DD206562A1 - PROCESS FOR THE PREPARATION OF PGB VECTOR PLASMIDES FROM STREPTOKOKKEN - Google Patents

Abstract

The invention relates to a process for preparing PGB-PLASMIDES from streptococci, in particular those which can be used to advantage in the production of recombinant DNA molecules. AFTER INTRODUCTION OF SUCH MANUFACTURED MANIPULATED DNA MOLECULES IN APPROPRIATE HOMES, HIGH-QUALITY PRODUCTS FOR USE IN HUMAN MEDICINE, COMMERCIAL PRODUCTION, AGGREGATE PRODUCTION, FOOD AND AGRICULTURAL INDUSTRY ARE EFFECTIVELY PRODUCED BY TECHNICAL-MICROBIOLOGICAL WAYS. OBJECTIVE OF THE INVENTION IS THE PREPARATION OF PGB PLASMIDES WHICH CAN BE USED AS CLONING VECTORS FOR GENETIC ENGINEERING WITH GRAMPOSITIVE MICROORGANISMS. THE TASK IS BASED THAT, AFTER TRANSFORMATION OF STRAINS FROM SPECIFIC STREPTOKOKEN SPECIES WITH A NATURALLY OBTAINING ANTIBIOTIC RESISTANCE PLASMIDE, A REDUCED PLASMID PGB 301 IS ISOLATED AND THEREFORE A MODIFICATION OF A SERIES OF ABECOMMING OF THIS PLASMIDS PGB 301 IS OBTAINED.

Description

235197 .2

Title of the invention

Method of producing pGB vector plasmids from streptococci

Field of application of the invention

The invention relates to a terpene for the preparation and isolation of pGB vector plasmids, in particular those which can be used advantageously for the production of recombinant DNA molecules, which may contain the genetic information for extracellular microbial products Manipulated DNA molecules into suitable hosts can be used to produce high-quality products for use in human medicine, livestock production, active ingredient production, the food industry and the fermentation industry in a technically and microbiologically effective manner. "

Characteristic of the known technical solutions

Process for the preparation of plasmid and bacteriophage for recombinant DNA technology for genetic manipulation of microorganisms since the early 70s known (S. Cohen, A _♦> Chang, Η »Boyer, R» Helling, Construction of biologically functional plasmids in vitro, Proc. Natl. Acad., 7, 3240 (1973)) · Such preparation methods initially concerned only the standard objects of microbial genetics, the Gram-negative E. coli bacterium, and its bacteriophages. As a result, also from Gram-positive microorganisms, especially those of the genera

27. ^ 1981 * 974165

K 1 O7

Bacillus, Staphylococcus and Streptomyoes, isolates naturally occurring plasmids that are used by themselves or after conversion as vector plasmids for these bacterial genera (K, Keggins, P. Lovett, E. Duvall , Molecular donation of genetically active fragments of Bacillus subtilis DNA in Bacillus subtillis and properties of the vector plasmid pUB110 Proo. Natl. AOAD, Be "75> 1423 (1978), M _r Bibb, Ly _ ^ hotte_l, S, Cohen» A DNA oloning system for interspecifio gene transfer in antibiotic-producing Streptorayces, Nature £ 84,526 (1980) *

In streptococci, the first plasmid was detected in 1972, (P *., .. Cpurvalin, C. Parlier, Y, Chabbert, Plasmidlinked tetracycline and erythromycin resistance in group D Streptococci, Ann Inst Past 123 (1972), 755). · Further work showed that a whole range of streptococcal species

Plasmids harbored "(D, Olewell, Y, Yagi, G. D unnyy £.

