NZ204501A - Isolation of plasmids from methylomonas clara;recombinant dna method,with obligate methylotrophic bacteria as recipients;hybrid plasmids - Google Patents

Description

2 04 Priority Datejs): Complete Specification Filed: .f ciass: c^/r^/ac.... • JUN1983 * * Publication Date; £ 5 J?!rP.

P.O. Journal, fJo: ..•••••••• • N.Z.No.

NEW ZEALAND Patents Act, 1953 COMPLETE SPECIFICATION "PROCESSES FOR THE PREPARATION OF OBLIGATE METHYL-OTROPHIC BACTERIA WHICH EXPRESS FOREIGN DNA, AND PLASMIDS AND HOST ORGANISMS SUITABLE FOR THESE." We, HOECHST AKTIENGESELLSCHAFT, a corporation organized under the laws of the Federal Republic of Germany, of D-6230 Frankfurt/ Main 80. Federal Republic of Germany, do hereby declare the invention, for which we pray that a Patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - d 204501 The invention relates to the genetic nanipula-tion of methylotrophic microorganisms, in particular processes for the preparation of obligate methylotrophic bacteria which express contained foreign D N A , plasmids 5 for introducing the foreign DNA, and host organisms. One of the processes according to the invention comprises a) isolating a plasmid which originates from an obligate methylotrophic bacterium, b) preparing from this and from a plasmid with selection 10 markers a hybrid plasmid with a replicon inherent to the C3 obligate methyIotrophic bacterium, c) introducing this hybrid plasmid by transformation into a host organism and amplifying it there, d) after selection, treating the clones with a suitable 15 conjugative pLasmid and abolishing the mobiIizabiIizy defect of the hybrid plasmid, e) conjugating the clones thus obtained with obligate methylotrophic bacteria as the recipient and f> selecting the desired clones.

The plasmid employed in process step a) is pre- w ferably isolated from a bacterium of the"genus Kethyl- omonas, in particular of the species fiethylomonas clara. The plasmid pBE 3 from the Methylotnonas clara strain which is deposited in the German Collection of Microorgen-25 isms under the number DSK 2397 and appropriate plasmids having the same replicon are particularly preferred. '<^VThe invention also provides a process for isolating plasmids £from the strain M. clara DSM 2397 which comprises lysing the '22SEPI986/ vbacteria and subjecting the lysate to a cesium chloride r ; density gradient centrifugation. <• . 204501 The plasmid with selection markers which is employed in process step b) can be the plasmid pER 322 which is described in Gene 2 (1977) 95-113. The hybrid plasmids thus obtained are denoted pRKx in the text 5 which follows.

A hybrid plasmid which contains the genetic infoi— mat i on for the expression, of insulin, as is described in U.K. Patent No 2,067,574, is also suit able as a plasmid with selection markers which can be 10 employed in process step b) .

The hybrid plasmid obtained by process step b) is then introduced into a suitable host organism, advantageously Escherichia coli, by transformation, and amplified there.

In process step d) h suitable conjuaative plas- s(see A E Jacob et al., Nature 255(1975) 504-506)/ m i d , advantageously R P 4, is introduced into this host organism which contains the hybrid plasmid. In addition, the mobiIizsbi I ity defect is complemented by in-,(sce I G Young et al., Gene 14 (1978) 175-179), . troduction of suitable plasmids, such as Col Klend Co I V (see M M Binns et al., Nature 279 (1979) 778-781).

The host organism thus prepared can then/in pro cess step e), transfer by conjugation the hybrid plss-mid introduced in process step c) to a/preferably plas-mid-free, obligate methylotrophic bacterium as recipient. Preferred recipients are bacteria of the genus 25 Methylononas, in particular of the species Methylononas clara, above all o-r the strain ATCC 31 226. This strain is described, for example, in US Patent A,166,004.

Finally, in process step f), the desired clones 2045 01 o are selected by culturing in a medium which contains methanol as the carbon source and on the basis of the transferred resistance or sensitivity towards antibiotics. The obligate rncthyLotrophic bacteria thus ob-5 tained are capable of expressing the contained foreign DNA, and thus, for example, of producing insulin.

DetaiLs of these process steps are expanded on within the framework of the examples.

