NO173452B - PROCEDURE FOR PREPARING POLYPEPTIDES IN STREPTOMYCETS - 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

The present invention relates to a method for producing polypeptides from genetically codeable amino acids.

In the German patent application P 33 31 860.3, a method for the production of tendamistat by fermentation of Streptomyces tendae was already proposed, in which the method is characterized by the use of tendamistat-producing S.tendae strains and treatment with sublethal doses of Acriflavin. From strains thus formed, a DNA fragment with the gene for tendamistat, namely a 2.3 kb Pst I fragment, was isolated. By incorporating this fragment into Pst I-cut pBR 322, this DNA could be amplified in E. coli and again isolated.

It was now found that on this 2.3 kb fragment immediately before the structural gene for tendamistat, a signal peptide (prepeptide) of formula I is encoded

in which X means a hydrophobic region comprising 17-20 amino acids.

It was further found that this signal peptide is also able to export other peptides from the host cells which contain a corresponding signal peptidase.

The present invention is therefore characterized by expressing in a host cell from the species Streptomyces a gene structure which, in the reading frame with the structural gene for the desired polypeptide, contains the DNA sequence B of a prepeptide, obtainable from the tendamistat-producing Streptomyces tendae strain, which is preferably treated with sublethal doses of acriflavine, through isolation of total DNA, digestion with Pst I, Southern hybridization with DNA sequence A isolation of the 2.3 kb Pst I fragment, digestion with BamHI, Southern hybridization with sequence A , isolation of the 0.94 kb-Pst-I-BamHI subfragments, digestion with Sau 3a, Southern hybridization with sequence A, isolation of the 0.525 kb-BamHI-Sau 3a subfragments and sequencing of the DNA, and

a) lies immediately before the structural gene,

b) codes at the amino terminus for Met-Arg-Val-Arg-Ala-Leu-Arg,

c) codes at the carboxy terminus for Ala-Ser-Ala

d) codes in the middle for a hydrophobic region X, comprising 10

to 25 amino acids.

This DNA sequence is referred to in the following as sequence B or signal peptide.

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

Instead of the sequence A, a desirable sequence can be selected for the Southern hybridization, for the tendency stage or the antisense complementary sequence.

For the various steps to characterize DNA sequence B, in practice respectively DNA is brought into a suitable vector, this is transformed in a host cell, amplified where by colony hybridization with sequence A the transformants are determined and the DNA is reisolated. These steps are known per se.

DNA sequence C, whose coding strand is laid down in the appendage, shows the nucleotide sequence of the tendamistat structure from S. tendae.

The aforementioned gene structures thus contain the DNA sequence B in reading frame with a structural gene, which for example codes for tendamistat, and preferably the DNA sequence C.

DNA sequence B for the prepeptide is isolated from S. tendae. Plasmids which are characterized by DNA sequence B, in reading frame with a structural gene, which for example codes for tendamistat, especially for DNA sequence C, can contain a replicon active in E. coli and are then able to amplify and also to express DNA in E. coli.

Preferred plasmids additionally contain a replicon active in streptomycetes. If a streptomycete is transformed with such a plasmid, it becomes able to express the peptide determined by the structural gene in the form of a propeptide of formula II, which is then cleaved within the framework of the process with the help of a signal peptidase and secretes the desired peptide into the culture medium.

Also advantageous are so-called "Shuttle" plasmids, which contain a replicon active in E. coli as well as in Streptomycetes. These "Shuttle" vectors can be amplified in E. coli and, after re-isolation, transformed into streptomycetes, where the production of the island-secreted polypeptide then takes place.

While for gram-negative bacteria there are a number of vectors, for gram-positive bacteria only a few vectors have been described, vectors for bacteria of the type S. tendae are not yet known. The invention thus enables an approach for the utilization of S. tendae as a host organism.

A particular advantage of the invention lies in the fact that transformed Streptomyces strains, especially the S. lividans strain, form spores optimally, i.e. the content of the recombinant plasmid does not affect this strain in its generative phase. The transformed organisms are thus also suitable for further strain improvements, for example for the production and selection of metabolic mutants, in the production of which spores are used. _

Compared to the known strains of S. tendae, the transformed strains, especially S. lividans, produce no melamine. Separation thereof is therefore omitted and this facilitates the isolation of the desired peptide, for example tendamistat, and prevents loss of yield.

