formation, i.e., the content of the recombinant plasmid does not generate this strain in its generative phase. The transformed organisms are therefore also suitable for further stanuna improvements, e.g. for the preparation and selection of metabolic mutants by whose preparation is worked on spores.

25 Unlike the known strains of S. tendae, the transformed strains, especially S. lividans, do not form any melanin. Therefore, the separation thereof lapses, which facilitates the isolation of the desired peptide, e.g. tendency loss, significantly and prevent loss of dividends.

30

A further advantage of the invention is that foreign genes are also expressed in S. lividans and the corresponding polypeptides are secreted, which also gives 35

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5 DK PR 172458 B1 many possibilities for strain enhancement and also for modification of the polypeptide thus produced.

However, according to the invention, other Streptomyces species can also be transformed, e.g. S. ghanaensis or 5 aureofaciens, If plasmid-free strains capable of synthesizing a specific signal peptidase are transformed with the hybrid plasmids of the invention, stable transformants are expressed which express and secrete the encoded peptide, particularly preferred embodiments of the invention will be elucidated. explained in the following examples. The percentages here are by weight, unless otherwise stated. The figures in the drawing, which produce hybrid plasmids, show the cut sites in the correct scale conditions.

In the examples, the following vectors known from the literature are used; single strand phages M 13 mp 8 and M 13 mp 9; Brass et al., Gene 19, 269 (1982), pUC 8: Vierra et al., Gene 19, 259 (1982), pAC 177 and 184: Chang et al., J. Bacteriology 134, 1141 (1978) ), pIJ 102 and 350: Kieser et al., Mol, Gen. Genet. 185, 223 (1982),

How the strain S, tendae is obtained, is. disclosed in U.S. Patent No. 4,226,764, The isolation of the tendon mist may in principle occur from any tendamistat-producing strain. However, particularly advantageous is the method of German Patent Application No. P 33 31 860.3, in which Example 3 the isolation of DNA is described. This isolated total DNA is the starting material for Example 1 below.

Example 1 5 µg DNA is completely digested with the restriction enzyme Pst I and transferred after separation to a 0.8% agarose gel on nitrocellulose filter (Southern transfer).

The filter with it. bound denatured DNA is pre-hybridized for 6 hours in 5 ml of pre-hybridization medium (0.6 molar NaCl, 0.06 molar Na-EDTA, 0.1% sodium dodececyl sulfate solution,

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6 DK PR 172458 B1 100 µg / ml ultrasound treated calf DNA and 4 times concentrated Denhardt solution. Then, again, treat with 5 ml of pre-hybridization medium, to which, however, 500,000 cpm / ml of radiolabeled DNA is added. This radiolabeled sample is obtained as follows: The DNA Sequence A is chemically synthesized by the phosphite method. It contains 20 nucleotides (molecular weight approx. 13,000) and is complementary to the putative DNA sequence for tendamistat, derived from the amino acid sequence, from tendamistat from amino acid 37 of tendamistat using the E. coli preferred triplets.

The PNA sequence A is radiolabeled at the 5 'end by 32 of γ-β-ATP and nucleotide kinase.

To hybridize this radioactive sample to the complementary DNA sequence of the total DNA, the mixture is left for 24 hours at 37 ° C. Then, the unbound radioactive DNA is removed and the filter is washed at 37 ° C for 5 x 30 minutes each time with 200 ml of hybridization medium and then autoradiographed. After 24 hours of exposure, the hybridization signals show that the gene is on a 2.3 kb Pst I fragment. This fragment is recovered by sweep electroelution on preparative agarose gel of a sectional portion corresponding to this fragment size to partition the Pst1 digested total DNA. The 25 eluted DNA is cloned into the Pst I cut site in plasmid pUC 8.

These hybrid plasmids are transformed into E. coli JM 103 and propagated. The detection of such clones carrying the searched insert with the tendamistat gene is accomplished by colony hybridization using the radioactive DNA sample A. The hybrid plasmids pKAI1a and lb thus obtained are prepared in FIG.

1 a and b.