Furthermore, the invention relates to a variant 10 of the a bovementioned process, in which the process steps d) and e) are simplified as follows: After selection, in step d), the hybrid plasmid is isolated and, in step e), the isolated plasmid is introduced by transformation into spheroplasts of the obligate me-15 thylotrophic bacterium.

Thus the invention also relates to spheroplasts of methy lotrophic bacteria, particularly of obligate me-thylotrophic bacteria, especially of bacteria of the genus Methylomonas, preferably the species Methylononas 20 clara. The strain Methylomonas clara ATCC 31226 is particularly preferred.

The spheroplasts according to the invention can p* be prepared by culturing the bacteria in a glycine-rich medium containing an osmotic stabilizer. This medium 25 is preferably slightly hypotonic. Further preferred embodiments of this preparation process are explained in more detail below.

Furthermore, the invention relates to the use of the new spheroplasts for inserting external DNA into 2 04 5 0 1 o these bacteria. This external DNA is primarily introduced in the form of a plasmid. Suitable pLasmids are the abovementioned hybrid pLasmids.

In order to produce the spheroplasts, the methy-5 lotrophic bacteria are cultured in a suitable medium, advantageously in a minimum medium, up to a suitable cell density. Suitable cell densities are in the OD^qq range of, preferably, 0.5 to 1.8, in particular 0.9 to 1.4. A defined volume of these cultures is then intro-10 duced into about 5 times the amount of the medium mentioned, which is moreover glycine-rich and contains an osmotic stabilizer. The content of glycine can be as much as the saturation concentration, a content of 2 to 4* by weight being preferred.

Suitable osmotic stabilizers are sugars, such as sucrose, sugar base alcohols, such as sorbitol, ond polyglycols, such as polyethylene glycol 6000. The medium is preferably slightly hypotonic, this being brought about by suitabLe concentrations of the osmotic 20 stabilizer. For example, s sucrose concentration of 10% by weight or 1 molar sorbitol is suitable.

The bacteria ere shaken in this medium until essentially no rod-shaped methyLotrophic bacteria are detectable under'the phase contrast microscope. In gene-25 ral, after about 1 hour of shaking <100 to 180 rprr.) at about 37°C, the production of the spheroplasts as immobile spherical structures can be observed. This process is generally complete after about 4 hours.

The produced spheroplasts are carefully 2 04." 0 1 centrifuged down, for example at 2600 x g and A°C for 10 minutes, and resuspended in suitable media. This re-suspension medium likewise contains an osmotic stabilizer at a suitable concentration, for example 15% by weight of polyethylene glycol of molecular weight 6000, to stabilize the spheroplasts.

A mixture which comprises equal parts of the medium used to resuspend the spheroplasts and the carrier for the DNA to be introduced, in a suitable buffer, is added to this suspension at ari approximate volume ratio of 1:5- A suitable DNA carrier is, as detailed above, primarily a hybrid plasmid. A buffer suitable for this purpose comprises an aqueous solution containing 10 mmol of TRIS (tris(hydroxymethyl)aminor.ethane) end 1 mmol of sodium ethyIenediaminetetraacetate (TE buffer) per liter. An osmotic stabilizer is added to this mixture (for example 4Y. by weight of polyethylene glycol 6000 solution, three times the volume of the suspension of spheroplasts) and it is carefully and thoroughly 20 mixed. After standing briefly at room temperature, the } resuspension medium, about ten times the volume of the suspension of spheroplasts, is added and the mixture is carefully centrifuged C3600 x g). ^ The precipitate is then taken up in resuspension o medium, twice the volume of the suspension of spheroplasts, and streaked onto agar plates to check whether transformation has taken place. These agar plates have a composition corresponding to the resuspension medium and contain, in addition to 1.5JS by weight of Bacto agar, 3C/ 204501 an antiobiotic suitable for selection, for example 50 pg /ml of ampicillin or 10 pg/ml o"f tetracycline, in the case where the hybrid plasmids employed contained the appropriate resistance genes. The pLatcs are then incubated at 3 7 0 C for 1 to 3 days, and resistant colonies, after being cultured in the abovementioned liquid medium which contains a suitable antibiotic, are investigated -for the vBiochin. Biophys. Acta, presence of plasmid DNA (Humph reys et a I. ,j 385 (1575) 457-463). By this means, the plasmids employed ■for transformation can be isolated from the methylotro- phic bacteria.