A further advantage of the invention lies in the fact that foreign genes are also expressed in S. lividans and the corresponding polypeptides are secreted, which likewise offers multiple opportunities for strain improvement and also modification of the polypeptide thus produced.

According to the invention, however, other streptomyces types can also be transformed, for example S. ghanaensis or aureofaciens. If plasmid-free strains capable of synthesizing a specific signal peptide are transformed with hybrid plasmids, stable transformants are obtained, which express and secrete the coded peptide.

Particularly preferred embodiments of the invention are explained in more detail in the following examples. The postal information here refers to weight, if nothing else is noted. The figures showing the hybrid plasmids show the cut sites to the correct scale.

In the examples, the following vectors known from the literature were used: Single-stranded phage M 13 mp 8 and M 13 mp 9: Messing et al., Gene 19 (1982) 269, pUC 8: Vierra et al., Gene 19

(1982) 259, pAC 177 and 184: Chang et al., J. Bacteriology 134

(1978) 1141, pIJ 102 and 350, Kieser et al., Mol. Gen. The gene. 185 (1982) 223.

Storage of S. tendae is discussed in US patent 4,226,764. The isolation of the tendamistat gene can in principle take place from any tendamistat-producing strain. However, it is particularly advantageous to proceed according to German application P 33 31 860.3, in whose example 3 the isolation of DNA is discussed. This isolated pooled DNA is the starting material for the following Example 1.

Example 1

5 µg DNA from S. tendae is completely digested with restriction enzyme Pst I and transferred after separation on a 0.8% agarose gel to a nitrocellulose filter (Southern-Transfer). The filter with the bound, denatured DNA is prehybridized for 6 hours in 5 ml prehybridization medium (0.6 M NaCl, 0.06 M Na_EDTA, 0.1% sodium dodecyl sulfate solution, 100 pg/ml ultrasound-treated calf thymus DNA and 4-fold concentrated Denhardt solution) . It is then treated again with 5 ml of the pre-hybridization medium, to which, however, 500,000 cpm/ml radioactively labeled DNA has been added. This radioactively labeled sample is obtained as a probe: the DNA sequence A is chemically synthesized according to the phosphite process. It contains 20 nucleotides (molecular weight about 13,000) and is complementary to the putative DNA sequence for tendamistat, derived from the amino acid sequence of tendamistat from amino acid 37 of tendamistat using the E. coli preferred triplet. This DNA sequence A is radioactively labeled at the 5' end by means of \/<32>P-ATP and nucleotide kinase.

For hybridization of this radioactive sample to the complementary DNA sequence in the total DNA, the mixture is allowed to stand for 24 hours at 37°C. The unbound radioactive DNA is then removed and the filter is washed at 37° C 5 times for 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 located on a 2.3 kb Pst I fragment. This fragment is recovered from a preparative agarose gel, by electroelution of a section corresponding to this fragment size, on which the PstI-digested total DNA was divided. The eluted DNA is cloned into the Pst I cut site of the plasmid pUC 8.

These hybrid plasmids are transformed and amplified in E. coli JM 103. Detection of the clones that have the sought-after insert with the tendamistat gene is carried out by colony hybridization using the radioactive DNA sample A. The thus formed hybrid plasmids pKAI I a resp. 1 b is shown in fig. 1 a and 1 b.

By further Southern hybridizations against the isolated 2.3 kb Pst I fragment and its subfragments, the exact location of the gene can be determined (Fig. 2a to 2c).

Example 2

In the individual cut sites of plasmid pIJ 102 for Pst I, a 2.3 kb Pst I fragment from S. tendae is cloned. The thus formed hybrid plasmids pAX 1 a and 1 b, which differ in the orientation of the insert, enable the S. lividans strain to produce tendamistat after transformation.

Fig. 3 shows the plasmid pAX 1 a.

Example 3

The commercial strain S. lividans TK 24 (John Innes Institute, Norwich, England) is transferred in a known manner in protoplasts and 1 jjg hybrid plasmid pAX 1 a is mixed with 10^ protoplasts while adding 2056 polyethylene glycol 6000. The transformed protoplasts are grown on regeneration media R2YE ( Thompson et al., Nature 286 (1980) 525) at 30°C for 5 days.

Detection of the extracellular amylase inactivator was formed can be done with the help of a plate test.