By further Southern hybridizations against the isolated 2.3 kb Pst I fragment and its 35 sub-fragments, the gene can be accurately located (Figs. 2a to 2c), DK PR 172458 B1 o 7

Example 2 In the particular sectional sites of plasmid pIJ 102 for Pst I, the 2.3 kb Pst I fragment of S. tendae is cloned. The hybrid plasmids pAX 1a and lb thus obtained, which differ in orientation, insert after transformation into S, lividans strains to produce tendamistat. In FIG. 3, the plasmid pAX 1a is seen.

Example 3

The market strain S. lividans TK

24 (John Innes Institute, Norwich, England) is known in known manner to protoplasts and 1 µg hybrid 8 'plasmid pAX1a is added with 10 protoplasts with the addition of 15 20% fs polyethylene glycol 6000. The transformed protoplasts are grown on regeneration medium R2YE [Thompson et al., Nature 286, 525 (1980)] at 30 ° C for 5 days.

The detection of an extracellular amylase inactivator can be established by means of a pla-20 test:

The regenerated colonies are poured with 5 ml of a 0.4-1 mg / ml pancreatin-containing aqueous solution and incubated for one hour at 37 ° C. The solution is then removed and 5 ml of a 2% starch agar is added. After 2 hours of incubation at 37 ° C, the plates are poured with 5 ml of an iodine-potassium iodide solution to develop. The blue ring colonies show,. that the clones synthesize and secrete tendamistat.

As a control, from the tendamistat-producing well-spore-forming strains, the plasmid DNA can be isolated and mapped. All tendamistat-producing strains present the inserted plasmid DNA.

Example 4 The procedure of Example 2 is used, however, using the plasmid pIJ 350, which as the selection marker

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8 DK PR 172458 B1 in streptomycetes carries a thiostrepton resistance gene.

Hereby the hybrid plasmids pAX 350 a and b are obtained (different in orientation of the insert).

FIG. 4 shows the plasmid pAX 350 a.

After transformation of Example 3, select on minimal medium [Hopwood, Bacteriological Reviews 31, 373-403 (1967)] in the presence of 50 µg / ml thiostrepton resistant clones and these are tested either directly on minimal medium or after scrubbing on non-selective R2YE -10 agar for tendamistat production.

Example 5

Hybrid plasmids containing the 2.3 kb Pst X fragment and, in addition to the streptomycet replicon, also contain an E. coli replicon, have as shuttle vectors a number of advantages: because of the E. coli replicon and the resistance markers active in E. coli can be propagated well in these organisms. After isolation and transformation in streptomycetes, especially S. lividans, 20 exhibit high stability. Due to their selection markers active in streptomycetes and the streptomycet replicon, they can also propagate well in these organisms and express and secrete tendamistat.

Plasmid pAC 184 is fully digested with the restriction enzyme Sal I and the enzyme is removed by phenol / chloroform extraction. The protruding 5 'ends are filled in the presence of ATP, CTP, GTP and TTP by the enzyme DNA polymerase ( Klenow fragment). To the blunt ends, at a ligase reaction 30 at 16 ° C, a Pst-I glue piece of structure 5'TCG AGC TGC AGC TCG A 3 '3' AGC TCG ACG TCG AGC T 5 '35 (2 µg intermediate car Q, 4 µg DNA), the DNA is extracted with phenol / chloroform and, after precipitation, digested with

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9 DK PR 172458 B1 the enzyme Pst I to recover the ligatable Pst I ends.

The DNA is then dephosphorylated, extracted again with phenol / chloroform and ligated with the partially digested plasmid pAX 1a with Pst I. The ligation mixture is transformed into E. coli (HB 101 or MC 1061). Chloramphenicol resistant clones are purified from the plate and the DNA isolated. S. lividans TK 24 is transformed with 1-2 µg DNA and tested for tendamistat production,

Clones that respond positively to the tendamistat-10 assay are isolated, the plasmid DNA is isolated by alkaline flashlight and back-transformed into E. coli HB 101 or MC 1061. The post-proliferated isolates do not differ from the S. lividans strains isolated plasmids. Depending on the orientation of the insert carrying the tendamistat gene, the recombinant plasmids are designated as pSA 2a or b (Figs. 5a and b).