Furtherrnope, the invention relates to the plas-visolated by the process of the present invention/ mids\"from the Met hy lomonas clara strain DSf' 2397 ana the hybrid plasmids with a repLicon "inherent to an obligate Hit t ii y IC't r o p h "i c I* a c t c r" u n'i, in ether words p L r* s m '> c s which are rcpliccted in the bacteria of the genus M e t h y Lomonas, preferably of the species Methylomonas clara, in particular the strain ATCC 31226, in which prokaryotic or eukaryotic DNA is integrated, in particular that for the expression of insulin.

Furthermore, the invention relates to the host organisms which contain the plasmids mentioned, and the host organisms which additionally contain the conjuc;6-tive plasmid and, furthermore, those which contain additional plasmids for the abolition of the mobilization defect. Preferred host organisms contain RP 4 as the conjunctive plasmid and Col K or Col V as plasir.ids for the abolition of the mobilization defect.

The advantage of the invention comprises use 204SOl being made of a replicon of a plasmid fron an obligate methyIotrophic bacterium, in other words one with a very narrow range of hosts. Thus the process according to the invention is distinguished by a high degree of safety, so that it cen be applied to recombinant DNA which contains hazardous information.

The invention is illustrated in more detail in the following examples. In these, percentage data relate to weight unless otherwise specified.

In the examples, the following abbreviations are ATP = adenosine triphosphate EDTA = ethylenediam inetetrsacetic acid or -acetate (Na> ®^60C = °PT''ca^ density at 600 nm TE-boffer = aqueous solution containing 10 mmol of tris HCl per liter, adjusted to pH 8, and 1 rrmol of EDTA per liter, also adjusted to pK 8.0 Tris ( H C I > = trishydroxymethylaminomethane (hydrochloride) .

Examples 1. Isolation of plasmid fron M. c 1 a r ? DSK 23 97 The plasmid was isolated essentially according ^Biochiir.. Biophys. Acta ,■ to the method of Humphreys et a I. ( j 383 4 57—463) .

For this purpose, the bacteria fron 1 liter of culture medium having an ° f about 1.0 were centrifuged down and the precipitate of bacteria was resuspended in v 5 ml of sucrose solution (Z5X sucrose in 5 D minoL/l ®\iof tris HCl solution of pH 8.0). While cooling in ice, •n R •ft/ 1 ml of lysozyme solution (5 mg/ml of lysozyme in 250 2 04 5 mmol/l of tris HCL solution, pH 8.0) and 2 ml of 0.2 molar EDTA solution of pH 8.0 were added. The mixture was incubated on ice for 5 minutes with occasional swirling. Lysis was then brought about by the addition 5 of 8 ml of a mixture containing 50 mmol/l of tris HCL, 75.5 mmol/l of EDTA and 0.25.' of a non-ionic surfactant ((R)Triton X-10C) of pH 8.0. The highly viscous mixture was centrifuged at 48,000 x g for 30 minutes, a clear lysate being obtained as the supernatant. 10 Per 10 ml of supernatant, 1.1 ml of 5 molar saline and 1 g of polyethylene glycol, of mean molecular weight 6,000 were added. The mixture was incubated overnight at 4 0 C . The flocculent precipitate was collected by centrifuga-tion at 1500 x g for 5 minutes and dissolved in 3.5 ml 15 of buffer solution (50 mmol/l of tris HCl, 5 mir.ol/l of NaCl, pH 8.0), and the volume of the solution was measured. After addition of 1 g of CsCL per ml of solution and of 1/15 ml of a 1% strength aqueous ethi-dium bromide solution, centrifugation was carried out 20 for 10 minutes at 16,000 x g. The supernatant from this was centrifuged to equilibrium (20 hours at 47,000 revolutions per minute, 18°C, in a vertical rotor) and showed two bands which were clearly visible, even without irradiation with UV light, lying about one centime-25 ter apart. The fluorescent bands were accentuated by irradiation with UV light of wavelength 366 nm, and the lower plasmid band was harvested by piercing the gradient from the side with a needle and subsequently aspirating the band in a syringe. The ethidium bromide was 2045 01 removed by repeated extraction by shaking with i sopropa-nol saturated with CsCl. After dialysis against 2 liters of TE buffer 3 times, for at least 2 hours each time, the plasmid DNA was obtained in a purity which permitted its use in the following process steps.

