GB1588572A - Process for the production of filamentous hybrid phages - Google Patents

Abstract

Filamentous hybrid phages are prepared which are suitable as a vector for the preparation of synthetic recombinants having certain desired novel metabolic properties. For this purpose, the DNA of filamentous phages is randomly cleaved using an enzyme, the cleaved DNA is linked enzymatically in vitro to a first additional DNA, which contains the genetic information for the desired novel metabolic property, host cells are infected with the linked DNA, and the hybrid phages formed by the host cells are recovered. If desired, the process is repeated with the incorporation of a second additional DNA which is different from the first one.

Description

(54) A PROCESS FOR THE PRODUCTION OF FILAMENTOUS HYBRID PHAGES (71) We, MAX-PLANCK-GESELL SCHAFT ZUR FORDERUNG DER WISSENSOHAFTEN E.V., of 10 Bunsenstrasse, D-3400 Göttingen, Federal Republic of Germany, a Company of the 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: The present invention is concerned with a process for the production of filamentous hybrid phages, with the new filamentous hybrid phages themselves, with the use thereof and with the metabolic products obtained therewith.

More particularly, the present invention is concerned with a process for the production of filamentous hybrid phages, which may be used as a vector for the production of synthetic recombinants leading to the synthesis of RNA's, polypeptides and new metabolic products and properties resulting therefrom.

It is known that the biosynthesis of desired metabolic products is controlled by the genetic information contained in the deoxyribonucleic acid (DNA). Furthermore, it is known that by culturing micro-organisms, certain metabolic products produced by these micro-organisms, i.e. pharmaceuticals, including antibiotics, antigens and enzymes, can be obtained in a technically advantageous manner. These production procedures can, however, only be applied in those cases in which a microorganism which produces the desired metabolic product is found. Many valuable metabolic products cannot, therefore, be obtained in this fashion because no suitable micro-organism is known. In addition, in many cases microorganisms are known which produce the desired metabolic product but the micro-organism itself has highly unfavourable properties for culturing.

Therefore, efforts have been made to develop methods to introduce the genetic information for the synthesis of certain desired metabolic products into-micro-organisms which do not yet contain this information in such a way as to create new synthetic recombinants which now possess the desired metabolic property and produce the desired metabolic product which can then be recovered from them. This process, which is called "genetic engineering", makes it possible, in principle, to introduce any desired metabolic property into a micro organism especially suitable for culturing and processing, thereby significantly broadening the biosynthetic potentialities.

The principle underlaying this method is ro infect micro-organism host cells with so-called vectors, i.e. phages, phage DNA or plasmid DNA in which the genetic information for the desired metabolic product has been bio chemically introduced. The process of intro ducing and reproducing the newly introduced genetic information into the host cell is called cloning. The host cell infected by the vector and its integrated genetic information can then read off the newly introduced genetic informa tion and produce the corresponding metabolic product.

In practice, however, the realisation of such procedures has proved very difficult. In particular, it has proved to be very difficult to produce suitable viable vectors and to dis criminate between suitable and unsuitable vectors.

It is an object of the present invention to provide a process for the production of hybrid phages which does not suffer from the abovementioned disadvantages or only exhibits them to a significantly reduced extent.

Thus, according to the present invention, there is provided a process for the production of filamentous hybrid phages which are vectors for the production of synthetic recominants leading to the synthesis of RNA's, polypeptides and new metabolic products and properties resulting therefrom, wherein the DNA of filamentous phages is randomly cleaved by an enzyme, the cleaved DNA is enzymatically ligated in vitro to a first additional DNA containing the genetic mformation for a desired new metabolic property, host cells are infected with the ligated (hybrid) DNA and the resulting filamentous hybrid phages and/or the new RNA's, polypeptides or metabolic products produced by the host cell are obtained and, if necessary, the process is repeated with the introduction of a second additional DNA different from the first one into the filamentous hybrid phage DNA leading to additional metabolic properties which may or may not modify the effect of the first inserted DNA.

Restriction endonucleases are especially preferred as the cleavage enzymes.