Schultz "Characterization of 3 plasmid DNA molecules in a, * *, J, Baoteriol. 117 (1974) 283-289; T. Horodnioeanu u. a , H-plasmids in Streptococcus agalaotiae (group B), Antimicrob. Ag. Chemother, 10 (1976), 795-801; ff, Macrina.,. C. Scott , Evidence for a disseminated plasmid in Streptococcus mutants, Infect. Immun. 20 (1978), 296-302; D. Behnke , H. Malke , M. Hartmann "Pe ₁₁ Walter, post-transformationally rearrangement of an in vitro reconstructed group A streptococcal erythromycin resistance plasmid, Plasmid 2 (1979)? 605-616), some of these plasmids have relatively favorable natural conditions for their use for the production of vector plasmids ·

Vector plasmids that are to be used for recombinant DNA work must have the following properties:

w the ability to autonomously replicate,

the presence of at least one selective marker (eg antibiotic resistance),

- ³ "235197 2

The presence of at least one unique restriction endonuclease intersection, this intersection must be outside essential plasmid functions. "

In addition to these requirements, effective Fektorplasmide have other properties that either facilitate the cloning or increase the yield of the desired product. Such properties are:

the presence of a second selective marker which can be inactivated by inserting a Premd DNA into an existing unique restriction enzyme site (insertion inactivation) and thus facilitates the selection of recombinant plasmids ",

-A small molecular weight of the vector to clone as large pieces of foreign DNA,

<- a high copy number of the vector in the host cell, this is achievable by manipulation of the appropriate control functions of the plasmid or by variation of the external conditions »

None of the known naturally occurring streptococcal piasmids meets the minimum requirements for a cloning vector. • Only small cryptic plasmids (pVA 318, pVA 380-1) found in Streptococcus mutans and Streptococcus ferus strains ( P. Maorina C, Scott Maorina , K. Jones , P.W. ccd , Chimeric streptococcal plasmids and their use as molecular cloning vehioles in Streptococcus sanguis (Challis), J, Baoteriol, 143 (1980), (1978), supra. 1425-1435) can be used to clone selectable properties, in the present case of antibiotic resistances. " (D. Behnke, J. Ferretti , Physical mapping of pDB101a potential vector plasmid for molecular cloning in streptococci, Plasmid 4 (1980). 130-138; ?. Maorina , K. Jones, Pv Wood (1980) a.

These recombinant plasmids (pDB 201, p VA 680, pVA 637) and other streptococcal plasmids prepared by in vitro V / (pDB 101, pDB 103, pSM 10) (D., Behnke, H. Malke, M. Hartmann Walter, (1979) a "0, D. Behnke, J. Fferetti, Molecular cloning of an erythromycin determinant in streptococci, J. Baeteriol 144 (1980), 806-813, H. Malke, L 'Burman , S, Holm , Molecular cloning in streptococci, physical mapping of the vehicle plasmid pSM 10 and demonstration of intergroup DNA transfer, MoI. Gen. Genet 181 (1981), 259-267) fulfill the minimum requirements for vectors, However, they are characterized by the following disadvantages

- lack of a second selective marker

not usable for cloning via insertion inactivation

- Too large molecular weight and in part presence of redundant and inverted DNS> ~ Seqtienzen

- in part to low number unikaler intersections for Re striktionsenzyme

- no copy number mutations ·

Recently described ways of reducing some of these disadvantages are the opening of two complete üasmide with different antibiotic resistances (S1asmidChimäre pSM 10221) (H.JUalke, l. .. ,, Barman, S. Holm (1981) aa 0.) .. or in the elution of further selectable markers into existing vector plasmids * (ff, _T Maerina, K. Jones, P. Wood (1980) a. a, 0 »)

Object of the invention

The invention serves for the production of pGB vector plasmids which, owing to their properties, can advantageously be used for the production of recombinant DNA molecules for introduction into suitable hosts. The invention is intended to overcome the disadvantages of the previously known processes for

235197 2

Production and Use of Vector Plasmids from Streptococci Must be Avoided «

Explanation of the essence of the invention

The invention has for its object to provide an effective method that allows to obtain from streptococci such vector plasmids, which can be used advantageously for the production of recombinant DNA molecules ^