Example 6

The procedure of Example 5 is used, however, starting with plasmid pAX 350a selected in E. coli for chloramphenicol resistance and in S. lividans for thiostrepton resistance and tendamistat production, thereby obtaining the plasmids pSA 351 a and b (Fig. 6 a / b).

25

Example 7

The procedure of Example 5 is used, however, starting with plasmid pAC 177 instead of pAC 184, thereby obtaining plasmids pSA 3 a and b (Fig. 7 a / b).

For this, plasmid pAX 1a is partially incised

Pst I, and the enzyme is heat inactivated at 15 minutes heating to 68 ° C. The DNA is ligated into the Pst I section dephosphorylated and deprotected plasmid pAC 177. After transformation of E. coli HB 101 or 35 MC 1061, they are purified against kanamycin resistant clones from the plate, the plasmid DNA is isolated and S, lividans TK

DK PR 172458 B1 o 24 is transformed with 1-2 µg of this DNA. Tendamistat-producing clones are selected and the plasmids are characterized.

Example 5

Proceed according to Example 7, but instead of plasmid pAX 1, plasmid pAX 350 a is used and selected in S. lividans for thiostrepton resistance to obtain plasmids pSA 352 a and b 10 (Fig. 8 a / b).

Example 9

As shown in FIG. 2, the gene encoding tendamistat and the signal sequence is approx. 0.3 kb in size.

Thus, the 2.3 kb fragment used in the above examples can be used in abbreviated form to construct hybrid plasmids which produce tendamistat production;

The plasmid pKAI 1a is digested with Sal I and religated. Thus, it is available with approx. 750 base pairs abbreviated plasmid pKAI 2, This is cloned, isolated and sectioned with Pst I, the DNA dephosphorylated with calf intestinal alkaline phosphatase and deproteinized with phenol / chloroform, the plasmid pIJ 102 cut completely with Pst I, and the fragments ligated into Pst The I-site of pKAI 2 after heat inactivation of the enzyme. The ligation mixture is transformed into E. coli HB 101 or MC 1061. The plasmid DNA is isolated from clones resistant to ampicillin 30 by alkaline rapid lysis; and S, lividans TK 24 is transformed with 1-2 µg of this DNA is selected from tendamistat production and the plasmid DNAf in these clones is isolated by alkaline rapid lysis.

After reverse transformation in E. coli HB 101 or MC 1061 35 and after isolation of the plasmid DNA from the transformed E. coli strains, plasmid pSA 1 is determined by

II

DK PR 172458 B1 o of restriction analysis (Fig. 9). The plasmid shows no differences from the plasmids isolated from S. lividans-stairans, but the DNA work-up from E.coli is more abundant and occurs in a shorter time.

5

Example 10

The procedure of Example 9 is used, but instead of the plasmid pIJ 102, the plasmid pIJ 350 is used, whereby in S.lividans it can then be further selected for thiostrepton resistance to obtain plasmids pSA 350 a or b. FIG. Figure 10 shows the plasmid pSA 350a. In shake culture, this plasmid leads to higher yields of tendamistat than the plasmid pSA 350b, which contains the insert with the tendamistat gene in reverse orientation. By contrast, reducing the insert to 1.5 kb has no significant influence on product formation.

Example 11

To determine the structure of the tendamistat gene and the nucleotide sequence, the 0.94 kb Pst Ι-Bam HI sub-fragment (Figs. 2a and b) and the 295 kb Sau 3a - Bam HI sub-fragment (clone) are cloned ( Fig. 2c) into the single strand phages M 13 mp 8 and M 13 mp 9. As initiator of the di-deoxy sequencing reaction, the DNA sequence A, which comprises 20 nucleotides as well as a commercially available 15 kb starter (Bethesda Research Laboratories), is used. GmbH, Neu-I.senburg). The DNA sequence C is obtained

Example 12 In front of the tendamistat structural gene, there is an open reading frame on the DNA until the start codon ATG (met) for a protein that is stored seamlessly in front of the tendamistat amino terminus. This signal peptide corresponds to the DNA sequence B.