The present invention is based upon the discovery that, under certain conditions, filamentous phages are especially suitable for conversion into vectors if their DNA is cleaved in an appropriate manner, ligated to an additional DNA carrying genetic information leading to an easily identifiable metabolic product and then host cells are infected with it. A prerequisite for the successful integration of genetic information leading to the appearance of a new, easily identifiable metabolic property is the cleavage of the phage DNA at a site which is not essential to its viability. Since cleavage occurs randomly and thus may take place at essential as well as non-essential sites, a natural selection of DNA takes place during the process of the present invention which has not been cleaved at a site essential to viability, since an easily identifiable new metabolic property only appears in host cells which have been infected with a vector that can reproduce, i.e. in which the additional DNA has been integrated at a non-essential cleavage site of the DNA.

Essentially, two vector systems are currently known with which it is possible to clone prokaryotic and eukaryotic DNA in Escherichia coli, they being plasmids of the Col E 1 family or pSC 101 and the bacteriophage X genome (see M. So, R. Gill and S. Falkow, Mol. Gen.

Genet., 142, 239-249/1975).

According to D. A. Marvin et al. (Bacteriol.

Rev., 33, 172-209/1969), upon infection of the host cell, the single-stranded phage DNA of the filamentous phage is converted into a double-stranded supercoiled form (RF I) and increased to about 300 copies per cell. The phage-producing cells are not killed by the infection and can continue to reproduce. The growth of a bacterial cell infected with a filamentous phage is slightly retarded, thus lead ing to the formation of "turbid plaques", whereby infected bacteria can be detected rapidly and without selective pressure.

Although several procedures for cloning DNA are known (see M. So, R. Gill and S.

Falkow, Mol. Gen. Genet., 142, 239 249/1975), it has not been possible also to clone DNA in filamentous phages. That the cloning of DNA in filamentous phages is not possible with the methods of the current state of the art is shown by the following experiment. If, for example, as with plasmids, a double-stranded circular DNA of the filamentous phage M13 is cleaved once with the restriction enzyme Hind II and the single Hind II recognition site is used as a receptor for a DNA fragment which contains the information for the inactivation of the antibiotic kanamycin and Escherichia coli is transformed by known procedures (see M. So, R. Gill and S. Falkow, Mol. Gen. Genet. 142, 239-249/1975) with this in vitro product, kanamycin-resistant phages can be isolated. However, these are unstable and can only be reproduced in the presence of a helper phage. Consequently, they are lost after a short period.

It was, therefore, not to have been expected that filamentous phages can be used as vectors for the cloning of DNA. By using the process of the present invention, a site in the phage genome is found which is suitable for. the in vitro recombination and cloning of DNA and which leads to stable hybrid phages. These hybrid phages induce the expression of additional genes or the synthesis of additional metabolic products which the original phage cannot produce.

In the process according to the present invention, enzymatic cleavage is preferably effected with the restriction endonuclease Bsu I. For the enzymatic ligation, T4 DNA ligase is preferably used (see Weiss et al, J. Biol.

Chem, 243 (17), 4543--4555/1968).

Any DNA fragment can be used, in principle, as the first additional DNA which carries the complete information for the production of a desired new metabolic property. A DNA fragment is preferably used, the genetic information of which leads to the production of a metabolic product which is easily identifiable, for example by a colour reaction. As a result, in the case of the use of filamentous phage DNA as a vector ligated with this DNA fragment, host cells infected with DNA leading to viable hybrid phages can easily be detected because of the presence of the new metabolic product, for example due to a colour reaction.

According. to a preferred embodiment of the present invention, the lac Hind II fragment is used as additional DNA which contains the information for the synthesis of the ,obfragment of fi-galactosidase. If a host cell is used which is lacking this a-fragment, then upon infection with DNA of such a hybrid phage produced according to the present invention, ,ss- galactosidase activity can be easily detected by a known colour reaction.

If, in this preferred embodiment of the present invention, the strain Escherichia coli K12 strain 17-18 (ATCC 31274) is used as the host cell which lacks the fragment of ,ss- galactosidase, those host cells infected with viable hybrid phages can be easily detected and isolated because of the colour reaction and the host cells, in turn, can be used again to obtain the new hybrid phages.

If, in the preferred manner described above, the lac Hind II fragment is inserted into the DNA of the filamentous phage M13 using Bsu I as restriction enzyme and T4 DNA ligase, then the hybrid phage M 13 mpl (ATCC 31274-B) will be produced by using suitable host cells as described in the following.

The present invention thus also provides the hybrid phage M 13 mpl (ATCC 31274-B) which is characterised in the accompanying drawing by its physical and genetic features.

