CH621147A5 - Process for obtaining a cell culture which produces a desired polypeptide by genetic modification of microorganisms - Google Patents

Description

The present invention relates to a method for obtaining a cell culture that produces a desired polypeptide by genetic modification of microorganisms. Regarding the state of the art, reference is made to the Journal of Bacteriology 1969 pp. 637-650: "current topics in microbiology and immunology" 1973 pp. 61-88: US Patents 3,853,467 and 3,905,767.

The process according to the invention is characterized by the following process steps a) splitting molecules of a DNA receptor material obtained from a plasmid which is suitable for infecting the cells of the said microorganism, by treating the plasmid DNA with an endonuclease,

b) splitting another genetic material containing DNA donor, coding for the synthesis of a desired polypeptide using a similar endonuclease, whereby DNA fragments are obtained, the ends of which are compatible with those obtained according to a),

c) Mixing the DNA fragments obtained from the various sources, thereby allowing the DNA fragments to come together at random

Influence of a DNA ligase takes place, whereby hybrid DNA molecules are obtained which contain the combined fragments from both DNA sources,

d) infecting the cells of the microorganism to be modified by contacting them with the hybrid DNA molecules,

e) have the infected microorganism metabolized in the presence of a substrate which is suitable for conversion into the desired polypeptide, and f) screening for the production of the desired polypeptide and production of a cell culture which produces the polypeptide.

The present invention thus relates to the genetic modification of a microorganism - i.e. an organism that is generally characterized by

that the individual cells are not organized in one tissue - through the introduction of foreign genetic material in such a way that the microorganism synthesizes polypeptides and / or proteins that either correspond entirely to the genetic information of the foreign genetic material or partially to the genetic information of the foreign one genetic material and that of the microorganism associated genetic material correspond.

In the present description, the term “DNA” is to be used to designate the genetic material (ie the material in whose replication the genetic information is transmitted from one generation to the next when the organism reproduces), since deoxyribonucleic acid is the most important representative of the represents materials mentioned. Usually, RNA, which is the more general genetic material, also includes RNA. The present invention therefore also relates to the genetic modification of organisms in which RNA is the genetic material; Inverse transcription to obtain a DNA copy of the RNA can facilitate the application of the method according to the invention to such organisms.

Double-stranded DNA can be cleaved by certain enzymes, which are generally referred to as endonucleases; the mechanism of action of some of these endonucleases seems to be that they "recognize and cleave" specific nucleotide sequences on the DNA double helix.

For the purposes of the present description, depending on the type of cleavage, these endonucleases can be classified into i) endonucleases which cleave both strands of the DNA double helix at exactly opposite sites (FIG. 1A),

ii) Endonucleases that cause staggered cleavages of the two complementary DNA strands (i.e., sites not opposite on the double helix) (Fig. 1B). By cleavage with the help of class ii) endonucleases, DNA fragments with two different but complementary single-stranded ends are obtained, i.e. In accordance with the interaction of the nucleotides with one another, such single-stranded ends will have an affinity for one another and a tendency towards specific attachment to the corresponding complementary partner (FIG. 2). In the treatment of the two different DNA preparations (receptor or donor material) with the same endonuclease of class ii) and after extraction or inactivation of the endonuclease, after mixing the two DNA fraction preparations obtained in this way, random, but complementary attachment of corresponding single-stranded ends of the DNA fragments either from the two different preparations or attachment of fragments of the same preparation. According to the hypotheses valid today, the stability of the fragments obtained in this way depends primarily on the strength of the hydrogen

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Bonds between the complementary bases in the single strands of the two fragments. However, the two fragments can also be covalently bound to one another by the action of certain enzymes known as DNA ligases, which results in stronger binding.

In addition to the DNA, which is the genetic core material of the living cell, there is also a large group of extranuclear DNA molecules, commonly referred to as "plasmids", which are capable of autonomous self-replication. In their biologically active replicable form, however, these DNA molecules only manifest themselves if they are in a sensitive host organism or a suitable in vitro system, production of daughter plasmid DNA molecules taking place and corresponding formation of polypeptides and / or Proteins, according to the genetic information of the plasmid DNA. Typical plasmids are bacteriophage DNA, resistance factors and colicinogenic factors. The method according to the invention relates in particular to the use of bacteriophages.

Many plasmids are able to assume a wide variety of states in a host cell, e.g. the E. coli sex factor “F” can either be present as a self-replicating DNA molecule in the host cell or integrated into the core DNA of the host cell. Bacteriophage (X) DNA can also be in an integrated "lysogenic" form if the phage's activities are "dormant", but can be converted from this state into an active self-replicating form, which is used in the synthesis of phage particles (DNA + protein envelope ) and possible lysis of the host cell and release of the daughter phage results.

Both naturally occurring and derived plasmid DNA molecules could be identified, which could be cleaved at a specific point by the action of a specific endonuclease. Examples of such endonucleases are restriction enzymes, e.g. "Eco RI" and "Hind III". The treatment of DNA molecules with a cleavable region on the molecule by e.g. Eco Rl enzyme, results in the formation of two linear Rl “cohesive” ends, which can be attached to each other again to form the original molecule, or to another DNA fragment with corresponding complementary cohesive ends, which are also linked by Rl- Treatment was formed, can be accumulated. Figure 3 illustrates the operation described above, in which dissimilar DNA molecules having both R1 recognition regions are cleaved by R1 treatment, brought together and reassembled, and treated with a DNA ligase enzyme , whereby hybrid DNA molecules are formed. Hind III shows a corresponding specific effect.

The scheme in Figure 3 describes the production of hybrid plasmid / non-plasmid DNA, but other combinations can occur in the same way. In particular, the above scheme illustrates how the genetic information of the plasmid can be changed by incorporating non-plasmid DNA into the plasmid DNA molecule. The infection of a host cell with the hybrid plasmid DNA results in the production of further hybrid DNA and of polypeptides and / or proteins, according to the genetic information of the hybrid DNA mentioned.

Of course, the hybrid DNA produced in this way will be composed at random and it is therefore necessary to use an appropriate screening in order to achieve a desired DNA composition, as is used for replication and reproduction.

The isolation of plasmid DNA molecules in which a specific foreign Rl fragment is incorporated can be carried out in various ways. Thus the Rl-treated "donor" DNA fragments can be analyzed using e.g. Acrylamide or agarose gel electrophoresis are separated. These fractions can then be used in separate integration and transfection experiments. On the other hand, CsCl density gradient centrifugation can be used to fractionate phage particles containing different proportions of foreign DNA. The fractions obtained in this way can then be used in separate infection experiments.

In order to achieve a particularly preferred DNA composition by treating the suitable host infected by DNA, a method is preferably used for the method according to the invention, which is called «immunoscreening». This method is not based on the extraction and identification of a specific DNA material, but on the identification of the desired product of the hybridization technique. For example, if a particular plasmid hybrid molecule is to be obtained by the hybridization, which in a bacterium synthesizes a specific polypeptide, e.g. Insulin induced, the said screening related to this specific polypeptide.

Although we describe the use of a concentration or separation method (the examples given, of course, are not exclusive in nature) followed by immuno-screening, this sequence is used only for practical reasons (each of a number of fractions is tested for activity before dilution , Screening of the diluted fractions shows their activity) and, if desired, the entire hybrid plasmid population can be screened without prior fractionation.

By immuno-screening we mean application to microorganisms that are either actively metabolizing or to their cleavage products, a specific antibody for a particular metabolite of the organism (usually a polypeptide or a protein) and the localization of retained antibody that indicates the presence of the metabolite displays.

The method according to the invention consists in particular in that in a first reaction step a receptor DNA material is cleaved, whereupon the fragments of the DNA material thus obtained are mixed with «donors> DNA fragments which are cleaved by another DNA mate -rials are brought in the same way, whereby a random attachment of the individual DNA fragments takes place, so that “hybrid” DNA molecules are obtained, whereupon suitable hybrid host cells are infected with the hybrid DNA molecules thus obtained, whereby these infected host cells undergo metabolism, and whereupon certain metabolites are determined by immuno-screening, and whereupon the desired metabolite can be produced by replication of the host cells and / or the corresponding DNA.

If an "active" fraction is found, it can be worked up by refractioning and re-testing until a pure strain e.g. from phages, which carries the desired “foreign” hybrid genetic material.

Bringing the selected pure fraction of hybrid DNA into a host cell will result in the production of daughter hybrid DNA and the polypeptides / proteins, according to the genetic information of that DNA.

The process according to the invention is to be explained in more detail by the following examples.

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example 1

Synthesis, detection and isolation of an A phage containing the gene for E coli trp A polypeptide.

1. Preparation of restriction enzyme Hind III.

Hemophilus influenza cells are located in a brain

Cardiac nutrient medium, enriched with 10% hemin / ml and 2% NAD / ml, produced on an E 550 of 0.7, crushed by centrifugation, washed once and in 0.002 vol. resuspended in 0.05 M Tris (pH 7.4), 0.001 M glutathione. The cells are disrupted by ultrasound treatment at 4 ° C. and centrifuged at 100,000 g for 30 min and the supernatant is placed in 1 M NaCl; this solution was then placed on a 2.5 x 49 cm Bio-Gel A 0.5 M (200-400 mesh) column, with 10 vol. Pre-washed with 1 M NaCl, 0.02 M Tris HCl (pH 7.4), 0.01 M mercaptoethanol. Elution was carried out at 1.2 ml / min using the buffer mentioned and 6 ml fractions were collected. Fractions 18-27 were pooled and mixed with 2 vol. diluted by 0.02 M Tris, pH 7.4. Subsequently, ammonium sulfate was slowly added up to 50% saturation and the precipitate formed was centrifuged off. The supernatant solution was subjected to a further precipitation, whereby a 50-60% precipitate and subsequently a further precipitation, whereby a 60-70% precipitate was obtained. The precipitation was given together and in 0.1 vol. of 0.05 M NaCl, 0.02 M Tris HCl (pH 7.4), 0.001 M mercaptoethanol and dialyzed against the same buffer. (All operations were done at 4 ° C).

A phosphocellulose column (Whatman P. 11), 0.5 cm x 9.5 cm, was equilibrated with 100 ml of 0.01 H potassium phosphate buffer (pH 7.4). The redissolved rainfall was 9 vol. of the buffer mentioned and added to the column at a rate of 5 ml / h. The protein was then eluted step by step with 5 ml portions of 0.01 M potassium phosphate buffer (pH 7.4), containing increasingly large amounts of KCl. The protein eluted at 0.3 M was precipitated with 75% saturation with ammonium sulfate and redissolved in 10 mM potassium phosphate (pH 6.8). 6 absorption units at 280 nm were placed on a hydroxyl apatite column (1x10 cm). Elution was carried out with a linear gradient of 160 ml each of 10 mM and 400 mM potassium phosphate (pH 6.8). Fractions 79-85 (at about 0.25 M phosphate) were used as Hind III restriction enzyme preparation.

2. Production of restriction enzyme R1

A strain of E coli containing an R factor determining the Rl restriction enzyme was grown in 10 L of L broth to an E 550 of 1.0. The cells were then broken up by centrifugation at 4 ° C., frozen in liquid nitrogen and stored at −20 ° C.

100 g of the frozen cells were sonicated in 200 ml of an extraction buffer (0.01 M KH2P04 pH 7.0; 0.001 M EDTA; 0.007 M 2-mercaptoethanol) for a total of 12 min. (MSE Atomix, full power in batches of 45 seconds with 30 seconds each cooling). The supernatant solution was then centrifuged at 35,000 rpm (MSE 8 x 50 rotor) for 1 h at 4 ° C. 100 ml of 5% w / v streptomycin sulfate in extract buffer was slowly added to the supernatant solution with constant stirring. After stirring for 15 min, the precipitate obtained was separated by centrifugation and ammonium sulfate was slowly added to the supernatant solution up to 50% saturation. After stirring for 30 min, the precipitate was separated by centrifugation (10,000 rpm; 10 min; 4 ° C.), dissolved in 45 ml of extraction buffer and dialyzed against 4 batches in 48 h of extraction buffer. A 4 M NaCl solution was then added to the dialyzed ammonium sulfate fraction to give a final concentration of 0.35 M NaCl and then the solution (at a flow rate of 40 ml per hour) onto a phosphocellulose-Pll column ( 15 cm x 3 cm), which were washed by H + and OH cycles ° and equilibrated with 0.35 M NaCl in extraction buffer, followed by a linear gradient (0.35-0.8 M) of NaCl in Extraction buffer was eluted. 10 ml / fractions were collected and active fractions at approx. 0.5 M NaCl were combined and dialyzed against extraction buffer, and then at a flow rate of 20 ml / h onto a DEAE cellulose DE52 column (5 cm × 1 cm) , which was equilibrated with extraction buffer. The column was washed with 0.15 M NaCl in extraction buffer and eluted with a linear gradient (0.15-0.50 M) of NaCl in extraction buffer at a flow rate of 10 ml / h. 4 ml fractions were collected and active fractions at approx. 0.3 M NaCl were combined and dialyzed against extraction buffer. The enzyme was concentrated by absorption on a small DE52 column (2x1 cm), which had been equilibrated with extraction buffer, and eluted with 3 ml of 0.5 M NaCl. The eluate obtained was used as the Rl enzyme preparation.

3. Preparation of A-phage DNA

1.5 liters of a culture of sensitive E coli host cells in L nutrient medium and in exponential growth phase (OD 550 = 0.5) were infected with X-phages at an infection multiplicity (m.o.i) of 1.0. This was followed by incubation at 37 ° C with vigorous shaking.

After lysis had occurred, the cultures were shaken with chloroform for 10 min (1 ml chloroform per 10 ml culture), then distributed in 500 ml polypropylene in centrifuge bottles and centrifuged at 4 ° C. for 20 min and at 7000 rpm. The supernatant solution containing the phages was decanted off and centrifuged for 2 h in a preparative L2 ultracentrifuge (Beckman) at 10 ° C. and 18,000 rpm (rotor 19).

The supernatant solution was poured off and the residue was retained. To each of the centrifuge bottles were added 1.5 ml of 10mM trishydroxymethylaminomethane (Tris) / HCl, 10mM MgSO4 buffer, pH 7.2, in which the residue was resuspended by mild homogenization after standing overnight at 4 ° C. The bottles were washed with two 0.5 ml portions of 10 mM Tris / MgSO4 buffer.

The phage suspension was collected in a 15 ml centrifuge tube and centrifuged for 15 min at 7000 rpm and 10 ° C. (rotor JA20, Beckman J21 centrifuge) in order to remove remaining bacterial material.

Water and cesium chloride crystals were added to the supernatant solution containing the phages to obtain a total of 12 ml with the refractivity of 8 ° 10 '. The suspension was then placed in 3 cellulose nitrate tubes Q4 x 2 ") and covered with 1 ml paraffin oil to the top of each of the three tubes.

The suspension containing cesium chloride and the phages were centrifuged at 30,000 rpm for 22 h at 10 ° C. in a SW50 rotor in the Beckman L2 ultracentrifuge. The centrifuge tubes were then punctured at their lower end and the visible phage band was collected by dropwise fractionation of the CsCl gradient. The phages were placed in a small screw-top container and stored at 4 ° C. The absorption (OD 260) was measured in 20 μl of a 1:10 diluted sample of the phage suspension in a 1 mm quartz microcell. 20 OD 260 units (calculated for undiluted phages in a 1 cm light path) of the phages were checked overnight

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three batches of 10 mM potassium phosphate buffer (KPB) dialyzed at pH 7.4 and at 4 ° C.

The dialyzed samples (approx. 1 ml) were placed in a 15 ml Corex centrifuge tube, 10 ml of 25% sodium dodecyl sulfate (SDS) were added and the solution was heated to 60 ° C. for 3 minutes. The opalescence caused by the phages disappeared and the solution became clear. The tube was then cooled in ice water.

A volume of phenol, saturated with 0.1 M KPB, precooled on ice, was added to the tube and its contents were mixed by inverting at room temperature for 1 min, and then centrifuged for 10 min at 7,500 rpm and at 5 ° C.

The lower phenolic phase was pipetted off and discarded and the phenol extraction repeated twice. The supernatant aqueous solution was then slowly added to a dialysis tube. This was followed by dialysis against three batches of 10 mM Tris HCl / 10 mM MgCl for 24 h. The product obtained was used as a phage DNA preparation.

Receptor phage DNA

The phage DNA produced, which was used as receptor A, was that of a derivative of X-phages in which parts of the normal complement of the X-phage chromosomes had been removed (approx. 20% total). In addition, the phage DNA had only one region, as can be cleaved by Hind III enzyme (region 3).

The essential properties of the phage can be summarized as follows:

h \ RI 1-2V, Hind III 3+, N21 i21, nin.

The host bacterium used to produce X phage A was E. coli strain C600.

The phage DNA prepared for use as receptor B was that of a derivative of X phage in which parts of the normal complement of the genome had been removed (approx. 15% total). These phages had two cleavage regions for cleavage by Eco Rl restriction enzyme. Removal of the fragment between the two cleavage regions by R1 treatment increases the capacity of the receptor to introduce DNA.

The essential properties of this phage can be summarized as follows:

h \ RI 1-2V, R13 +, nin.

The host bacterium used for the production of phage B was E coli KY Mei.

Donor phage DNA

The phage DNA produced as donor phage DNA was that of a derivative of X containing the genes for the tryptophan synthesizing enzymes (the trp operon) of E. coli. Essential properties:

h80, i1, trp +

E coli KY Mei was used as the host bacterium for the production of donor phage DNA.

4. Production of E coli DNA

Cells of the prototrophic strain W1485 from E coli, grown in L nutrient medium, were broken up and resuspended in 100 mM Tris-HCl (pH 8.0), 10 mM EDTA up to a cell concentration of 2 × 199 / ml. Lysozyme was added to a concentration of 100 µg / ml and the mixture was incubated at 37 ° C for 5 min.

Sodium dodecyl sulfate was added up to 0.5% w / v followed by proteinase K of 50 xg / ml and incubated overnight with gentle shaking at 37 ° C. The mixture was then treated with an equal volume of cold phenol, saturated with buffer (500 mM Tris HCl pH 8.0; 10 mM EDTA; 10 mM NaCl; 0.5% w / v SDS) and for 10 min at room temperature with 60 rpm rolled. The mixture was then at 7 ° C

cooled and centrifuged at 500 g for 10 min. The viscous aqueous phase was separated off with a pipette and the phenol treatment was repeated. The aqueous phase was then placed in a dialysis tube which had previously been immersed in 64% ZnCl2 for 20 minutes and then washed in 0.1 M HCl and in 5 mM EDTA. Dialysis was carried out against 50 mM Tris HCl (pH 8.0), 10 mM EDTA, 10 mM NaCl (buffer C) in a 1 liter measuring cylinder at room temperature until the absorption at 270 nm outside the dialysis tube was less than 0.05 units.

The solution was then placed in an untreated dialysis tube and RNA-Ase A (final concentration 50 ng / ml) and RNA-Ase T (final concentration 2 (ig / ml) were added. This mixture was stirred for 4 hours at 37 ° C. against buffer C. dialyzed.

The DNA solution was placed in a further untreated dialysis tube and SDS up to 0.5% w / v and Proteinase K up to 50 [ig / ml were added; this mixture was dialyzed against buffer C at 37 ° C. overnight.

This was followed by two more phenol extractions. The aqueous phase was finally placed in a pretreated dialysis tube and dialyzed against 10 mM Tris HCl (pH 7.5), 10 mM NaCl for 1 h at room temperature and then at 4 ° C. for 4 days.

This product was then used as a donor E coli DNA preparation.

5. DNA restriction

DNA for the restriction and the restriction enzyme were in the ratio 1 [ig DNA: 1 [il enzyme in 10 mM Tris HCl (pH 7.5), 10 mM MgCl2.10 mM 2-mercaptoethanol, 50 mM NaCl, mixed. The mixture was incubated at 37 ° C for 45 minutes, followed by heating to 70 ° C over 10 minutes.

6. DNA ligation

5] J, 1 T4 polynucleotide ligase (Miles Laboratories) were mixed with 495 µl ligase buffer, the same volume of 616 mM Tris HCl (pH 7.5), 93 mM MgCl2, 560 mM NaCl, 93 mM dithiothreitol, 0.933 mM ATP, 9.33 mM EDTA, 0.93 mg / ml bovine serum albumin.

4 [ig restriction receptor phages, prepared as described in 5. were heated to 70 ° C. for 10 min and then rapidly cooled on ice. The product was mixed with 8% restriction donor DNA, prepared as described in 3 or 4, the mixture was heated at 30 ° C. for 15 minutes and stored at 0 ° C. for 24 hours. The mixture was then added to ligase buffer and incubated at 10 ° C. for 6 h and then left to stand at 0 ° C. for 6 days. After this time the mixtures were used in the transfection experiments.

7. Transfection

0.1 ml of a one day culture of E coli host cells on L nutrient medium was inoculated in 10 ml P medium and grown overnight. 2 ml of this culture was added to 25 ml of fresh medium and grown to an E 550 of 0.6. The cells were then cooled on ice for 10 min, crushed by centrifugation and resuspended in 0.5 volume of 0.1 M CaCl2. After 20 min on ice, the cells were smashed a second time and resuspended in 0.1 volume parts of CaCl2. After a further 20 min on ice, the cells were used for transfection.

The DNA for transfection was diluted in SSC to 3 µg per ml and mixed with the cells in a ratio of 1.0 volume of DNA: 2.0 volume of cells. The mixture was heat shocked at 37 ° C for 30 seconds and during kept on ice for another 2 h before pouring onto 0.6% soft agar containing 10 ~ 3M MgSO4

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was layered in L-agar dishes. The dishes were incubated overnight at 37 ° C and eluted the following day by overlaying with 4 ml of L nutrient medium and allowing to stand at 4 ° C overnight. The eluates were crushed, shaken with 0.5 ml chloroform and stored at 4 ° C.

P medium

25 ml P medium buffer; 0.02 M potassium phosphate (pH 7.0), 0.015 M (NH4) 2S04 0.25 ml 0.1 M MgS04 0.43 ml IO- * M Feso4 0.125 ml 20% glucose 0.1 ml 20% acid hydrolyzed casein + L-tryptophan, 50 [ig / ml final concentration.

SSC 8.76 g / 1 NaCl

4.41 g / l tri Na citrate (pH 7.0)

L-Medium Per liter: Difco Bacto Trypton, 10 g;

Difco Bacto yeast extract, 5 g;

NaCl, 5 g;

Glucose 1 g.

pH to 7.2

L-Agar L-Medium + Difco Bacto Agar to 15 g / 1

Soft agar L medium + Difco Bacto agar to 6.5 g / 1.

Host bacterium

The host bacterium E coli used in the transfection experiment was a derivative of E coli strain W3110, in which a functional tryptophan operon was missing and which also had no functional tryptophan regulatory gene, of the following composition:

trp R-, trp A-EV

In other experiments, trp_ derivatives of E coli strains KY Mei and C600 were used as host cells for the transfection.

8. Production of the agar layers

100 Iii of the phage eluate from each transfection layer and 100 μl of a suitable indicator bacterium (E. coli W3110, trp R-, trp A-EV) were mixed. 4 ml of soft agar at 46 ° C. were added to each mixture and the contents were immediately poured into petri dishes (7.5 cm in diameter). By moving the dish gently, the entire area was covered by the agar. The dishes were then incubated at 37 ° C. overnight.

Following the incubation, the layers were observed and the approximate number of infected sites was recorded on them. This number varied according to the transfection mixture from a few to an innumerable number. Using a small spatula, the edges of the agar mixture were carefully detached from the rim of the dish, a filter paper the size of which corresponded to the diameter of the petri dishes was placed on top and carefully pressed down to remove air bubbles. If the filter paper was not completely wetted by the agar layer, a few drops of distilled water were added. The filter paper with the agar layer adhering to it was then carefully removed from the petri dish. The agar layer was then placed on the flat glass surface of a 10 cm diameter disc, taking care to ensure that no air bubbles could form. The glass panes were then heated in an oven to 60 ° C. until the agar layers provided with the filter paper had dried.

After the discs had cooled to room temperature, the filter papers were wetted with a few drops of distilled water and the filter papers were peeled off, leaving the agar layer adhered to the glass plate. Any excess water was removed and the agar was then fixed in 10 ml of ethanol for 20 min at room temperature. The ethanol was poured off and the agar was washed with 10 ml of distilled water for a few seconds. The agar was then dried in a warm air stream.

The sites on the agar containing the antigen (trp A) produced were determined using a double antibody fluorescence method [although other antibody labeling methods would have been appropriate [e.g. Radioactivity (e.g. I125), enzyme (e.g. peroxide-ase), electron density (e.g. ferritin)], either with one designated antibody or with two antibodies, the second was designated. In the double antibody method, the first antibody (unnamed) is specific for the antigen and is elevated in one species, while the second antibody (labeled) is elevated in another species and is specific for the elevated antibody in the first species].

Production of trp A

The production of pure trp A for use as an immunogen in the production of antibodies against trp A was carried out as described below. 10 ml of a one-day culture of E coli W3110 (trp R ~, trp A am88, Sup III) in L nutrient medium was added to 1 liter of L nutrient medium together with 10 ml of 0.1 M MgS04 and the mixture at 37 ° C incubated in air until an E 550 of 0.6 could be observed. 1 x 1012 phage-trp Am 1 (trp A-E +, sus Q, NV, nin) were then added and the mixture was incubated for a further 4 h. The cellular sections of phage product were crushed by centrifugation (30 min, 4 ° C, 8000 g) and resuspended in cold (4 ° C) 0.1 M potassium phosphate buffer pH 7.0 (1.3 ml / g wet weight). The cells were crushed by ultrasound (4-6 fi, 5 x 20 sec at 1 min intervals), the temperature being kept at 4 ° C. by immersion in ice. 100 jil mercaptoethanol and 800 {il from IM. MnCl2 was added and the solutions mixed. The cellular residues were removed by centrifugation for 1 h at 4 ° C. The residue was discarded and 0.2 mg of disodium EDTA was added to the supernatant solution, which was stirred in ice for 10 min. The pH of the mixture was then adjusted to 4.5 with cold 1M sodium trium acetate (pH 3.8) and the mixture was stirred at 4 ° C. for a further 15 min. The mixture was centrifuged at 4000 g for 25 min at 4 ° C., the residue was discarded and solid ammonium sulfate was added to the supernatant until a concentration of 33 g / 100 ml was reached. The mixture was then stirred at 4 ° C. for a further 20 min. This was followed by centrifugation at 40,000 g for 20 min at 4 ° C., the supernatant was discarded and the precipitate was resuspended and dissolved in a minimal volume of cold 0.1 M potassium phosphate buffer pH 7.0. The solution was dialyzed against 0.05 M potassium phosphate buffer, pH 7.0 at 4 ° C for 2 h. The precipitate formed during dialysis was removed by centrifugation (40,000 g, 4 ° C, 20 min).