The regenerated colonies are poured over with 5 ml of a 0.4-1.0 mg/ml pancreatin-containing aqueous solution and incubated for 1 hour at 37°C. The solution is then removed and replaced with 5 ml of a 2% starch agar. After 2 hours of incubation at 37°C, the plates are poured over with 5 ml of an iodine-potassium iodide solution for development. Colonies with a blue accumulation indicate that the clone synthesizes and extracts tendamistat.

As a control, plasmid DNA can be isolated from tendamistat-producing well-sporulating strains and mapped. All tensdamistat-producing strains have the plasmid DNA used.

Example 4

One proceeds according to example 2, but uses the plasmid pIJ 350, which Streptomyceter has as a selection marker a thiostrepton resistance gene. Hybrid plasmid pAX 350 a and b are thus obtained (which differ in the orientation of the insertion). Fig. 4 shows the plasmid PAX 350 a.

After transformation according to example 3, clones are selected on minimal medium (Hopwood, Bacteriological Reviews 31 (1967) 373-403 in the presence of 50 µg/ml thiostrepton resistant clones. These are examined either directly on minimal medium or after plating on non-selective R2YE agar for tendamistat production .

Example 5

Hybrid plasmids containing a 2.3 kb Pst I fragment and, in addition to the Streptomycet replicon also an E. coli replicon, have a number of advantages as "Shuttle" vectors: Due to the E. coli replicon and in E. coli active resistance markers, they can well amplified in these organisms. After isolation and transformation in Streptomyceters, especially S. lividans, they show a high stability. As a result of their selection markers active in Streptomyceter and of the Streptomyceter replicon, they can also be well amplified and expressed in these organisms and secrete tendamistat.

The plasmid pAC 184 is completely digested with the restriction enzyme Sal I and the enzyme is removed by phenol-chloroform extraction. The overhanging 5'-ends are fulfilled in the presence of ATP, CTP, GTP and TTP by means of the enzyme DNA polymerase (Klenow fragment). On the blunt ends (blunt ends) a Pst I linker of the structure is ligated in a ligase reaction at 16°C

(2 µg linker on 0.4 µg DNA). DNA is extracted with phenol chloroform and, after precipitation, is digested with the enzyme Pst I to extract ligable Pst I ends. The DNA is then dephosphorylated, extracted again with phenol-chloroform and ligated with partially Pst I-digested plasmid pAX 1 a. The ligation mixture is transformed into E. coli (HB 101 resp. MC 1061). Clones resistant to chloramphenicol are removed from the plate and DNA is isolated. S. lividans TK 24 is transformed with 1-2 pg DNA and examined for tendamistat production.

In the tendamistat sample, positively reacting clones are isolated, plasmid DNA is isolated by alkaline Schnell lysis and transformed into E. coli HB 101 resp. MC 1061. Plasmids reisolated after amplification do not differ from those isolated from S. lividans strains. Depending on the orientation of the insertion, which has the tendamistat gene, the recombinant plasmids are designated as pSA 2 a or b (Fig. 5 a and b).

Example 6

If you proceed according to example 5, however, starting from plasmid pAX 350 a, selected in E. coli for chloramphenicol resistance and in S. lividans for thiostrepton resistance and tendamistat production, you get the plasmids pSA 351 a resp. b (fig. 6 a/b).

Example 7

If you proceed according to example 5, however, instead of the plasmid pAC 184 from pAC 177, you get the plasmids pSA 3 resp. b (Fig. 7 a/b).

For this, the plasmid pAX 1a is partially cut with PST and the enzyme is heat-inactivated by heating for 15 minutes at 68°C. DNA is ligated into the Pst I-cut dephosphorylated and deproteinized plasmid pAC 177. After transformation of E. coli HB 101 resp. 1061 are taken against kanamycin-resistant clones from the plate, plasmid DNA is isolated and S. lividans TK 24 is transformed with 1-2 µg of this DNA. Tendamistat-producing clones are selected and charalterize the plasmid.

Example 8

If you proceed according to example 7, however, instead of the plasmid pAX 1, you use the plasmid pAX 350 a and select S. lividans for thiostrepton resistance, then you get the plasmids pSA 352 a resp. b (fig. 8 a/b).