According to the invention the object is achieved in that with the in a known manner from the Streptococcus faecalis strain JH 2-2 (pIP 501) isolated plasmid pIP 501 a recipient strain of one of the species Streptococcus sanguis (challis) Streptococcus milleri or Streptococcus pneumoniae transformed and screening for a transformant line containing a downsized plasmid (pGB 301, for example, in strain Challis 57 (pGB 301)). The scaled-down, new plasmid pG-B 301, which further carries the chloramphenicol and macrolide resistance of the starting plasmid pIP 501, is isolated and, after being characterized for cleavage sites of a series of restriction endonucleases, modified either by re-transformation with cleaved and religated ones Plasmid DNA and subsequent screening for further reduced descendants of pGB 301 or by removal of DNA sequences in vitro (disassembly of the plasmid pGB 301 by means of restriction enzymes into fragments and circularization thereof by means of DNA ligase) and subsequent transformation of the recipient strain, for example Streptococcus sanguis Challis 57 * After these process steps, new plasmids are further reduced in different transformant lines, which either have lost one of the two antibiotic resistances (pGB 302, pGB 303, pGB 304, pGB 305, pGB 306, pGB 307, pGB 308, pGB 309, pGB 310, pGB 321, pG B 351, pGB 352, pGB 353, pGB 354).

- 235197 2

Loss of chloramphenicol resistance: pGB 351, pGB 352, pGB 353, pGB 354 - loss of macrolide resistance) or in addition carry a targeted achievable genetic modification in the copy control mechanism (pGB 354), which leads to an increase in plasmid copy number to about 50 plasmid molecules per cell. At the same time, removal of redundant DNA sequences (along with the above-noted antibiotic resistance gene losses) results in the elimination of restriction sites so that unique restriction sites become available for other restriction enzymes that are useful for the production of recombinant DNA molecules.

The isolation of the starting plasmid pIP 501 takes place in such a way that a sufficient amount of the streptococcus strain JH 2-2 (pIP 501) is digested by lysozyme / Prognase treatment and lysed by the addition of the detergent sodium dodecylsulfate (SDS) oomosomal DNA selectively precipitated after addition of NaCl in the cold. After removal of the precipitate by low-speed centrifugation, the plasmid pin? 501 separated from the supernatant in a conventional manner by dye density gradient centrifugation ·

The novel method is carried out as follows: The purified plasmid pIP 501 thus obtained is used in the early stages of suitable excipient strains of the species Stu sanguis or Str. Milleri var, anginosus or Str pneumoniae, preferably Challis 57, Challis 6, MS 238 or DL 8 After reaching the late logarithmic growth phase, these strains are subjected to a selection pressure by wiping on solid antibiotic-containing nutrient media in such a way that only cells can survive which have taken up and functionally established an intact plasmid.

Ca "% # ^ f I <* J i £ a

In the following, a number of transformant clones are isolated and are each propagated in a larger volume of liquid nutrient medium until sufficient cell material is available for a rapid assay for the presence and size of the plasmids hosted. In this rapid test, overnight cultures of the transformant clones are centrifuged and the sedimented cells are resuspended in a small volume of a buffer solution containing lysozyme. After a sufficiently long incubation of the cells with the lysozyme, neutralized phenol is added so that the cells lyse and the high molecular chromosomal DNA precipitates, which can be separated during the subsequent centrifugation. "The supernatant containing the plasmid DNA is subjected to further phenol and chloroform isoamyl alcohol extractions and finally the plasmid DNA is precipitated by means of alcohol precipitation in the cold." The precipitated DNA is then incubated in a suitable buffer, preferably 10 mM Tris-HCl, pH 7.4, and used for analytical determination of the molecular weight for electrophoretic separation in agarose gels according to a known methodology.