The supernatant solution was purified on a Sephadex G100 column (2 x 50 cm) with 0.05 m potassium phosphate buffer pH 7.0 at 4 ° C and 50 4 ml fractions were collected. The trp A was determined by analyzing 50 μl portions of the individual fractions. The 50 ml portion was mixed with 950 ml of the test mixture mentioned below and incubated for 20 min at 37 ° C. The test mixture consisted of L-serine (21 mg / ml, 3 ml) pyridoxalphosphate (100 ml / 1 ml), 1 M Tris hydrochloride pH 7.8 (1 ml), saturated sodium chloride (300 [il], indole (0.5 [iMole / ml,

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800 ni), water 2.4 ml and «trp B solution» (0.5 ml). The «trp B solution» was made from a one-day 2L culture (37 ° C, air supply) of E coli W3110 (trp R ~, trp Av) in L medium. The cells were crushed by centrifugation at 8000 g for 30 min at 4 ° C. in 600 ml of 0.1 M potassium phosphate buffer pH 7.0 and the like. centrifuged at 8000 g for 30 min at 4 ° C. The cells were suspended in 40 ml 0.1 M potassium phosphate buffer pH 7.0, containing pyridoxal phosphate (8 (ig / ml) (at 4 ° C.) and at 4-6 | i for 6 x 15 sec with 1 min intervals with Ultrasound with simultaneous cooling in ice, the cell residues were removed by centrifugation at 30,000 g for 30 min at 4 ° C. and the supernatant solution was used as “trp B solution”.

After incubation with the test mixture, 1 ml of indole reagent (9 g of 4-dimethylamino-benzaldehyde, dissolved in 200 ml of ethanol and mixed with 45 ml of concentrated hydrochloric acid) was added and the mixture was left to stand at room temperature for 20 minutes. A yellow color indicates the presence of trp A and a red color indicates the absence of the above.

The fractions containing trp A were combined and 43 g of ammonium sulfate per 100 ml were added. The mixture was stirred at 4 ° C for 20 min and then centrifuged at 40,000 g for 45 min and at 4 ° C. The supernatant solution was discarded and the precipitate resuspended in a small volume of 0.5 M potassium phosphate buffer pH 7.0 at 4 ° C. The solution was dialyzed overnight against 0.01 M potassium phosphate buffer pH 7.0 at 4 ° C.

The final cleaning step was carried out on a column loaded with DEAE cellulose (2 x 100 cm), prepared in 0.01 M phosphate buffer pH 7.0 at 4 ° C. The dialysate was added to the column with 50 ml of the same buffer and elution was carried out using a linear gradient consisting of 600 ml of 0.01 M and 600 ml of 0.3 M phosphate buffer pH 7.0 at 4 ° C. The fractions containing the trp A protein were determined and pooled in the same manner as for the first column. Then 44 g of ammonium sulfate per 100 ml were added and the mixture was stirred for 15 min. The trp A was collected by centrifugation (40,000 g, 45 min, 4 ° C) and the resulting precipitate redissolved in 0.05 M phosphate buffer pH 7.0 at 4 ° C. The solution was dialyzed against the same buffer overnight. The protein concentration of the solution was determined by the Folin reaction and adjusted to 1 mg / ml by dilution with the same buffer. This solution was used to immunize rabbits to produce antiserum against trp A.

Anti-trp A

The immunization method was carried out by emulsifying 500 ml of the trp A solution with 1 ml of Freud's complete adjuvant, and subcutaneously injecting the mixture into a rabbit at various locations (at least 5). The injections were repeated at monthly intervals and the animals were bled 10-14 days after each injection. The serum was collected and examined for precipitation zones in an immunodiffusion system (in 1% agar) at various dilutions against trp A solutions. The antiserum was used in immuno screening at 1/10 of the dilution, which revealed a visible precipitation zone.

Immuno screening

500 ml of the diluted anti-trp A were added to the dried agar layer and distributed evenly thereon. The mixture was incubated at room temperature for 30 min and then with phosphate buffer saline

(0.1 M, pH 7.2, 9 g / L sodium chloride) (10 ml, 3x1 min) and water (1 min). The agar was then dried with warm air.