35

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12 DK PR 172458 B1

Appendix: DNA Sequence C (Encoding Size 5 10 5'-GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG 5 Nl -Asp Thr Thr Val Ser Glu Pro Ala Prc Ser Cys Val 15 20

ACG CTC TAC CAG AGC TGG CGG TAC TCA CAG GCC GAC

Thr Leu Tyr Gin Ser Trp Arg Tyr Ser Gin Ala Asp 25 30 35 10 AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC Asn Gly Cys Ala Glu Thr Val Thr Val Val Val Val «« o 5

TAC GAG GAC GAC ACC GAA GGC CTG TGC TAC GCC GTC

Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala Val 15 50 55 εο

GCA CCG GGC CAG ATC ACC ACC GTC GGC GAC GGC TAC

Ala Pro Gly Gin Ile Thr Thr Val Gly Asp Gly Tyr 65 70

ATC GGC TCG CAC GGC CAC GCG CGC TAC CTG GCT CGC

20

Ile Gly Ser His Gly His Ala Arg Tyr Leu Ala Arg TGC CTT TAG-3 '

Cys Leu Stp 25

Examples 1 and 3 of TV patent application P 33 31 860.3 Example 1 2

Mutation method: Approx. 0.25 cm of spore-forming mycelium 30 of strain DSM 2727 grown on oatmeal agar (EP-A 49 847) is seeded in 100 ml Erlenmeyer flasks loaded with the following culture medium:

Soybean meal 2% Ammonium citrate 0.1%

Glucose 3% Malt extract 0.5% Maize starch 0.2% KH2PO4 0.2%

Urea 0.1% DK PR 172458 B1 o 13

The medium is autoclaved for 30 minutes at 121 ° C and 1 bar, the pH value is 7.0. The seed flasks are incubated for 2 days at 30 ° C on a shaker. Then, 2 ml of the cultured preculture is inoculated into 20 ml of the main culture medium contained in the 100 ml Erlenmeyer flasks (EP-A 49847).

Soluble starch 2-4% Skimmed milk powder 0.7%

Soybean meal 0.4% glucose 1.0%

Fl. corn casting liquid 0.4% (NH4) 2HPO4 1.2% 10

After autoclaving, the pH of the medium is 7.6.

To this medium, acriflavine is added at a concentration of 10-25 µg / ml as a mutagenic agent. The culture is shaken for 5 days at 28 ° C and 220 rpm. The culture solution is then centrifuged (1300 G, 5 minutes) and the cell pellet in the main culture medium washed. The washed cells are fragmented with a glass homogenizer and smeared on agar plates with the following medium: Cane sugar 0.3% Tryptone Soybean Soup 0.5% 20 Stage Step 1.5% (Fa. Oxoid)

NaCl 0.05% Meat Extract 0.1% K2HPO4 0.05% (Fa. Difco)

FeS04 x 7 H2 O 0.001% Yeast extract 0.2%

Agar (Fa. 1.5% (Fa, Difco) 25 Merck)

The solution pH is 7.1 (autoclaving conditions as above).

The plates are grown for 5 days at 28 ° C, and single colonies are seeded in sloped glass with 30 oatmeal agar. These are grown as described until the mycelium has formed spores. The spore-forming mycelium is propagated as described above in the pre- and main culture, and the culture filtrate is tested after 5-day main culture for tendamistate content (cf. EP-A 49 847). In this way, strains 1-9353, 1-9362, 1-9417 and 1-9418 can be selected, which during their growth period under the described conditions PR PR 172458 B1 o 14 produce approx. 90,000-100,000 I.E., tendami state / liter culture solution corresponding to 1.5-1.8 g inactivator / liter.

Example 3

Isolation of deoxyribonucleic acid from the 5th tendae and its molecular biological characteristics._

The culture of cells is done according to Example 1.

After 3 days of growth in the culture medium, cells io are harvested in an Erlenmeyer flask by centrifugation for 5 minutes at 3000 G and 4 ° C and washed twice with 50 ml of a solution of 50 mmolar Tris / HCl buffer, 100 mmolar NaCl and 25 mmol EDTA.

In a typical preparation, shock-frozen 3 g solid-centrifuged cells are frozen in liquid nitrogen at -198 ° C and then homogenized with a chopping knife blender (Warring Commercial Blender) in the presence of N2 to a fine powder. This powder is taken up in 10 ml of the said buffer, dissolved on ice and incubated with 0.8 20 ml of lysozyme solution (30 mg / ml) for 20 minutes at 28 ° C with light shaking. Then 0.8 ml of a proteinase K solution (15 mg / ml) and 0.8 ml of a 20% solution of the sodium salt of N-lauroylsarcosine are added to the suspension.