If impurities were still evident in the DNA solution, it was extracted twice with identical volumes of phenol saturated with 0.1 molar tris HCl solution of pH 8.0 and then 3 times with absolute ether. The excess ether was blown off, 1/9 of the volume of three molar sodium acetate solution was added to the solution, and the DNA was precipitated by addition of the same volume of isopropanoI. After standing overnight, the mixture was centrifuped at 12,000 x g for 30 minutes, and the DNA precipitate was washed with 90S strength ethariol. Subsequently, the DNA was freeze-dried and taken up in TE buffer. 2. Isolation of DNA from agarose In the process which follows, the method described by Lang ri cge et al. (Analytical Biochemistry 103 (19 S 0) 264-271) was used.

The DNA was applied to a horizontal 0.5% strength agarose gel (low-melting agarose, type VII, No. A-4018, Sigma). After electrophoresis, the bands were visualized by staining with ethidium bromide and cut out of the gel. The agarose disk containing the DNA was m^ltod in a glass tube at 70°C, and the volume was measured, and it was cooled down to 37°C. To this were added equal volumes of the butanol and water phases described below. 2045 01 150 ml of ri-butanol were shaken with 150 ml of water in a separating funnel,and, after separation of the phases, 1 g of hexadecyItrimethyIammcnium bromide was dissolved in 100 ml of the water-saturated butanol phase. This solution was extracted by shaking with 100 ml of the water phase, it being possible for a defoamer to be added (50 1 of Antifoam A, Sigma). After separation of the phases overnight, they were collected separate ly.

The mixture obtained after addition of these bu-tanol and water phases was thoroughly mixed by carefully rotating the tube, and the phases were allowed to separate at 37°C. The upper butanol phase containing the DNA was separated off, and the remaining aqueous phase was extracted twice more with butanol in the manner described. One quarter of the volume of 0.2 molar Ha CI solution was added to the combined butanol phases, and thorough mixing was again carried out. After the aqueous phase had been separated off, another salt extraction was carried out and the same volume of chloroform (which had been purified over en aluminum c >: i d c column) was added dropwise to the combined aqueous phases. After standing on ice for half an hour, the lower chloroform phase was discarded and the remaining chloroform was blown out with air. The DNA was precipitated with i sopropanoI and, after separating off, resuspended in TE buffer. 3. Electrophores is on polyacryl-amide gel 8 - 15% strength polyacrylamide (PAA) gels were used for the visualization and characterization of DNA 2 045 01 O n fragments below 0.3 ND. The gels were 1 mm thick, 30 cm long and 14 cm wide. A mixture containing 89 mmol of boric acid, 89 mmol of tris HCl and 2.5 mmol of EDTA per Liter and having a pH of 8.2, served as the buffer. 5 In order to prepare an 8% PAA gel, 33 ml of a 24% strength PAA stock solution (23.22 g of acrylamide and 0.78 g of N,N'-methylenebisacrylamide in 100 g of aqueous solution), 10 ml of the electrophoresis buffer mentioned, which however contained all components in 10 10-fold concentration, 6.25 ml of a 6.4% strength 3-ethylaminopropionitrile solution and 50.75 ml of water were mixed. The mixture was degased on a water pump, and polymerization was started with solid ammonium peroxodi-sulfate. The gel was poured immediately thereafter, and 15 a minimum of 2 hours was allowed to elapse before the start of electrophoresis. Before applying the samples, a voltage of 100 V was applied for about one hour to the gel to remove residual ammonium peroxodisuLfate.

The electrophoreses were carried out at 100 V 20 and a current of 12 mA. 4. Characterization of the plasmid pBE 3 from the Hethylononf? clara strain DSM 2397 The plasmid pBE 3 was digested with the restriction endonucleases mentioned in Table 1, and the 25 fragments produced were separated by gel electrophoresis. The results recorded in Table 2 and Figure 1 were obtained by double digestions and by characterization of individual fragments cloned in pBR 322, as described be low. 2045 01 The plasmid pBE 3 is a deletion mutant of a larger plasmid. Besides this, smaller plesmids also exist. All these plasmids are equivalent in the sense cf this invention when they contain the replicon inhe-5 rent to Methy lomonas.