The enzymes Bsu I and Hind II are restriction endonuclases which have been described in detail by S. Bron, K. Murray and T. A.

Trautner (Mol. Gen. Genet., 143, 13 23/1975) and by P. Phillipsen, R. E. Streek and H. G. Zachau (Eur. J. Biochem, 45, 479 488/1974).

The physical order of the Bsu I fragments is shown outside of the circle. Fragments are indicated by capital letters and the Hind II cleavage site was taken as the zero point of the map. Inside the circle, the order of the genes is shown by Roman numerals. "X' represents the "X-protein" within gene II and I. R. represents the intergenic region with the initiation site for reproduction. Positions of the five G and the three A promoters are indicated by long bars. The map units are given as the distance from the Hind II cleavage site per length of M 13 wild-type RF. The direction of transcription is counter-clockwise on the genetic map (see L. Edens et al., Eur. J. Biochem., 70, 577-587/1976). The inserted lac frag ment is represented on an outer circle segment on the same scale as the inner one. Its orienta tion has been elucidated by the Bsu I and Hpa II cleavage patterns of M 13 mpl and M 13 wild-type RF: I' = part of the lac repressor gene p = lac promoter o = lac operator Z'( e zap region of fi-galactusidase The new hybrid phage M 13 mpl has been deposited with the American Type Culture Collection at 12301, Parklawn Drive, Rockville, Md./USA under ATCC/No. 31274-B, on March 17th, 1977. The Escherichia coli K12 strain 71-18 has also been deposited there under ATCC No. 31274, on March 17th, 1977.

As stated above, the process of the present 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 essential to the viability (reproduction) of the phage. By using the restriction enzyme Bsu I according to the present invention, the RF LNA of the filamentous phage M 13 can be randomly cleaved and a non-essential cleavage site could also be found among the numerous cleavage sites which are essential in the above-mentioned sense (G. 0.08, see the gene map in the drawing). The cleavage reaction is stopped in known manner, for example by the addition of a denaturing agent, such as urea or sodium dodecyl sulphate (SDS) and preferably with 1% by weight of SDS. The reaction products are purified, for example by gel-electrophoresis and preferably with agarose gels. The amount of restriction enzyme used can be chosen so that, for example, mainly the linearised complete RF molecule, i.e. the DNA-molecule which has been cleaved only once, is found on the agarose gels. As an appropriate means of recovery, this class of molecules is cut out of the gel and thereby separated from smaller fragments or partial digestion products. In order to remove the agarose, the DNA is, for example, subjected to equilibrium centrifugation in, for example, potassium iodide (density 1.4 to 1.6 g./ml.), the salt is then removed by dialysis and the DNA is precipitated with an alcohol.

The linearised phage DNA sequence obtained in this manner is the starting material for the integration of the foleign DNA with the regulatory region.

The ligation of the RF DNA, linearised (cleaved) with Bsu I as described above, and the lac Hind II fragment made according to the process of A. Landy et al. (Molec, gen. Genet., 133, 273-281/1974) from the A plac DNA by digestion with Hind II endonuclease, is achieved with T4 DNA ligase. The reaction is, for example, effected in a ligase buffer (40 to 60 mM NaCI, 8 to 12 mM MgCl2, 10mM DTT (dithiothreitol) or 8-mercaptoethanol, 0.3 to 2 mM ATP, 20 to 80 mM Tris-HCl, pH 7.2 to 7.8) at 4 to 15 C. over a period of 7 to 48 hours.

By heteroduplex mapping and restriction enzyme analysis, it can be shown that the in vitro insertion of the foreign DNA takes place at a site in the phage genome which neither interrupts the sequence of a structural gene nor affects initiation sites for the transcription of phage specific proteins.

The phage M 13 mpl according to the present invention exhibits the following advantages over the previously known filamentous phages: The bacterial chromosome of the Escherichia coli K12 strain 71-18 (ATCC 31274), for example, lacks the information for lactose metabolism. Instead, the lactose operon with the following modifications is on an episome (i.e. a special genetic element) in the cell, which is necessary for the infection with filamentous phages. The first structural gene of the lactose operon which carries the information for the uB-galactosidase has a deletion in the a-region. The bacterial cell produces a stable defective protein lacking the amino acids 11 to 41 so that it no longer possesses any enzymatic activity. A mutation also leads to the overproduction of lac repressor which represses the expression of the structural genes.

If Escherichia coli K12 strain 71-18 is infected with a normal filamentous phage, the production of defective 8-galactosidase is unaffected after derepression due to the addition of IPTG (isopropyl-thiogalactoside), which is an allosteric effector of the lac repressor, and ss-galactosidase activity is still not detected. If, however, the infection is effected with M 13 mpl, then, after derepression with IPTG, the synthesis of a p-galactosidase protein fragment is induced (controlled by the DNA inserted in vitro which is able to effect intracistronic complementation with the defective 8-galactosidase described above. The enzyme activity of the ,ss-galactosidase is shown by hydrolysis of Xgal (5 - bromo - 4 - chloroindolyl - galactoside), which is a colourless compound, to 5-bromo-4-chloroindigo, which is a deep blue compound. Whereas, upon infection of Escherichia coli K12 strain 71-18 with filamentous phages, the colonies seeded on indicator plates (with the addition of Xgal and IPTG) remain colourless, the colonies infected with M 13 mpl are coloured deep blue. With- out the addition of IPTG M 13 mpl, infected colonies also remain colourless, i.e. transcription of the integrated lac promoter DNA is specifically shut off.

These results show that filamentous phages can take up additional DNA in vitro by the process described and that the integrated DNA produces a peptide under the control of the regulatory region of the lactose operon, which the host cells do not possess.

These characteristics of the hybrid phage M 13 mpl according to the present invention may be used in a particularly advantageous way by subjecting the phage genome, i.e. its DNA, to a further cycle of the process according to the present invention and integrating a second DNA fragment which is different from the lac Hind II fragment integrated during the first cycle of the process leading to the formation of this phage. Cleavage with a suitable enzyme, for example another restriction enzyme, again takes place in such a way as not to interrupt any essential base sequence. The second DNA fragment which, for example, carries the information for the synthesis of a desired pharmaceutical or hormone is integrated in an analogous manner. In principle, there are two different possibilities of doing this. The DNA is linked in an additive manner so that, besides the first metabolic property, a second one can be expressed or the second DNA fragment is inserted within the first integrated DNA fragment. As a rule, the metabolic property directed by the first fragment is thereby destroyed or modified. In the case of the hybrid phage M 13 mpl, this would be of advantage since it-would no longer produce any blue colouring so that the integration of the second fragment is easily detectable. In the case of the infection of host cells with the vector produced in this way, in which the new DNA was inserted, host cells are obtained which can be easily found with the help of the change in the colour reaction described above and also produce the desired metabolic product controlled by the newly integrated second DNA fragment.

The utilisation of filamentous phages for the production of vectors, which is now possible according to the present invention, is, for several reasons, more advantageous than the spherical phages hitherto used. In the case of a spherical phage, for example, only a certain amount of DNA can be incorporated because of the predetermined volume of the particle.

There is no such strict limitation in the case of filamentous phages. By integrating new DNA, they become longer so that probably a multiple of the molecular weight of their DNA can be integrated therein. All the strains described in the publication of D. A. Marvin et al. (Bactenol. Rev., 33, 172-209/1969), for example, Escherischia coli bacteria, Pseudomonas and Salmonella, may be used as host cells within the scope of the present invention.

The following Examples are given for the purpose of illustrating the present invention: Example 1.

A. The starting lac Hind II fragment is made according to the method of A. Landy, E. Olchowski and W. Ross (Molec. gen. Genet., 133, 273-281/1974).

The following materials are required for this and in the specific Examples: lac-operon DNA (e.g. A-plac DNA), M 13 RF DNA, the restriction enzymes Hind II and Bsu I, T4 DNA ligase, the Escherichia coli K 12 strain 71-18 (A(lac, pro), F' lac I9ZaM 15 pro+), isopropylthiogalactoside (IPTG), 5-bromo-4-chloro indolyl-83-D-galactoside (Xgal) and lac repressor.

Approximately 200 ,ug. A-plac DNA are digested with Hind II endonuclease and the reaction is stopped by heating (10 minutes at 60 C.). 20 ,ag. lac repressor are added to the reaction mixture and the solution is slowly filtered through nitrocellulose filters (pore size 0.45). Filter-bound DNA is eluted with a buffer containing 1 mM IPTG, treated with phenol to receive the lac repressor protein, precipitated with alcohol and the precipitate dissolved in 50 ,ul. TES buffer (20 mM NaCI, 1 mM EDTA, 20 mM Tris-HCl; pH 8.0).

B. Hybrid phage M 13 mpl (ATCC 31274-B).

20 g. of M 13 RF DNA, prepared as described by D.A. Marvin et al. (Bacteriol. Rev., 33, 172-209/1969) is treated with Bsu I at ambient tempeiature and the reaction is stopped by adding 1% by weight SDS. The reaction products are electrophorised with the use of agarose gels. The amount of enzyme is adjusted so that mainly the linearised complete RF molecule is found on the agarose gels. This class of molecule is separated on the gel from smaller fragments or partial digests and cut out of the gel. The DNA is separated from the agarose by equilibrium centrifuging in potassium iodide (density 1.5 g./ml.), the salt is removed by dialysis and the DNA is precipitated with alcohol. After resuspending in 50 ,awl. TES buffer, 2 ,ag. Bsu I linearised RF DNA and 0.5 ag. lac Hind II fragments are diluted to 100 us. in ligase buffer (66 mM NaCI, 10 mM MgCl2, 10 mM DTT (dithiothreitol), 0.5 mM ATF, 50 mM Tris-HCl; pH 7.5) and T4 DNA ligase (1 ,awl., 8 mM final concentration) is added to the mixture. The ligase reaction is carried out at 12.5 C. for 16 hours.

Example 2.

Verification of the hybrid phage in the bacterial cell (identification of the hybrid phage by the expression of the a-fragment of '8-galactosidase).

The reaction mixture with the hybrid phage M 13 mpl (ATCC 31274-B) is brought to 30 mM calcium chloride and mixed in ice with calcium chloride (30 mM) - washed, exponentially growing Escherichia coli K 12 strain 71-18 (ATCC 31274). After one hour, the cells are heated to 42"C. for 2 minutes.

The cells are then mixed with IPTG (1 mM), Xgal (40 ,ag/ml.) and 5 ml. top agar and plated on standard media. The agar plate is incubated at 37"C. for 24 hours. Blue colonies or blue-turbid plaques are removed in a sterile manner and seeded for single colonies.

The present invention is also concerned with metabolic products, for example pharmaceuticals of a protein character, such as insulin, antigens and antibodies, whenever produced by infection of an appropriate host cell with a phage obtained by the process of the present invention, including the hybrid phage M 13 mpl (ATCC 31274-B).

WHAT WE CLAIM IS:- 1. A process for the production of filamentous hybrid phages which are vectors for the production of synthetic recombinants leading to the synthesis of RNA's, polypeptides and new metabolic products and properties resulting therefrom, wherein the DNA of filamentous phages is randomly cleaved by an enzyme, the cleaved DNA is enzymatically ligated in vitro to a first additional DNA containing the genetic information for a desired new metabolic property, host cells are infected with the ligated hybrid DNA and the resulting filamentous hybrid phages and/or the new RNA's, polypeptides or metabolic products produced by the host cells are obtained and, if necessary, the process is repeated with the introduction of a second additional DNA different from the first one into the filamentous hybrid phage DNA leading to additional metabolic properties which may or may not modify the effect of the first inserted DNA.

2. A process according to claim 1, wherein the cleaning enzyme is the restriction endonuclease Bsu I.

3. A process according to claim 1 or 2, wherein T4 DNA ligase is used for the enzymatic ligation.

4. A process according toany of the preceding claims, wherein the lac Hind II fragment is used as the first additional DNA.

5. A process according to claim 4, wherein a-compensation as specified by the use of Escherichia coli K12 strain 7118 (ATCC 31274) is used.

6. A process according to claim 1 for the production of filamentous hybrid phages, substantially as hereinbefore described and exemplified.

7. Filamentous hybrid phages, whenever produced by the process according to any of

Claims (1)

1. claims 1 to 6.

   8. The hybrid phage M12 mpl (ATCC 31274--B) as hereinbefore described and with reference to the accompanying drawing.

   9. Antigens and antibodies, whenever produced by infection of an appropriate host cell with a phage according to claim 7 or 8.

   10. Metabolic products, whenever produced by infection of an appropriate host cell with a phage according to claim 7 or 8.

   11. Pharmaceuticals of a protein character, whenever produced by infection of an appropriate host cell with a phage according to claim 7 or 8.

   12. Insulin, whenever produced by infection of an appropriate host cell with a phage according to claim 7 or 8.