CH640268A5 - Process for the preparation of filamentous hybrid phages, novel hybrid phages and their use - Google Patents

Description

The invention relates to a method for producing filamentous hybrid phages, the new filamentous hybrid phages themselves and their use. In particular, the invention relates to a process for the production of new filamentous hybrid phages which are suitable as a vector for the production of synthetic recombinants with certain desired new metabolic properties.

It is known that the biological synthesis of desired metabolic products is controlled by the genetic information contained in the DNA. It is also known that certain microorganisms produced by these microorganisms, such as e.g. Antibiotics. Medicines, antigens and enzymes, can gain in a technically advantageous manner. However, these recovery methods can only be used where a microorganism that produces the desired metabolite is found. Many valuable metabolic products can therefore not be obtained in this way, since no suitable microorganisms are known. In addition, microorganisms are often known which form the desired metabolic product, but the microorganism itself has extremely unfavorable properties for breeding. Therefore one tries to develop methods to the genetic information for the

Formation of certain desired metabolic products in microorganisms, which do not yet contain this information, in such a way that new synthetic recombinants are formed which now have the desired metabolic properties and form the desired metabolic product, which can then be obtained therefrom. In principle, this process, which is referred to as “genetic engineering”, makes it possible to introduce any desired metabolic properties into a microorganism that is particularly suitable for cultivation and processing, thereby decisively expanding the possibilities of biosynthesis. The principle of these methods is that microorganism host cells with so-called vectors, i.e. infected with phage, phage DNA or plasmid DNA into which the genetic information for the desired metabolite has been introduced biochemically. The process of introducing and increasing the newly introduced genetic information in the host cells is called cloning. The host cell infected by the vector along with built-in genetic information can read the new genetic information introduced and1 form the corresponding metabolic product.

In practice, however, the implementation of such methods has proven to be very difficult. In particular, it has proven very difficult to obtain suitable viable vectors and to distinguish suitable vectors from unsuitable vectors.

The invention is therefore based on the object of providing a method for producing hybrid phages which does not have the disadvantages mentioned above or does so only to a considerably reduced extent.

This object is achieved according to the invention by a process for the production of filamentous hybrid phages which are suitable as vectors for the production of synthetic recombinants with certain desired new metabolic properties, which is characterized by the fact that the DNA of filamentous phages [D. A. Marvin et al., Bacteriol. Rev. 33 (1969) 172-209] statistically cleaves with an enzyme, the cleaved DNA with a first additional DNA, which contains the genetic information for the desired new metabolic property, is enzymatically linked in vitro, infected with the linked DNA host cells and that of the hybrid phage formed in the host cells wins. The process can be repeated with the incorporation of a second additional DNA different from the first. Restriction endonucleases are particularly suitable as the enzyme for cleavage.

The invention is based on the knowledge that, under certain conditions, filamentous phages are particularly suitable for being converted into vectors if their DNA is suitably cleaved, an additional DNA associated therewith, which carries genetic information which leads to an easy leads to detectable metabolites and then infects host cells with them. The prerequisite for the successful incorporation of genetic information, which leads to the appearance of the new, easily detectable metabolic property, is now a cleavage of the phage DNA at a point that is not essential for the viability of the phage. Since the cleavage takes place statistically and thus occurs both at essential and at nonessential locations, the method of the invention naturally selects DNA that has been cleaved at a location that is not essential for viability, since the easily detectable new metabolic property only occurs in such host cells infected with a replicable vector, ie in which the additional DNA was inserted into a nonessential cleavage site of the DNA.

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Essentially two vector systems have become known with which prokaryotic and eukaryotic DNAs can be cloned in E. coli, namely plasmids of the Col E 1 family or pSC 101 and the bacteriophage - X genome [cf. M. So, R. Gill and S. Falkow (1975) Mol. Gen. Genet. 142, 239-249],

According to D. A. Marvin et al. [Bacteriol. Rev. 33, 172-239 (1969)] in filamentous phages, the single-stranded phage DNA is converted into a double-stranded twisted form (RFI) after infection of the host cell by the phage and increased up to 300 copies / per cell. The phage-producing cells are not killed by the infection and can continue to multiply. The growth of a bacterial cell infected by a filamentous phage is slightly delayed, this delay leads to the formation of “cloudy plaques”, with which infected bacteria can be identified quickly and without selection pressure.

Although some methods for cloning DNA [cf. M. So, R. Gill and S. Falkow (1975) Mol. Gen. Genet. 142, 239-249], it has so far not been possible to clone DNA even in filamentous phages. The experiment described below shows that DNA cloning in filamentous phages is not possible with the means of the current state of the art. If you e.g. a double-stranded circular DNA of the filamentous phage M13 as in the case of the plasmids with the restriction enzyme Hind II, the only Hind II recognition site as a receptor for a DNA fragment which contains the information for the inactivation of the antibiotic kanamycin, and Escherichia coli after known methods [cf. M. So, R. Gill and S. Falkow (1975) Mol. Gen. Genet. 142, 239-249] with this in vitro product, Kanamycin-resistant phages are isolated, but are unstable and can only be propagated in the presence of a helper phage. They are therefore lost after a short time.

It was therefore not to be expected that filamentous phages could be used as vectors for cloning DNA. By using the new method of the invention, a location is found in the phage genome which is suitable for the in vitro recombination or cloning of DNA and leads to stable hybrid phages. These hybrid phages cause the expression of additional genes or the synthesis of additional metabolic products which the starting phage is unable to produce.

In the method according to the invention, the enzymatic cleavage is preferably carried out using the restriction enzyme Bsu I. For cloning (enzymatic linkage), preference is given to using T4 DNA ligase.

In principle, any DNA fragment can be used as the first additional DNA, which carries the complete information for the formation of the desired new metabolic property. Such a DNA fragment is preferably used, the genetic information of which leads to the formation of an easily detectable metabolic product, for example, detectable by a color reaction. As a result, when the filamentous phage DNA cloned with this DNA fragment is used as a vector, the host cells infected with viable hybrid phage DNA can be easily recognized by the presence of the new metabolite, for example by forming a color.

According to a particular embodiment of the invention, the Lac Hind II fragment is used as additional DNA, which contains the information for the synthesis of the a fragment of the β-galactosidase. If a host cell which lacks this a fragment is used, DNA β-galactosidase is formed when infected with such a hybrid phage according to the invention and can be easily detected by a known color reaction.

If the strain E. coli 17-18 (ATCC 31274) is used as the host cell in the context of this special embodiment of the invention, and this a fragment of the β-galactosidase is missing, the host cells infected with viable hybrid phages can be easily detected using the color reaction. isolate and with the help of these host cells in turn the new hybrid phages can be obtained.

If, in the preferred manner described above, the Lac Hind II fragment is used to clone filamentous phage DNA using Bsu I as a restriction enzyme and T4-DNA ligase for cloning, the host cell, such as that described below, is used to clone the new hybrid phage M13 mpl (ATCC 31274-B) was formed.

The present invention thus also relates to the new hybrid phage M13 mpl (ATCC 31274-B), which is characterized in the accompanying drawing with its physical and genetic characteristics. The enzymes Bsu I and Hind II are restriction endonucleases, which are described in detail in Bron, S., Murray, K. & Trautner, T.A. (1975) Mol. Gen. Genet. 143, 13-23; and Phillipsen, P., Streeck, R.E. & Zachau, H.G. (1974) Eur. J. Biochem. 45, 479-488.

The physical order of the Bsu I fragments is given outside the circle. The individual fragments are indicated by large letters and the Hind II fission point is chosen as the zero point of the map. Within the circle, the genetic order is indicated by Roman numbers. "X" represents the "X protein" in Gen II and I.R. represents the intergenetic region with the starting point for replication. The positions of the five G and the three A promoters are represented by long lines. One card unit corresponds to the length of 6400 base pairs. The map units are shown as the distance from the Hind II fission point per length of the M13 wild-type RF. The direction of the transcription on the genetic map runs counterclockwise [cf. L. Edens et al. (1976) Eur. J. Biochem. 70, 577-587]. The Lac fragment used is shown on an outer circle segment with the same scale division as the inner circle. Its orientation was clarified by the split pattern of the M13 mpl and M13 wild type RF with Bsu I and Hpa II:

I 'part of the lac repressing morning p lac-promotor o lac operator

Z '(a) a region of the β-galactosidase.

The new hybrid phage M13 mpl was launched on March 17, 1977 at the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Md / USA under ATCC no. 31274-B. The Escherichia strain 71-18 was also on March 17, 1977 under the ATCC no. 31274 deposited there.

As mentioned, the method of the invention makes it possible to find a cleavage site on the DNA of the filamentous phage (phage genome) which does not interrupt a base sequence which is essential for the viability (replication ability) of the phage. By using the restriction enzyme Bsu I according to the invention, it was possible to statistically cleave the RF-DNA of the filamentous phage M13 and, among many cleavage sites essential in the above sense, also a non-essential cleavage site (G. 0.08, see the gene map on the Drawing). The cleavage reaction is stopped in a manner known per se, e.g.

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by adding a denaturing agent such as urea or sodium dodecyl sulfate (SDS), preferably with 1% SDS. The reaction products are purified, e.g. by gel electrophoresis, preferably with agarose gels. The amount of restriction enzyme used can be metered in such a way that on the agarose gels e.g. mainly the linearized complete RF molecule, i.e. the DNA molecule that has been cleaved only once is recovered. This class of molecules is expediently cut out for recovery with the gel and thereby separated from smaller fragments or partial digestion products. For separation from the agarose e.g. the DNA of an equilibrium centrifugation, e.g. in potassium iodide (density 1.4 to 1.6 g / ml), the salt is dialyzed out and the DNA precipitated with alcohol. The linear DNA sequence of the phage obtained in this way represents the starting material for the incorporation of the foreign DNA with the regulatory region.

The linkage of the RF DNA linearized (cleaved) once with Bsu I in the manner described above to that by the method of A. Landy et al [(1974) Molec. genet. 133, 273-281] Lac Hind II fragment obtained from X-plac DNA by digestion with Hind II endonuclease is achieved with T4 DNA ligase. The reaction is e.g. in a ligase buffer (40 to 60 mM NaCl, 8 to 12 mM MgCl2, 10 mM DTT or ß-mercaptoethanol, 0.3 to 2 mM ATP, 20 to 80 mM Tris-HCl, pH 7.2 to 7.8) 4 to 15 ° C over a period of 7 to 48 hours.

Heteroduplex mapping and restriction enzyme analysis can show that the in vitro introduction of the foreign DNA takes place at a point in the phage genome that does not interrupt the sequence of a structural gene, nor does it start the transcription for the phage -specific proteins impaired.

The phage M13 mpl obtained according to the invention now has the following advantages over the already known filamentous phages:

The bacterial chromosome of E. coli strain 71-18 (ATCC 31274) e.g. the information for the lactose metabolism is missing. Instead, the lactose operon is located on an episome - a special genetic element in the cell - which is necessary for the infection of filamentous phages, with the following modifications: The first structural gene of the lactose operon, which contains the information for the ß -Galactosidase carries a deletion. The bacterial cell produces a stable trunk protein, but lacks the amino acids 11-41, so that it no longer has any enzyme activity. Furthermore, a mutation leads to the overproduction of lac repressor, which represses the expression of the structural genes.

If E. coli 71-18 is infected with a normal filamentous phage, the production of the trunk ß-ga-lactosidase is maintained after de-depression by adding IPTG (iso-propylthiogalactoside), which is an allosteric effector of the lac repressor. but ß-galactosidase activity is still not detected. However, if infection with M13 mpl occurs, the synthesis of a protein fragment of β-galactosidase is induced (controlled) by the in vitro built-in DNA after derepression with IPTG, which fragment can intracistronally complement the above-described trunk β-galactosidase. Enzyme activity of ß-galactosidase is indicated by hydrolysis of Xgal (5-bromo-4-chloro-indolyl-ß-D-galactoside), a colorless compound to 5-bromo-4-chloro-indigo, a deep blue dye. While after infection of E. coli 71-18 with filamentous phages, colonies remain colorless after sowing on indicator plates (with the addition of Xgal and IPTG), M13 stain

mpl infected colonies deep blue. Without the addition of IPTG, M13 mpl infected colonies also remain colorless, i.e. Transcription of the built-in DNA from the lac promoter is specifically switched off.

These results show that filamentous phages can absorb additional DNA in vitro using the described method and that a peptide that the host cells do not have is produced by the built-in DNA under the control of the regulatory region of the lactose operon.

These properties of the hybrid phage M13 mpl obtained according to the invention can be exploited in a particularly advantageous manner by subjecting the phage genome, that is to say its DNA, to a further cycle of the method according to the invention and thereby inserting a second DNA fragment which is different from that in the first process cycle to form it Phage built-in Lac-Hind II fragments is different. Cleavage with a suitable enzyme, e.g. another restriction enzyme, then again takes place in such a way that no essential base sequence is interrupted. The second DNA fragment, which for example carries the information for the formation of a desired drug, hormone, etc., is inserted in an analogous manner. There are two fundamentally different options. You add the DNA additively, so that in addition to the first one a second metabolic property can develop or you insert the second piece of DNA within the first stored piece of DNA. As a rule, the metabolic properties that depend on the first piece are destroyed. This has advantages in the case of the hybrid phage mpl, because it no longer produces a blue color, so that the installation of the second piece is easily recognizable. When host cells are infected with the vector obtained in this way, in which the new DNA has been cloned, host cells are obtained which, on the one hand, can be found very easily by the color reaction described above and, on the other hand, also the desired one which is controlled by the newly incorporated second DNA fragment Manufacture metabolic product.

The use of filamentous phages that has become possible according to the invention for the production of vectors is advantageous over the spherical phages previously used for several reasons. E.g. only a certain amount of DNA can be incorporated into the spherical phage for the given volume of the particle. This narrow limitation does not exist with filamentous phages. Rather, they become longer when new DNA is inserted, so that a multiple of the molecular weight of their DNA can probably be inserted into it.

In the context of the invention, host cells are understood to be all in the publication by D.A. Marvin et al. [Bac teriol. Rev. 33, 172-209 (1969)] described strains such as e.g. E. Colibacteria, pseudomons, salmonella, etc.

example 1

A. The starting lac Hind II fragment is obtained according to the instructions from A. Landy, E. Olchowski and W. Ross (1974) Molec. genet. 133, 273-281.

The following materials are required for this and in the exemplary embodiments:

DNA of the lac operon (e.g. X-plac DNA), M13 RF DNA, the restriction enzymes Hind II and Bsu I, T4 DNA ligase, the E. coli K12 strain 71-18 (A [Iac, pro], F 'lac Iq ZAM15 pro +), isopropylthiogalactoside (IPTG), 5-bromo-4-chloro-indo-lyl-ß-D-galactoside (Xgal), lac repressor.

Approx. 200 (ig X-plac DNA are digested with Hind II endonuclease and the reaction is ended by a heating step (10 minutes at 60 ° C.). 20 [ig lac repressor are added to the reaction mixture, and the solution is slow

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filtered through nitrocellulose filter (pore size 0.45 (i). Filter-bound DNA is eluted through an IPTG-containing solution (1 mM), phenolized, precipitated with alcohol and the precipitate in 50 μl TES buffer (20 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0) was added.

B. Hybrid phage M13 mpl (ATCC 31274-B)

The Ml3 RF DNA (20 (ig) is prepared as described [Marvin: Bacteriol. Rev. 33, (1969) 172-209], treated with Bsu I at room temperature and the reaction is stopped by adding 1% SDS. The reaction products are electrophoresed through agarose gels. The amount of enzyme is adjusted so that mainly the linearized complete RF molecule is found on the agarose gels. This class of molecules is cut out with the gel and separated from smaller fragments or partial digestion products. The DNA is removed from the agarose separated by equilibrium centrifugation in potassium iodide (density 1.5 g / m), the salt dialyzed out and the DNA precipitated with alcohol. After re-suspension in 50 μl of TES buffer, 2 μl of RF DNA linearized with Bsu I and 0, 5 (ig of the lac Hind II fragment

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diluted to a volume of 100 [xl in ligase buffer (66 mM NaCl, 10 mM MgCIa, 10 mM DTT (dithiothreitol), 0.5 mM ATP, 50 mM Tris-HCl, pH 7.5) and T4DNA ligase (1 (il , 8 nM final concentration) to the mixture 5. The ligase reaction is carried out at 12.5 ° C. for 16 hours.

Example 2

Verification of the hybrid phage in the bacterial cell io (identification of the hybrid phage by expression of the a fragment of the β-galactosidase).

The reaction mixture with the hybrid phage M13 mpl (ATCC 31274-B) is brought to 30 mM CaCl2 and exponentially growing E. coli 15 71-18 (ATCC 31274) 'washed with CaCl2 (30 mM) is mixed at ice temperature. After one hour, the cells are treated at 42 ° C for 2 minutes. The cells are then mixed with IPTG (1 mM), Xgal (40 (ig / ml) and 5 ml top agar and plated on standard nutrient medium. The agar plate is incubated for 24 hours at 20 ° C. Blue colonies or blue cloudy plaques become removed sterile from the agar and sown on individual colonies.

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Claims (12)

640268 1. A process for the production of filamentous hybrid phages, which are suitable as a vector for the production of synthetic re-combinants with certain desired new metabolic properties, characterized in that the DNA of filamentous phages is statistically cleaved with an enzyme, the cleaved DNA with a first additional DNA, which contains the genetic information for the desired new metabolic property, is enzymatically linked in vitro, infected with the linked DNA host cells and the hybrid phages formed by the host cells are obtained. 2. The method according to claim 1, characterized in that one uses as restriction enzyme Bsu I. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that one uses T4 DNA ligase for the enzymatic linkage. 4. The method according to any one of claims 1 to 3, characterized in that the Lac Hind II fragment is used as the first additional DNA. 5. The method according to claim 4, characterized in that the host cell used is E. coli 71-18 (ATCC 31274). 6. The method according to claim 1, characterized in that said method is repeated with the incorporation of a second additional, different from the first DNA. 7. Use of hybrid phages obtained according to claim 1 for the synthesis of metabolites by infection of host cells. 8. Use according to claim 7 for the synthesis of medicaments of protein or peptide character. 9. Use according to claim 7 for the synthesis of insulin and insulin derivatives. 10. Use according to claim 7 for the synthesis of antigens or antibodies. 11. Use according to one of claims 7 to 10, characterized in that the hybrid phage M13 mpl or ATCC 31274-B is used. 12. Hybrid phage M13 mpl or ATCC 31274-B obtained by the method according to claim 1.