After gentle mixing, the mixture is incubated for 30 minutes on ice and further for 15 minutes at room temperature. After adding and dissolving 22 g of solid calcium chloride, the volume of the above buffer is adjusted to 22 ml and the suspension is centrifuged at 12,000 G and 4 ° For 25 minutes, to the clear centrifugate is added ethidium bromide and the refractive index is refractometrically adjusted with CsCl to n - 1.3920. After preparative ultracentrifugation (120,000 G, 36 h, 15 ° C), the bands of chromosomal DNA are removed from the centrifuge tubes and centrifuged under the same conditions. The isolated bands are freed by shaking with n-butanol for ethidium bromide and dialyzed to completely remove CsCl. The DNA thus obtained may be cut e.g. with the enzymes Pst I,

BamHI, Sau 3A, Cla I, Bgl II and Xho I and cannot be cut with e.g. the endonucleases Eco RI and Hind III.

It for example. DNAs extracted from mutants 1-9353 and 1-9362 are characterized relative to DNA from ATCC 31210 and DSM 2727 by an amplified genetic element whose single fragments appear clearly visible after staining with the fluorescence dye ethidium bromide on the basis of non-selectively propagated fragments.

The size of the reinforced element as well as the characteristic single fragments after digestion with restriction endonucleases are reproduced below:

Fragment Fragment size (kbp)

No.__ Pst I_Xho I_Bam Η I

15 1 9.0 13.5 7.2 2 7.0 11.5 6.1 3 6.8 9.3 5.3 4 4.6 2.8 2.9 5 3, ¾ 0.7 2, 6 20 6 3.1 2.5 7 2.3 2.15 8 1.1 2.1 9 1.5 10 1.2 25 11 1.1 12 1.0 13 0.8 1¾ 0.5 15__0, H8 30 total 37T3 37 ^ 8 37, i 3 35

Claims (8)

1. Peptide, characterized in that it has the formula 2. DNA Sequence B, which KAP is derived from tendamistat-producing Streptomyces tendae strains, which are preferably treated with sublethal doses of acriflavin, by isolation of the total DNA, digestion with Pst I, southern hybridization with the DNA sequence -15 late A 5 '- (32P-) CCT TCA GTG TCG TCT TCG TA-3' A * isolation of the 2.3 kb Pst I fragment, sectioning with BamHI, southern hybridization with sequence A, iso-2 0lation of the 0.94 kb PstI-BamHI sub-fragment, sectioning with Sau 3a, southern hybridization with the sequence A, isolation of the 0.525 kb BamHI-Sau 3a sub-fragment, and sequencing of the DNA, characterized by the characteristics: (A) it encodes immediately before the tendamistat structure gene, (b) it encodes at the amino-terminus of Met-Arg-Val-Arg-Ala-Leu-Arg, (c) it encodes at the carboxy terminus of 30 Ala-Ser -Ala, and (d) it encodes in the center of a hydrophobic region comprising 10-25, preferably 17-20 amino acids. 35 DK PR 172458 B1 3. A gene structure, characterized in that it contains the DNA sequence B, preferably immediately in the reading frame with a structural gene, in particular the structural gene for tendamistat, in particular the DNA sequence c. A plasmid characterized by a DNA sequence according to claim 2 or 3, preferably with a streptomy site replicon and / or an E. coli replicon. Host organism of the genus Streptomyces, especially the species S. tendae or S. lividans, characterized in that it contains a plasmid according to claim 4. Met-Arg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala where X is a hydrophobic region comprising 10-25, preferably 17-20 amino acids. A method for producing a polypeptide, characterized in that a host organism according to claim 5 is used which contains a signal peptidase which separates the prepeptide corresponding to the DNA sequence B. Method according to claim 6, characterized in that the polypeptide is removed from the cell after separation of the prepeptide. A method according to claim 6 or 7, characterized in that a host organism with a plasmid containing a gene structure according to claim 3. is used.