Table 1: Survey of the enzymes used for the characterization of pBE 3 : Enzyme Number of fragments Acc I 1 Xor II 1 Eco RI 5 Hinc II 5 Aval 12 Bal I at least 19 No c leavage with Bam HI Bgl II Bst EII Eco RV Hind III Hp a I Kpn I Nru I Fst I Pvu II Sal I Sma I J o Table 1 (continued) Sph I Sst I Sst II Stu I Xba I Xho I Xmn I 2045 C1 Table 2 : Size of the fragments (in MD) which were produced by cleavage of pBE 3 with restriction nucleases: EcoR I : Hinc II: Av a I : F1 : 4.14 H1 : : 6.8 A1 : 2.0 F2 : 3 .09 H2 : : 1 .42 A2 : 1 .72 F3 : 1 .27 H3 : : 0.S7 A3 : 1 .26 F4 : 0.91 H4 : : 0.31 A4 a : 1 .03 F5 : 0.63 H5 i CO CVI • o A4 : 1 .03 A5 : 0.85 A6 : 0.66 A 7 : 0.55 A8 : 0.50 A 9 : 0.13 A1 0: 0.08 A1 1 : 0.07 EcoR 1+ EcoR 1+ A va I: Hinc II: D1 : 1 .35 El : 2.29 • D2 : 1 .22 E2 : 1 .83 I \ o Table 2 (continued) 2 04301 D3 a : 1 .03 E3 : 1 .42 D3 : 1 .03 E4 : 1 .27 D4 : 0.85 E5 : 0.91 D5 : 0.70 E 6 : 0.63 D 6 : 0.63 E7 : C .46 D 7 : 0.55 E8 : 0.40 D8 : 0 .54 E9 : 0.31 D9 : 0.53 E10 : 0.28 D1 0: 0.50 D11 : 0.40 D12: 0.14 D1 3 : 0.12 D1 4: 0.1 D1 5 : 0.1 D1 6: 0.1 . Restriction digestions Individual digestions were carried out in a to-20 tal volume of 50 to 100 jx I with the buffers recommended by the manufacturers. In order to ensure complete digestion of the DNA, the samples were incubated overnight. r For double and multiple digestions, the approp-25 riate enzymes were usually added together to the DNA.

In these cases, the buffer comprised a solution containing 50 mmol of sodium chloride, 5 mmol of tris HCl (adjusted to pH 7.5), 6 mmol of magnesium chLoride, 6 mmol of 2-mercaptoethanol and 100 mg of bovine serum \ r*\ -16 - 2 °4r albumin per liter. Control experiments showed that the enzymes provide the same results in this buffer as in those recommended by the manufacturers. The exceptions were enzymes which have an above average salt requirement. In these cases, digestion was initially carried out at a low salt concentration with one enzyme, and the second enzyme was only added after increasing the salt concentration. 6. Partial digestions with Eco R I 2 p. g of plasmid DtJA were incubated with 1 U of the restriction enzyme at 37°C in a total volume of 50 jj^l. Identical mixtures were incubated for different times, and the reaction was stopped at the various times by heating the mixture at 70°C for ten minutes. Ana-15 lysis of the particular pattern of bands by gel electrophoresis showed the extent to which the digestion had advanced after the defined times. 7. Growth conditions All Escherichia coli strains used were cultured 20 in L broth (10 g of Dactro Trypton, 5 g cf yeast extract and 5 g of sodium chloride per 1 liter of water). The Methylomonas clara strains were cultured in one of the two following minimum media: M 36 : 1.5% of methanol 0.1% of H3PO4 0.083% of K2S04 0.018% of Na2S0A . 10 H20 0.036% of MgS04 . 7 H20 '0.004% of C3CO3 \ o 2 045 0 1 0.015% of citric acid 0.005% of (NHA)2Fe (SO^)2 • 6 H2° 80 ju I /I of trace element soLution V 135 L : 1.5% of methanol 0.1 6% of K2S04 0.06% of MgSO^ . 7 H20 0.02 5% of Na2S0/, 0.014% of CaC03 0.01% of Fe2Cs0/,)3 0.2% of H3PO4 0.28% of NHj (25%) 0.3% of KN03 0.3% of N a H CO3 1 irl/L of trace element solution 15 Trace element solution: 0.05 9/I of HJBOJ 0.01 g/l of KI 0.04 g/l of MnSO^ . 4 H20 0.04 g/l of ZnSO^ . 7 H20 20 0.02 g/l of (NH4)6Mo702a 8. Construction of the hybrid vectors The plasmid pBE 3 was partially'digested with the restriction enzyme Eco R I so that the major part of the plasmid was only cut once and was present in the 25 linear form. pBR 322 DNA was completely digest&d with Eco R I and then subjected to treatment with alkaline phosphatase. The DNAs thus prepared were mixed end in-cubated* with T4 DNA ligase at 14°C overnight. !30 jig of this Li gated mixture then served for transformation into ^ • 4 "• 2 the E. coli strain HB 101 made competent by treatment with CaCl2.