Example 9

As can be seen from fig. 2, it is for tendamistat and signal sequences coding gene ~ 0.3 kb large. The 2.3 kb fragment used in the above-mentioned examples can thus be used in abbreviated form for the construction of hybrid plasmids which effect the production of tendamistat: The plasmid pKAI 1 a is digested with Sal I and religated. You thus get around approx. 750 base pair shortened plasmid pKAI 2. This is cloned, isolated and cut with Pst I. DNA is dephosphorylated with alkaline phosphatase from calf intestine and deproteinized with phenol-chloroform.

The plasmid pIJ 102 is completely cut with Pst I and the fragments are ligated after heat inactivation of the enzyme in the Pst I cut sites of pKAI 2. The ligation mixture is transformed into E. coli HP 101 or MC 10611. Plasmid DNA from ampicillin-resistant clones is isolated by alkaline Schnell-Lyse method and S. lividans TK 24 is transformed with 1-2 pg of this DNA. One selects for tendamistat production and isolates plasmid DNA from this clone by alkaline Schnell-Lyse. By back transformation in E. coli HB 101 resp. MC 1061 and after isolation of plasmid DNA from transformed E. coli strains, plasmid pSA 1 is characterized by restriction analysis (Fig. 9). The plasmid shows no difference to the plasmids isolated from S. 1 ividans strains, however, the DNA processing from E. coli is more productive and possible in a shorter time.

Example 10

If you proceed according to example 9, however, instead of the plasmid pIJ 102, the plasmid pIJ 350 is used, since it can be selected in S. lividans then additionally for thiostrepton resistance, then you get the plasmids pSA 350 a resp. b. Fig. 10 shows the plasmid pSA 350 a. This plasmid leads in shaking cultures to higher yields of tendamistat than the plasmid pSA 350 b, which contains the insertion with the tendamistat gene in reverse orientation. In contrast, reducing the insertion to 1.5 kb has no clear effect on product formation.

Example 11

To determine the structure and nucleotide sequence of the tendamistat gene, the 0.94 kb Pst I-Bam HI subfragment (Fig. 1 a and b) as well as the 295 bp Sau 3a-Bam HI subfragment (Fig. 2 c) were cloned into the single-stranded phages M 13 mp 8 and M 13 mp. 9. As a primer for the dideoxy sequence reaction, they served 20 nucleotides comprising DNA sequence A as well as a commercially available 15 bp primer (Bethesda Research Laboratories GmbH., Neu-Isenburg). The DNA sequence was determined to be C.

Example 12

Before the structural gene of tendamistat there is an open reading frame on the DNA until the start codon ATG (Met) for a protein, which stores the amino-terminal end of tendamistat. This signal peptide corresponds to DNA sequence B.

Addendum:

DNA sequence C (coding strand)

Examples 1 and 3 from German application P 33 31 860.3:

Example 1

Mutation procedure: Approx. 0.25 cm<2> sporulated mycelium of the strain DSM 2727, which is grown on oat bran agar (EP-A 49 847), is inoculated into a 100 ml Erlenmeyer flask, which is filled with 25 ml of the following culture medium: the medium is autoclaved for 30 minutes at 121 °C and 1 bar, the pH value is 7.0. The inoculation flasks are incubated for 2 days at 30° C on a shaking machine. Then 2 ml of the washed pre-culture is inoculated into 20 ml of the main culture medium, which is in a 100 ml Erlenmeyer flask (EP-A 49 847):

After autoclaving, the medium's pH value is 7.6.

Acriflavine is added to this medium in a concentration of 10-25 pg/ml as a mutagenic substance. The culture is shaken for 5 days at 28°C and 220 rpm. The culture solution is then centrifuged (1,300 g, 5 minutes) and the cell pellet is washed in the main culture medium. The washed cells are fragmented in a glass homogenizer and plated on agar plates in the following medium:

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

The plates are grown for 5 days at 28°C and single colonies are inoculated into slanted tubes with oat bran agar. These are cultivated as described until the mycelium is pre-spurred. The pre-tracked mycelium is propagated as mentioned above in the pre- and main culture and the culture filtrate is examined after 5 days of main culture for the content of tendamistat (compare EP-A 39 847). In this way, strains 1-9353, 1-9362, 1-9417, 1-9418 can be selected during their growth period under the mentioned conditions producing approx. 90,000-100,000 IU tendamistat/1 culture solution, corresponding to 1.5-1.8 g inactivator/1.

Example 3

Isolation of deoxyribonucleotide acid from S. tendae and its molecular biological characterization.