500 (il of the second antibody (fluorescein designated 5 anti-rabbit immunoglobulin, derived from sheep, from Wellcome Reagents Limited) [in a dilution (approx. 1/20) determined in the same experiments and using anti-A phage as first antiserum) were then added sorely, evenly distributed over the layer, and incubated for 30 min. Excess antiserum was washed away as previously described and the layers were dried.

Prior to microscopic examination of the agar, it was covered with 1 ml of a lifting agent (3.16 g Na2C03 15 + 0.6 g NaHC03 in 100 ml water + 43 ml glycerol). The microscope was equipped with a quartz halogen lamp, a primary interference filter (Zeiss HP-500) and a pale yellow filter. The samples were observed using a dark field capacitor. 20 samples with positive reaction showed a fluorescent green color against a red / black background and samples without reaction were black. Positive reactions were initially observed with a frequency of approximately 1%.

Isolation of trp A phage 25 receptor phages A and B containing the trp A gene were isolated by two methods. First of all, the phage eluates were distributed from the transfection layers in such a way that about 200 infected sites were obtained per layer, the infected sites were then removed individually and 30 were eluted in 1 ml of L nutrient medium, and an aliquot of each was placed in a layer , with approximately 200 infected sites. Each layer was examined using the antibody technique as described above and positive reaction eluates were isolated. The second method of isolation was essentially the same, but an initial purification was by a dilution method. Aliquot parts of the eluates from the transfections were distributed in layers in such a way that approximately 10 infected sites were achieved per layer and each layer 40 was then eluted with 3 ml of L nutrient medium. Aliquots of these eluates were then placed in layers, yielding approximately 200 infected sites per layer, and these layers were then examined using the antibody method. Positive reactions were observed on some layers with a frequency of approx. 10%. The phages containing the trp A gene were then isolated from those eluates in which a higher frequency of positive reactions could be observed using the first isolation method. Finally, the phages with a positive reaction in the antibody test to the presence of the trp A gene in such a way that their property for transduction of suitable E coli trp A ~ auxotrophs and in the presence of trp A (by the trp A test as described above) in the supernatant solution after the lysis of suitable E coli trp A ~ Mutants was observed.

Examples 2 and 3 The procedure as described in Example 1 was repeated using E. coli strains KY Mei and C600 as host cells for the transfection.

Of course, the conditions in the above examples can be varied within a wide range, but without changing the basic procedure for carrying out the process according to the invention. Essential requirements are that the plasmid is capable of replication in the host organism and that the plasmid DNA in their

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biologically active form can be introduced into the host organism, in addition that the plasmid DNA has no more than 3 and preferably only 1 region shown for cleavage by a restriction enzyme of type 2, that the gene of interest has no region suitable for cleavage by the endonuclease used or this region is specifically protected; that the integration in the plasmids or in the bacterial genome has no manifestation of the specific gene of interest and that the poly-peptide product of the foreign gene which is introduced in the plasmids can be determined at very low concentrations by the described screening can. All of these requirements are more or less a matter of course for those familiar with the matter.

5 The specific application of the method according to the invention are the production of mammalian polypeptides and proteins, the production of bacteria by "foreign" enzymes and secondary metabolites, e.g. Glucose isomerase, and the production of compounds such as citric acid and io cephalosporin by modified tissue.

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Claims (5)

621147 1. Process for obtaining a cell culture which produces a desired polypeptide by modification of microorganisms, characterized by the following process steps a) splitting molecules of a DNA receptor material obtained from a plasmid which is suitable for infecting the cells of the said microorganism by treating the plasmid DNA with an endonuclease, b) splitting another genetic material containing DNA donor, coding for the synthesis of a desired polypeptide using a similar endonuclease, whereby DNA fragments are obtained, the ends of which are compatible with those obtained according to a), c) Mixing the DNA fragments obtained from the various sources, thereby randomly collapsing the DNA fragments under the influence of a DNA ligase, whereby hybrid DNA molecules are obtained which extract the combined fragments contain both DNA sources, d) infecting the cells of the microorganism to be modified by contacting them with the hybrid DNA molecules, e) have the infected microorganism metabolized in the presence of a substrate which is suitable for conversion into the desired polypeptide, and f) screening for the production of the desired polypeptide and production of a cell culture which produces the polypeptide. 2. The method according to claim 1, characterized in that the receptor DNA is a bacteriophage DNA. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that the donor DNA comes from humans or from mammals. 4. The method according to claim 1, characterized in that said infected microorganism is metabolized, whereupon the cell culture obtained is divided into fractions in which specific metabolites are detected by immunoscreening. 5. The method according to claim 4, characterized in that said immunoscreening step is followed by a refractionation and a further screening.