The bacteria were smeared onto L broth plates with 20jj.g/rnl of tetracycline ahd incubated overnight. 5 Resistant colonies were reinoculated on fresh pLates and well-grown clones were investigated for the presence of plasmid DNA having a higher molecular weight than pBR 322 by "single colony lysis". Clones which contained plasmid DNA of this type were finally cultured in 100 ml 10 of L broth with or without tetracycline, and, after chic ramphenicol stimulation of the bacteria, the plasmid DNA was obtained.

Restriction digestions with the enzymes Eco R I and Ava I showed that the hybrid plasmid pRM 21 shown 15 in Figure 2 and the hybrid plasmid pRM 54 shown in Figure 3 had been obtained.

In addition, it is possible to identify hybrid plasmids containing fractions of pBE 3. 9. Lipase reaction 20 Ligase reactions were carried out ir. a volume of 0 I at a total DNA concentration of 2 0 g / n I. The buffer contained 30 mmol of tris HCl (adjusted to pH 7.5), 4 mmol of magnesium chloride, 10 mmcl of dithio-crythritol and 0.2 mmol of ATP per liter. 1^>.l of T4 25 ligase, corresponding to 400 U , was added to t h e ? e mixtures (in this context, 1 U corresponds to the amount of enzyme necessary to Ligate 50% of lambda DNA, which has been digested with Hind III, in 30 minutes ot -16°C in a volume of 20 Ji-I . The concentration of the DNA "in r 204501 this mixture is about 330 |i g/m L) . The ratio of the two DNAs to one another which is optimum for a particular ligase experiment was calculated by the method of Du- pj. Mol. Biol. gaiczyk et al., > 9_o (19750 171. The incubation was 5 carried out at 1 4 0 C for at least 16 hours. 10. Conjunction Recipient and donor were cultured overnight to N give an OD^qq of 1.0 - 1.? (recipient) end 1.4 - 1.6 (donor), and equal volumes of recipient and donor were 10 mixed so that the mixture had a large available surface area. The mixture was incubated, without shaking, at *\ D j 37 C for 2 hours, and then 20 0 I of it was smeared on to agar plates, the composition of which permitted selection against the donor and for the receptor, the latter being provided with a new nropr-rty which v: 2 s supplied by the p I & ? n i d to be transferred. For the 15 conjugation between E. co li HB 101 and ft. clara DSM 2397, these were nethanol-ninimum medium plates with the addition of an antibiotic (determined by the type of the plasmid to be transferred).

In other experiments, recipient and donor were b/" incubated as described above and 200 j> I of this mixture was smeared onto metha nol-minimum medium plates without antibiotic. These plates were incubated at 37°C over-25 night, and the lawn of bacteria produced was rinsed off with 2 ml of methanol -minimum medium. 200 I of this suspension was then again smeared on rriethanol-miniivium ?*a1 /'"X l >medium plates with the appropriate antibiotic. Clones emerged after incubation of the plates at 37°C for p . 20 . 2 0 4 5 01 48-72 hours. 11. Expression of eukaryotic DNA in K. clara a) The plasmid pBE 3 was cloned or recloned by the processes described in derivatives of pBR 322 5 which contained, as the c-DNA sequence of a eukaryotic gene, that of monkey insulin.