Cultivation of the cells took place similarly to example 1. After 3 days of growth in the main culture medium, the cells are shaken from an Erlenmeyer flask by centrifugation for 5 minutes at 3000 g at 4°C and washed twice with 50 ml of a solution of 50 mM Tris/HCl buffer , 100 mM NaCl and 25 mM EDTA.

In a typical preparation, 3 g of fixed-centrifuged cells are shock-frozen in liquid nitrogen at -198°C and then homogenized with a cutting knife homogenizer (Warring Commercial Blender) in the presence of N2 to a fine powder. This powder is taken up in 10 ml of said buffer, dissolved on ice and incubated with 0.8 ml of lysozyme solution (30 mg/ml) for 20 minutes at 28°C with gentle shaking. 0.8 ml of a proteinase K solution (15 mg/ml) and 0.8 ml of a 20 µg solution of the sodium salt of N-lauroylsarcocine are then added to the suspension. After careful mixing, the mixture is incubated for 30 minutes on ice and a further 15 minutes at room temperature. After the addition and dissolution of 22 g of solid cesium chloride, the volume with the above-mentioned buffer is adjusted to 22 ml and the suspension is centrifuged at 12,000 g at 4°C for 25 minutes. The clear centrifuge is mixed with ethidium bromide and the refractive index is adjusted with CsCl refractometrically to n = 1.3920. After preparative ultracentrifugation (120,000 g, 36 hours, 15 °C), the bands of chromosomal DNA are removed from the centrifuge tubes and recentrifuged under the same conditions. The isolated bands are freed from ethidium bromide by shaking with n-butanol and dialyzed to completely remove CsCl. The DNA thus recovered is cleavable, i.e. with the enzymes Pst I, Barn H I, Sau 3 A, Cia I, Bgl II and Xho I, and not cleavable, for example with the endonucleoses Eco R I and Hind

III.

The DNA produced from the mutants 1-9353 and 1-9362, for example, is compared to DNA from ATCC 31210 resp. DSM 2727 characterized bed an amplified genetic element, the individual fragments of which appear clearly visible after staining with the fluoro-essence dye ethidium bromide from a background of non-selectively amplified fragments. The size of the amplified element as well as the characteristic individual fragments after digestion with restriction endonucleases are reproduced in the following:

Claims (9)

1. Process for the production of polypeptides from genetically coded amino acids, characterized by expressing in a host cell from the species Streptomyces a gene structure which, in the reading frame with the structural gene for the desired polypeptide, contains the DNA sequence B of a prepeptide, obtainable from tendamistat-producing Streptomyces tendae -strain, which has been preferentially treated with sublethal doses of acriflavine, through isolation of total DNA, digestion with Pst I, Southern hybridization with DNA sequence A isolation of the 2.3 kb Pst I fragment, digestion with BamHI, Southern hybridization with sequence A, isolation of the 0.94 kb Pst I BamHI subfragments, digestion with Sau 3a, Southern hybridization with sequence A, isolation of the 0.525 kb-BamHI-Sau 3a subfragments and sequencing of the DNA, and a) lies immediately before the structural gene, b) codes at the amino terminus for Met-Arg-Val-Arg-Ala-Leu-Arg, c) codes at the carboxy- terminus for Ala-Ser-Ala d) encodes in the middle a hydrophobic region X, comprising 10 to 25 amino acids. 2. Method according to claim 1, characterized in that DNA sequence B of the prepeptide in the middle codes for a hydrophobic region X comprising 17 to 20 amino acids. 3. Method according to claim 1 or 2, characterized in that DNA sequence B for the prepeptide is isolated from S. tendae. 4. Method according to claims 1 to 3, characterized in that the host cell is of the species S.tendae or S.lividans. 5. Method according to claims 1 to 4, characterized in that the host cell contains a signal peptidase which cleaves the prepeptide and which secretes the desired polypeptide into the culture medium. 6. Method according to one or more of the above-mentioned claims, characterized in that the gene structure is brought into the host cell with a plasmid containing a replicon that is active in streptomycetes. 7. Method according to claim 6, characterized in that the plasmid, apart from the replicon which is active in streptomycetes, contains a replicon which is active in E.coli. 8. Method according to one or more of the above claims, characterized in that the structural gene codes for tendamistat. 9. Method according to claim 8, characterized in that the structural gene exhibits DNA sequence C (appendage).