After selecting a Transformantenklons, which contains a suitable small plasmid, the new plasmid pGB 301, is from the same a greater amount of cell mass by culturing in a I ¹ Iussignährmedium, the P, L-threonine is added to made and in the following manner worked up to obtain the plasmid DNA. Washed cells are subjected to liysozyme / pronase treatment and then gently lysed with a detergent. High-speed centrifugation in the cold selectively removes most of the high-molecular-weight chromosomal DNA. The supernatant is purified by phenol-chloroform and chloroform-isoamyl alcohol shaking. After precipitation of the DNA with pure alcohol, the DNA re-absorbed in a buffer is analyzed in the dye-density

separated, after which the plasmid pGB 301 can be obtained in purified form ·

The characterization of the new plasmid with regard to the molecular weight and the location of cleavage sites for restriction endonucleases is carried out according to the known standard procedures. The new plasmid pGB 301 (see FIG. 1 and 2) has a molecular length of 9.8 kb and lies in a copy number of about 10 copies per cell. It has arisen as a result of a single deletion event. "The region in which the deletion endpoints were fused lies in the region of the Hind II fragment C (s _e Abb" ΐ) _β The Slasmid encodes resistance to CMoramphenikol- and macrolide antibiotics as well as against Lincosamine "Unusual cleavage sites were detected for the following restriction endonucleases * Ava I, I, BspR I, BstE II, Hpa. I, Hpa II, Epn I, and Pvu II. The restriction endonucleases Ava II, Hind II, Hind III, and Hae II break pGB 301 into 2, 3, 4, or 4 fragments (Figure 1). The location of the biological punctures (Resistence, replication, copy control) coding sequences is known by the analysis of the descendants of pGB 301 described below (see Fig. 2) "Of the unique cleavage sites is the BstE II site in Ghloramphenikolresistenzgen · insertion of foreign DNA in these Cleavage site leads to inactivation of the resistance property. "According to current knowledge, the Hpa I and Epn I sites as well as the Pvu II site are located in the copy control region or in a puncture essential for replication (Pvu II). All other unique restriction sites lie in functionally nonessential recesses of the pGB 301 molecule.

The process is continued by cleaving purified pGB 301 'DKS with the restriction enzymes BspR I or BstE II and subsequently reassembled with or without the addition of appropriately cut foreign DNA by the enzyme DNA ligase of the bacteriophage Te.

5 197 2

After the subsequent transformation, such transformant clones are isolated which no longer express chloramphenicol resistance. Using the described screening method, these transformant clones are examined for the plasmid content and the size of the plasmid housed. The plasmids pG-B 302, pGB 303, pGB 304, pGB 305, P.GB 306, pGB 307, pGB 308, pGB 309, pGB 310, pGB 321 obtained in this manner are up to 4.1 kb smaller than pGB 301 and have all the chloramphenicol resistance gene and sometimes also redundant DNA sequences were lost (see Fig. 3).

Modified pGB 301 plasmids that have macrolide resistance are prepared by transforming the recipient strain, for example, Streptococcus sanguis Ohallis 57, either in the presence of the dye ethidium bromide with intact pGB 301 DNA or in vitro modified pGB 301 DNS is used for normal transformation. The plasmid pGB 301 is converted in vitro in such a way that it is first completely cleaved with the restriction enzyme BspR I and then treated briefly with the enzyme Hind II, so that incomplete digestion products are formed. The smooth interfaces caused by both enzymes are then mixed with the enzyme DNA lyase reassembled. Prior to transformation, all reconstituted pGB 301 plasmids are inactivated by re-digestion with BspR I and addition of the Hpa II enzyme for transformation.

Among the transformant clones, those which are nooh resistant to chloramphenicol but no longer resistant to macrolide antibiotics (erythromycin) are selected, and in the described screening procedure the plasmid content and size of the plasmids in these transformant clones are subsequently examined »

The plasmids pGB 351, pGB 352, pGB 353 and pGB 354 thus prepared are all sensitive to macrolide antibiotics and to 1.7; 2.75; 2.15 or 3.6 kb smaller than pGB 301 (see Fig. 3).

° - 235197 2

The exact location of the DNA sequences missing in comparison to pGB 301 can be seen from FIG. 3. In the modification set in the plasmid pGB 354, the number of restriction sites is reduced so much that the enzymes Hind II, Hind III and Ava II are only have unique cleavage sites (see Fig. 3), which are now additionally useful for cloning experiments · The plasmid pGB 354 also has such a modified copy number control mechanism that this plasmid in increased copy number / cell compared to the other pGB plasmids (50 copies compared to 10) · The plasmid pGB 351 has a unique Ava II cleavage site.

From these data, which characterize the plasmids of the pGB series above and in the figures, it can be seen that these plasmids, especially pGB 301 and pGB 354, possess all the essential and additionally desirable properties that cloning vectors should possess. Thus, it is to be expected that these plasmids can be used effectively for producing such recombinant DNA molecules which, after conversion into suitable hosts, in particular gram-positive microorganisms, enable them to produce biologically syntheses of industrially and medically important products. Thus, the .gamma.GB plasmid family is arrested alternative vector system for cloning work in Gram positive hosts.

The biologically pure cultures of the strains used in these procedures were under the registration numbers

ZIMET 10 722 for strain Str. Faecalis IH 2-2 (pIP 501),

ZIMET 10 718 for tribe st. Sanguis Challis 57,

ZIMET 10 717 for trunk Str · sanguis Challis 6,

ZIMET 10 719 ¹ for strain Str · milleri var · anginosus MS 238,

ZIMET 10 720 for strain Str · milleri var, anginosus DL-8,

- Hi

- ^ 35197 2

ZIMET 10 721 for the plasmid-carrying strain

St. sanguis Ohallis 57 (pGB 301)

in the ZIMET Depository for Microorganisms, Central Institute for Microbiology and Experimental Therapy of the AdW of the GDR, 6900 Jena, Beutenbergstr. 11, deposited.

AuaführungsbeispieIe

For the transformation with plasmid DNA the strain Streptococcus sanguis Ohallis 57 (strain ZIMBT 10 718) is prepared as follows: · An overnight culture of Challis 57 in Brain Heart Infusion Broth (BHI / Difco) + 2.5 % heat-inactivated horse serum (BHI-HS ) is added to 25% with sterile glycerol, divided into small amounts (0.3 to 0.5 ml), frozen at -70 ⁰ O and stored at -20 ⁰ C. These stock cultures serve as starting material for the production of competent challis. The compartment is at least twice overnight in BHI-HS, the cells are sedimented, washed once with fresh BHI-HS and then 1: 200 in transformation medium (according to Le Blanc DJ and Hassell PP, Transformation of Streptococcus sanguis Challis by plasmid DNA from Streptococcus faecalis J. Bacteriol., Λ2 & (1976) 34-7-355) diluted · After 105 min incubation at 37 ⁰ O, the cells have reached the maximum of competence. After addition of pIP501 plasmid DNB (5-20 / μl pIP501 DNA of a concentration of about 1 / ug DTTB per 20 / μl) to 0.5 ml of Challis 57 culture, this is incubated for a further 4 hours at 37 ⁰ C. The culture is then in 10 increments diluted · 0.1 ml of each dilution is added to 3 in tempered soft agar (50 ⁰ C, BHI + 0.7% agar) and (on BHI Agar base layers BHI + 1.5% agar ) containing 10 / μg / ml of erythromycin for selection. The mixture is then incubated at 37 ^° C. for 48 hours. Transformant clones are isolated and assayed for chloramphenicol resistance by streaking on BHI agar plates with 5 / ug / l ghloramphenicol. Double-

21.JUIi1982 * ül7359

'(Ery Canr) -reslatente clones are subjected to a rapid test for the presence and size of plasmid DNA • For this purpose, these EL are one overnight in 20 ml of BHI-HS + 10 mM DL-threonine at 37 ° ® incubated · After sedimentation and washing the cells with 10 mM Tris-HCl (pH 7.4), they are taken up in 0.5 ml of 10 mM Tris-HCl (pH 7.4) V + 4 mg / ml Eysozyme and incubated for about 90 to 120 min at 37 ⁰ C 'incubated. Then 0.1 ml of an activated (30 min at 37 ⁰ C) DNase-free pronase solution (5 mg / ml in 10 mM Tris-HCl, pH 7.4) was added and incubated for a further 30 min at 37 ⁰ C. Addition of 0.6 ml of neutralized phenols (pH of the phenol adjusted to pH 6.8 by shaking with 100 mM Tris-HCl (pH 8.0)) causes lysis of the cells and precipitation of the chromosomal DNA together with the denatured protein. After a short (5 min) high centrifugation (Eppendorf centrifuge, full speed), the aqueous upper phase can be recovered, which is then extracted once more with 0.4 ml of phenol and 1.5 ml of chloroform-isoamyl alcohol (mixed in the ratio 24J1) becomes. For the aqueous phase obtained after the last extraction, the volume is determined and then the plasmid DNA contained therein is precipitated by the addition of 2 volumes of cold 96% ethanol. For quantitative precipitation samples are ca · 5 to 10 minutes kept at -70 ⁰ C and then centrifuged for 5 to 10 minutes at full speed in an Eppendorf centrifuge. The sedimented DNA is dried in vacuo and then dissolved in 50 μl of 10 mM Tris-HCl (pH 7.4).

The resulting plasmid preparations are used for analytical agarose gel electrophoresis. For this purpose, 20 μl of the preparations are mixed with 4 μl of a dye-glycerol solution and loaded onto a 0-, 8% agarose gel. The electrophoresis is carried out in a manner known per se (Helling, Goodman, Boyer, J. Virology 22 (1974) 764-775). After staining the gels with an ethidium bromide solution (1 μg / ml)

21.:JÜM9.8Z*Ül.7-.350

- 2 3b 1 y 7 l

For example, the plasmid bands can be identified by fluorescence in short-wave UV light. Such clones containing plasmids, which are actually smaller than the starting plasmid pIF 501, are selected and further characterized by restriction enzyme analysis in a manner known per se, thus obtaining the plasmid pGB 301.

2 For the transformation with plasmid DNA the strain Streptococcus sanguis Challis 6 (strain ZIMET 10 717) is prepared; otherwise the procedure is as in Example 1,

3. For transformation with plasmid DITS, prepare the strain Streptococcus milleri var. Anginosus MS 238 (strain ZIMET 10 719); otherwise the procedure is as in Example 1,

4 ·· For transformation with plasmid MS, prepare the strain Streptococcus milleri var _e anginosus DL-8 (strain ZIMET 10 720); Otherwise, the procedure is as in Example 1.

Claims (3)

- ι jb ι y / invention claim 1. A process for the preparation of pGB vector plasmids from streptococci after isolation of the plasmid ρΓΡ 501 from the strain Streptococcus faecalLis JH 2-2 (plF 501) (strain ZIMET 10 722), characterized in that the plasmid pIP 501 a recipient strain of a the species Streptococcus sanguis (Ohallis), Streptococcus milleri or Streptococcus pneumoniae is transformed, screening for a transformant clone containing a reduced plasmid pGB 301, the plasmid pGB 301, which further contains the chloramphenicol and the macrolide resistance of the starting plasmid pIP 501 pJfcasmid pGB 301 du ch modifications according to a known methodology, preferably by removing redundant DNA sequences and resistance genes and / or restriction sites and / or restriction sites and / or or by introducing a directed g Enetic change of the copy control mechanisms, into the PlasmidabkBmmlinge pGB 302, pGB 303, pGB 304, pGB 305, pGB 306, pGB 307, pGB 308, pGB 309, pGB 310, pGB 321 and pGB 351, pGB 352, pGB 353, pGB 354 is converted · 2. The method according to item 1, characterized in that as a recipient of either the strain Streptococcus sanguis Challis 57 (strain ZBffET 10 718) or the strain Streptococcus sanguis Challis 6 (strain ZBSEO? 10 717) is used. 3. The method according to item 1, characterized in that as a recipient strain either the strain Streptococcus milleri var. Anginosus MS 238 (strain ZIMET 10 719) or the strain Streptococcus milleri var. Anginosus DL-8 (strain ZIMET 10 720) is selected For this & pages drawings Λ JUN, 1982 * 017359