The monkey insulin c-DfJA had been incorporated in the Pst I cleaving site of pBR 322 so that this foreign information was expressed under the control of 10 the 'lactamase pronotor in E. coli. This produced a fusion protein which could be Detected with anti-insulin antibodies (U.K. Patent No. 2,067,574).

Monkey insulin c-DKA contains two internal Pst I cleaving sites. Thus the coding information cannot be 15 removed in En intect forrr from the plasmid by Pst I digestion. Thus in order to make possible replication of this plasmid in clara, the procedure was such that DNA sequences from the F.. clara plasmid pBE 3 were incorporated in this pBR 322 derivative expressing insu-20 lin information. The experimental procedure was as described above. By this means, a number of hybrid plasmids were obtained which differ in their structure from the hybrid vectors described above only by the monkey insulin t-DNA incorporated in the pBR 322 part. ?5 The arrangement of the insulin gene in the pF-R 322 part of these vectors still remains in phase, so that these v A ' i?^ clones also express insulin -antigenic determinants in E. ' 'is' j : coli. One of these clones, which contains the entire -T,; 'Pg*. S- ^ sequence, was given the name plnNc 68.

' * V r- <■ o - 21 - 204501 b) The mobiIizabi I ity defect found in pBR 322 can be complemented by plasmids such as Col K and Col V (Young and Poulis, Gene A (1978) 175-179). Thus, in addition, the plasmid Col K was. introduced by transformation into those donor strains with the conju-gative p la sr. id RP A and the hybrid vectors of the pRK series which had been made competent by treatment with calciur chloride and are described above. The co lie in-sensitive strain AB 1157 was used as the indicator strain to detect the plasmid Col K (Warren et pMol. Gen. Genet. al., I 170 (1979) 103-107). In experiments on conjugation between the E. coli donor strains HB 101 (RP A, Col K, pRK 54) or HB 101 (RP A, Col K, pRTI 21) and the recipient K. clara ATCC 31226, in order to select for clones which contained the hybrid vector, selection was carried out in the presence of high doses of tetracycline (50j.ig/ml) on methanol-minimun medium (N, 36).

Analysis of a large number of M. clara clones showed that, in about 10*' of the cases, clones had been obtained which only contained the hybrid vector. in an experiment on conjugation between the E. coli donor strain HB 101 (RP A, Col K, plnMc 68) and the M. clara recipient ATCC 31226, in the same r,i e n n e r , M. clara clones were obtained which only contained the p Las-mi d plnMc 68. c) The ft. clara clones with the plasmid plnKc 68 obtained were then cultured and checked fcr their insulin content by a radioimmunoassay or a fat-cell assay.

Consistent with the observations made on E. coli in the ■ f> - 22 - • 204501 U.K. Patent No. 2,067,574, insulin values between ^ and 5 IU per liter were also measured in the case of M. clara ATCC 31226. Thus the insulin information contained in the pBR 322 part of th® hybrid vector 5 plnftc 68 is also correctly and efficiently expressed in K.. clara. 12. The strain Methylomonas clara ATCC 31226 is cul-^ tured in the minimum medium K 36 to an OD^gg of 0.9 to 1.4 ml of these cultures are introduced into 100 ml of 10 K 36 medium which additionally contains 4 ~ glycine and 10X sucrose. The bacteria are shaken at 100 to 180 rpm in this medium ot 37°c for 4 hours. No oblong mobile bacteria are then any longer detectable under the phase contrast microscope.

The spheroplasts produced are centrifuged down at 2600 x g and 4°C for 10 minutes and resuspenci'ed in 1 ml of M 36 medium which additionally contains 10% sucrose.

The media mentioned can also contain, in place 20 of 10X sucrose, 1 mole per liter of sorbitol or "\5% Ty polyethylene glycol of mean molecular weight 6000.

The spheroplasts can also be separated out by centrifugation at 3600 x g and 4°C for 10 minutes. 13. 0.1 ml of a mixture comprising equal parts of —' 25 resuspension medium and TE buffer and containing the plasmid p R K 21 is added to 0.5 ml of the suspension ob~ tained as in Example 12. 1.5 ml of 4% strencth poly-ethylene glycol 6000 solution is edded to this mixture 2 \ - Tiij and it is carefully and thoroughly mixed. This mixture ?/ 2 o i r, n V - 23 - ~ is allowed to stand at room temperature for 2 minutes, 5 ml of resuspension medium are added and it is centrifuged at 3600 x g.

The precipitate is taken up in 1 ml of resuspen-5 si on medium and streaked on agar plates, the composition of which corresponds to the resuspension medium and uhich additionally contain 1.5 2 Bacto agar and 50jJ.g/ml of ampi-cillin. The plates are incubated at 37°C for 3 days, and the colonies are cultured in M 36 medium which con-10 tains 50j^g/ml of ampicillin. It was possible to isolate the plasmid pRM 21 employed for transformation from *._ the bacteria thus obtained.

It is also possible to employ in this process tetracycline at a concentration of 10 ^g/ml in piece of 15 ampicillin.

The same result is also obtained when, in place of the abovementioned plasmid, the plasmid p R M 54 is employed. 14. If the procedure is carried out as in F.xample 13, but the plasmid plnMc 68 is employed and selection is by means of 50j^g/ml of tetracycline, then Met hy.lomo-nas clara clones having this plasmid and producing insu~ li n are obt ai ned. o

Claims (14)

- 2A - 204501 ■WHAT */WE CLAIM IS: - -

1. A hybrid plasmid with a replicon inherent to an obligate methyIotrophic bacterium.

2. A plasmid as claimed in' claim 1, which is replicated in a bacterium of the genus Methylomonas, prefei— ably of the species Kethylomonas clara, in particular of the strain ATCC 31226.

3. A plasmid as claimed in claim 1 or 2, having integrated eukaryotic DNA, in particular for the expression of insulin.

4. A plasrrid as claimed in clair. 1 or 2 having integrated prokaryotic DNA.

5. A process for the preparation of obligate methy-lotrophic bacteria, which express contained foreign DNA, which comprises -.which originates a) isolating a plasm id|from an obligate m e t h >• lotrophic bacterium, b) preparing from this and from a plasmid with selection markers a hybrid plasmid with £ replicon inherent to the obligate methyIotrophic bacterium, c) introducing this hybrid plasr. id by transformation into a host organism and amplifying it there, d) after selection, treating the clones with a suitable conjugstive plesnid and abolishing the mcbilizability defect of the hybrid plasmid, e) conjugating the clones thus obtained with, preferably plasmid-free, obligate methylotrophic bacteria as the recipient and f) selectina the desired clones. 204501

6. The process as claimed in claim 5, wherein the tbacteria of step e) are, obligate methyIotrophic ^ of the genus Methy lomonas, preferably of the species Methylomonas clara, in parficular of the strain ATCC 31 226.

7. The process as claimed in claim 5 or 6 , wherein the bacterium from which the plasmid is isolated is of the strain DSN, 2397 of the species Methylomonas clara,

8. The process es claimed in one or more of claims 5 to 7, wherein the plasmid having selection markers is pDR 322 and/or contains the genetic information for the expression of insulin.

9. The process as claimed in one or more of claims 5 to 8, wherein the host oroanism is Escherichia coli, the r(as herein defined) conjugative plasmid is TP 4|c.nd the mob i I i z ab i I i t y defect is abolished by introduction of the plasmid Col or Col V (as herein defined).

10. The process as clained in one or more of cltirr.s 5 to 8, wherein, in place of the process steps d) and e), the obligate methylotrophic bacterium serving as the recipient is converted into spheroplasts and the hybrid plasmid is inserted by transformation.

11. A host organism containing a plasmid as cleimcd - 26 - 204501 in one or more of claims 1 to 4 and, where appropriate, a conjugative plasmid and, where appropriate, a plasmid abolishing the mobilizability defect of the hybrid plasmid.

12. The organism as claimed in claim 11 which contains RP 4 (as herein defined) as the conjugative plasmid and, where appropriate, the plasmid Col K or Col V (as herein defined).

13. A process for isolating plasmids from the strain M. clara DSM 2397 which comprises lysing the bacteria and subjecting the lysate to a cesium chloride density gradient centrifugation.

14. Plasmids^containing replicon inherent to a bacterium of the genus Meth^omonas ^isolated by a process according to claim 13. HOECHST AKTIENGESELLSCHAFT By Their Attorneys HENRY HUGHES LIMITED By: