GB2024229A

GB2024229A - Process for producing proteins by the expression of the corresponding ADN in bacteria, modified DNA and vectors applicable in such processes

Info

Publication number: GB2024229A
Application number: GB7920103A
Authority: GB
Original assignee: Institut Pasteur de Lille
Current assignee: Institut Pasteur de Lille
Priority date: 1978-06-08
Filing date: 1979-06-08
Publication date: 1980-01-09
Also published as: BE876881A; CA1203492A; CH652744A5; GB2024229B; NL7904517A; IT1166872B; DE2923297A1; IT7923414A0; FR2428075A1; FR2428075B1; JPS5550892A

Abstract

The invention concerns a vector, particularly a plasmid, containing a DNA sequence inserted in its genome, said DNA sequence coding for the major portion of a nutritional protein, particularly ovalbumin. The modified vector containing said DNA can be expressed in bacteria or yeasts. The proteins formed can be recovered from said microorganisms. The modified vector containing said DNA may in turn form a vector for the insertion therein of another DNA corresponding to a pre-determined protein, the expression of which may be sought in a micro-organism.

Description

SPECIFICATION Process for producing protein by the expression of the corresponding ADN in bacteria, modified DNA and vectors applicable in such processes The invention relates to a process for the production of proteins by micro-organisms, notably nutrient proteins, for example of the type which comprise the amino acid sequence of ovalbumin, this process bringing into play genetically modified plasmids. It also relates to these genetically modified plasmids themselves, as well as to the proteins produced.

It is known that the insertion in vectors such as plasmids of functional messenger RNA transcripts into their natural host cells and expressed in the form of a predetermined protein, has already been done successfully. For example, Humphries et coll.

(NUCLEIC ACIDS RESERACH, 1977, vol. 4, p. 2389) have described the insertion into a plasmid (pCR1) of a double strand DNA derived from the transcription of a purified RNA messenger of chicken ovalbumin.

The thus purified plasmid pCR1 ov 2-1 could be introduced into strains of Escherichia coli K12 by gene construction methods which have become conventional. No ovalbumin sythesis has however been demonstratable among the metabolism products of the bacterial strains used. In this regard, this experience confirms the difficulties encountered until now, and not yet overcome in practice, at the level of the expression of a higher eukaryot gene in bacteria harboring a vector including such a gene inserted in its genome. It must however be stressed that there is one exception to this still negative general situation.It relates to the expression which has been carried out recently of somatostatin in Escherichia coli bacteria converted by a plasmid comprising in its genome the product of the genetic fusion of a synthetic gene of somatostatin and of a gene of the lactose operon of E. coli. The experiments relating thereto have been described in an article entitled "Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin", published by Keiichi Itakura et coil. in SCIENCE, Vol. 198, pp 1056-1063 of 9 December 1977.

It may however be noted that the authors of this publication knew how to provide themselves with conditions particularly favorable for inducing the production by E. coli of a hybrid protein including a fragment formed from the 14 amino acids from which somatostatin is normally constituted. They resorted to a synthetic gene, constructed chemically so as to avoid the difficulties to which the translation by the bacterium of a natural gene could give rise.

In addition, these authors have themselves indi cated that the synthesis of somatostatin only takes place in practice if the above-mentioned synthetic gene is linked to the major portion of the Z gene of the ss-galactosidase of the lactose operon. If the gene of the somatostatin is only linked to the portion corresponding to the fragment of the lactose operon (comprising the "p", "o" genes and the 8 coding base triplets for the first amino acids of the bacterial P-galactosidase), hereafter designated as "lac", no somatosynthesis is detected.

As a result, the fragment corresponding to the somatostatin only represented, under the most favorable experimental conditions described by these authors, a small part of the expressed hybrid protein, the largest part of the latter consisting of a fragment having the amino acid sequence of fr galactosidase. It is possible to consider that the somatostatin fragments occurred, with respect to the whole of the hybrid protein formed, in a ratio of the order of 14 amino-acids (those of somatostatin) to a figure of the order of 1000 amino-acids.

It is an object of the invention to provide a process for the production of a prokaryot or eukaryot protein, bringing into play the expression of a suitable DNA of much larger size than that of the above-indicated synthetic gene of somatostatin, of greater economic efficiency in that the largest part of the protein expressed will be constituted by the protein fragment sought. It is also an object of the invention to provide a process enabling the expression by a bacterium either of DNA produced by enzymatic synthesis, notably from an RNA, or of a natural gene.

It is also an object of the invention to produce vectors, more particularly of the plasmid type, into which may be incorporated gene fragments whose expression is sought at the level of a microorganism.

It obviously also relates to the final vectors thus obtained.

A further object of the invention is to provide particular DNA fragments capable of coding the production of an eucaryotic or procaryotic protein subject to their preliminary insertion into an appropriate vector including vectors capable of transforming yeasts.

The plasmid according to the invention comprises in a non-essential part of its genome, a DNA fragment derived itself from aX phage fragment, linked to a bacterial operon fragment, notably of a lactose operon, comprising at least the bacterial promotor and a fragment of the gene linked to this promotor, such as the Z gene coding the production of ss-galactosidase in the lactose operon, this gene fragment being possibly limited to the first one or more nucleotide triplets coding the first one or more amino acids of the corresponding protein, ss- galactosidase in the case of the lactose operon, this plasmid being also characterized in that it is provided, at the level of said gene fragment (n Z), with a cleavage site by a predetermined endonuclease, preferably to the exclusion of any other cleavage site by the same endonuclease in the other parts of its genome.

Advantageously, the cleavage site concerned is a site cleavable by the EcoRI enzyme.

In the preferred plasmid of the invention, the bacterial operon fragment is a lactose operon fragment and it includes at least the sequence of the Z gene adapted to code the 7 first amino-acids of the bacterial ss-galactosidase (or the 8 first amino acids if one takes into account formyl-methionine as the first amino acid).

A preferred plasmid according to the invention is contituted by a recombinant of, on the one hand, the DNA of the pBR322 plasmid described by Bolivar et al, 1977, GENE, 2,95, from which a non-essential fragment was separated by the action of EcoRI and Hind III enzymes, and, on the other hand, a phage fragment coupled to a lactose operon fragment, the latter comprising the promotor, the operator and the Z gene fragment coding the 7 (or 8) first amino-acids, as defined abdve. Preferably the operator is not sensitive to catabolic repression.

It is possible to incorporate into such a vector an DNA fragment corresponding to the protein whose expression is sought, which gene fragment may be either synthesized chemically, or synthesized by an enzymatic route, notably from an RNA messenger, or also be a natural gene, this gene being in addition capable of having a very much greater size than that of the gene fragment forming part of the bacterial operon as defined above. The vector obtained forms also as such a part of the invention.

In a preferred embodiment of the invention as hereafter described and aiming at the production of a hybrid protein comprising most of the sequence of the ovalbumin amino-acids, use is made preferably of an DNA fragment synthesized by the enzymatic route starting from the corresponding messenger RNA. The latter may be obtained for instance through enzymatic transcription according the method described by HUMPHRIES et al, referred to hereabove. The possibility of causing said DNA fragment to be expressed in a bacterium, even in a yeast, after its preliminary insertion into an appropriate vector, is all the more remarkable as it seems that the same technique is not applicable to the natural ovalbumin genes. It has already been indicated that the genes coding for ovalbumin are formed of DNA sequences separated from one another by noncoding sequences called "introns".

Thus it is possible to incorporate into the vectors according to the invention the greater portion of the gene of ovalbumin and to cause the recombinant thus obtained to be expressed in a microorganism, such as a bacterium, notably E.coli so that it is possible to obtain the production of a hybrid protein of which the major part corresponds to the aminoacid sequence of ovalbumin.

The invention concerns more generally a plasmid as hereabove defined wherein the foreign DNA fragment comprises a fragment capable of coding the production of a nutritive protein in translation phase with the above said Z gene fragment.

More preferably the invention concerns the so modified plasmids obtained, particularly those resulting from the recombination of the abovesaid preferred plasmid and of a DNA fragment capable of coding the major part of ovalbumin. Preferably that modified plasmid comprises a DNA fragment resulting from the combination of the sequence comprising the triplets of those nucleotides which are capable of coding the translation of the 8 first amino-acids, inclusive formly-methionine, of the bacterial ss-galactosidase, on the one hand, and a sequence capable of coding the major portion of ovalbumin, possibly except for the fist amino-acids thereof (particularly the first 5 amino-acids), linked to the preceding sequence, either directly, or by means of a whole number of triplets selected among those which are not liable of interrupting the translation, on the other hand.

The invention further relates to the vectors formed by plasmids capable of transforming yeasts, the latter plasmids comprising inserted in their own genome at least part of the preceding plasmid, it being understood that said part of plasmid will necessarily include the hereabove defined DNA recombinant.

The invention is applied particularly advantageously to the synthesis of nutrient proteins, of which ovalbumin is only one example. The process is particularly advantageous in that the major part of the protein thus capable of being expressed is constituted by "useful" protein from the nutrient point of view, without it being necessary to proceed with the separation of this "useful" fragment from the fragment corresponding to the first amino-acids of p-galactosidase.

In particular, it will be noted that in the case of ovalbumin which is formed from about 385 aminoacids, 380/387 (expressed in number of amino-acids) of the hybrid protein finally obtained will be constituted by the protein sought.

Thus invention concerns in a general manner plasmids of the type as hereabove defined in which the DNA sequence corresponding to ovalbumin may be replaced by any sequence capable of coding the translation of any other nutritional protein, it being understood that if need be one or two base pairs (if need be completed by a whole number of triplets) may have to be inserted between on the one hand the fragment comprising the triplets coding for at least part of the amino-acids of ss-galactosidase and, on the other hand, the DNA sequence coding for the production of the nutritional protein, for the sake of causing the 2 fragments to be in proper phase when the later translated.

The invention therefore also provides a process for manufacturing a hybrid protein of which a part belongs to a protein normally coded in a prokaryot or eukaryot natural host-cell by the corresponding gene belonging to the genome of this latter cell, which process consists of introducing into a microorganism, such as a bacterium, notably E. coli, or as a yeast, such as Saccharomyces cerevisiae, a recombinant plasmid resulting from the insertion by genetic fusion of the above-mentioned predetermined DNA in the plasmid according to the invention, more particularly in the Z gene fragment (or the corresponding gene fragment in the case of a different bacterial operon) or immediately contiguouslyto the latter, the hybrid protein containing the desired fragment then being recoverable from the cellular proteins formed by said bacterium.

Advantageously, the DNA fragment characteristic of the desired protein has a size, expressed in number of nucleotides, higher than that of the fragment of the Z or similar gene fragment. This ratio is advantageously at least equal to 50 (of the order of 387/7 in the example concerned).

The invention also concerns the new products obtained, namely: - the proteins comprising a sequence of amino-acids as contained in ovalbumin, however essentially free of glycosylated groups of the type contained in the natural protein; - the corresponding DNA fragment which comprise, on the one hand a DNA sequence corresponding to at least part of the ss-galactosidase protein, starting with the first amino-acid thereof, inclusive (formyl methionine, more particularly those comprising the triplets corresponding to the 8 first mentioned amino-acids of B-galactosidase and, on the other hand, the whole or the major part of the DNA sequence such as the whole ovalbumin except for the first amino-acids, notably the 5 first amino acids thereof), the latter DNA sequence being linked to the former DNA sequence, either directly or through a whole number of triplets.

More generally the invention concerns a DNA sequence in which are associated, on the one hand, A DNA fragment corresponding to a portion of the lactose operon including the promotor and a fragment of the associated gene Z and, on the other hand, a DNA sequence capable of coding a nutritional protein in phase with the preceding fragment.

Preferably the DNA fragment which codes for the major part of the nutritional protein, particularly ovalbumin, consists of a DNA fragment resulting from the enzymatic transcription of the corresponding messenger RNA sequence.

Another characteristic of the modified protein according to the invention resides in its capacity to be detected by the antibodies of the natural protein, in current radio-immunological assays.

The invention also relates again to the strains of micro-organisms, particularly bacteria, notably E.

coli, or yeasts, notably Saccharomyces cerevisiae, containing the plasmids according to the invention and permitting their replication simultaneously with their own division.

Other characteristics of the invention will appear also in the course of the description of an example of the production of plasmids according to the invention and of their applications to the production of a nutrient protein, essentially characterized by a sequence of amino acids substantially identical to a part at least and preferably to the greater part of chicken ovalbumin.

Reference will be made in the course of this description to the drawings in which the transformations to which the initially treated plasmid gives rise are represented diagrammatically.

EXAMPLE: 1) Construction of the vector, It is proposed to incorporate in a non-essential part of the plasmid pBR322 (Figure 1a) already mentioned, a DNA including a X phage fragment bearing a lactose operon segment, with a site of cleavage by EcoRI situated at the level of the 7th amino acid of the & alactosidase (formylmethionine not included).

The latter DNA was obtained in the following manner: As a start there is used X phage in which there has been incorporated by genetic fusion, a lactose operon segment such as has been described by BACKMAN et coll. in PROC. NATL. ACAD. SCI. U.S.A. (sol. 73, No. 11, pp 4174-4178, November 1976, "Genetics"). The genome of this modified phage comprises an EcoRI-Z site in the Z gene of its lactose operon segment.

In figure 1 is shown a diagrammatic map of the X phage, showing therein the principal non-essential part NE of the natural phage, which has been modified. The numbers indicated in Figure 1 refer to the indication of the scale used, expressed in percentages of length starting from the genes of the head of the phage.

The abovesaid lactose operon segment contains the operator, the promotor and the coding information for the first amino-acids of the ss-galactosidase (with L8 and UV5 mutations). This fragment had been inserted in vitro into the EcoRI site situated at the end of the Z gene of aX plac5 phage (this phage had no other site sensitive to EcoRI).

Shown diagrammatically in Figure 2, is the approximate positioning of the lactose operon segment (rectangle Z) in this phage.

By the technique described by the abovesaid authors, there follows the insertion in this EcoRI-Z site (shown diagrammatically by the corresponding arrow of Figure 2) of an OP Hind lil 203 fragment also described by these authors (Figure 3a). The latter fragment includes, on opposite sides of a central portion OP, on the one hand, a fragment Z' coming from the start of the Z gene and corresponding to the seven first amino-acids capable of being coded by the Z gene, and on the other hand, a terminal I" fragment of the lactose operon repressor.When the OP Hind lit 203 fragment (shown at a reduced scale in Figure 3b) is inserted in the same sense as the homologous OP fragment and close to the terminal Z" fragment of the Z gene of the phage (indicated by the reference "op" in Figure 3b and close to a terminal fragment I" of the I gene of the lactose operon repressor), intramolecular recombination creates a deletion (Figure 3c) of almost the whole of the Z gene. This permits, also, the creation of an EcoRI site (EcoRI- A Zsite) in the place of the Hae Ill site, very close to the origin of the Z gene at a site corresponding to the 7th amino-acid of the ss- galactosidase. Of the initial Z gene there only subsist finally the fragments Z" and Z', on both sides of the EcoRI AZ site.

In Figure 2, there is also shown the site of cleavage by the Hind III enzyme closest to the lactose operon segment on the DNA of bacteriophage 2. This site is called s Hind Ill X2.

The vector thus obtained has been cut according to techniques known in themselves by the enzymes EcoRI and Hind III. By a method known in itself, the phage fragment is collected, including here the Z' fragment, having one end (on the side oft'), corresponding to the EcoRI-n Z cleavage site and whose opposite end corresponds to the sHind ill cleavage site. This identification can be carried out for example by taking advantage of the capacity of this fragment to act as an inductor of the lactose operon in a E.coli K12 Z strain, on a colored medium, notably on a chromogen substrate based on 5-chloro 4-bromo-3-indolyl-ss-galactoside.

The DNA of the plasmid pBR322 is itself cleaved by the enzymes EcoRI and Hind Ill atthe level of its corresponding cleavage sites.

The recombination in vitro follows of the fragments of DNA of the plasmid pBR322 and of the X phage fragments bearing the lactose operon segments, in the presence of a DNA-ligase, notably of that extracted from the phage T4 and one isolates by cloning in E.coli K12, notably E.coli K12 C600 rk mk+ according to a conventional method, for example that described in Humphries et coll. already cited, the desired vector shown diagrammatically by Figure 4b. This vector comprises the essential part A of the DNA of the initial plasmid and the inserted part B of the DNA comprising the fragment of AX phage and the fragment of lactose operon (E Z, o,p(i)) ((i) denoting a fragment only of the gene (i)).

The novel plasmid thus obtained, named p1397, bears the lactose operon segment indicated above, with a single site of cleavage by EcoRI at the level of the 7th amino acid (or of the 8th if counting formyl-methionine as such) of the ss-galactosidase.

The novel plasmid will also hereafter be referred to as "p OMPO".

2) Construction ofthe ovalbumin recombinant It is formed from the above-mentioned modified plasmid and the fragment Hha ov, defined in Humphries et al., 1977. It is a bicatenary DNA fragment cut up by the restriction enzyme Hha 1 from the pCRI ov 201 plasmid and isolated by sedimentation in a saccharose gradient (this fragment Hha ov, which comprises about 2400 pairs of bases, carries the greater part of the sequence corresponding to the messenger RNA of the ovalbumin). It is shown diagrammatically in Figure 4c in the drawing (reference Hha ov).

The plasmid p1397 is opened by EcoRI and treated by the specific nuclease of single strand SI DNA, so as to trim the cohesive short ends produced by EcoRI (Humphries etal, 1977).

The DNA obtained shown diagrammatically at 4d, is treated with DNA polymerase I of E. coli in the presence of the 4 deoxytriphosphates dATP, dCTP, dGTP and dTTP (as described in Humphries et al, 1977).

The Hha ov fragment is, in the same way, treated by DNA polymerase I of E. coli in the presence of the 4 deoxytriphosphates.

These enzymatic treatments enable the production of modified DNA molecules devoid of the cohesive ends and lending themselves to ligaturation by the DNA ligase of the T4 phage applied in excess (Itakura K., Hirose T., Crea R., Riggs A.D., Heynecker, H.L., Bolivar F., Boyer H.W. (1977) SCIENCE, 198, 1056).

Among the recombinant molecles obtained, there are isolated those whose coding sequence for ovalbumin (with the exception of the 15 first pairs of bases coding the 5 first amino acids) is fused with the 7 first amino acids of the bacterial ss- galactosidase (or the 8 first amino acids if one counts formyl-methionine (Figure 4e)). The strain which has finally been retained for the further experimentation hereinafter disclosed has been designated as p OMP 2.

This isolation is advantageously carried out by resorting to the sorting method which consists of introducing the recombinant molecules into a lysogenestrain of E.coli K12, bearing a thermoinducible prophage, and when this strain has formed colonies, inducing the lysis of a portion of the individuals which compose said colonies, the lysed individual DNA then being transferred by contact to a separate support, notably a cellulose filter, on which the desired DNAs are then detected in situ by hybridation with a DNA radioactive probe, at a spot which is then correlatable with the location in the initial culture of the micro-organism which has produced this intracellular constituent.This isolation can naturally be carried out by any other known method of detection.

The non-lysed strains belonging the the colonies responding positivieiy in this system (hence bearers of the ovalbumin sequence) are isolated and the plasmids retransferred by transformation into another non-lysogenic strain of E.coli K12 (C600 rkmk+), according to the technique described by Rougeon F., Kourilsky P., Mach B. (1975), NUCLEIC ACIDS RESEARCH, 2, 2365). These strains are cultivated in a broth and the extracts are made by sonication.

To this end, the bacteria are frozen in the culture medium, then thawed and subjected to ooh to an ultrasonic treatment sufficient to open them (three times 20 seconds under our conditions). A crude extract is thus-obtained, which can if necessary then be fractionated.

The presence of antigens similar two ovalbumin has been detected by radio-immunological analysis. In this assay, there is made to enter into competition the bacterial extract of certain strains with the I 125-marked ovalbumin, to form complexes with a very specific anti-ovalbumin antibody.

Radio-i mmunological assay indicates the presence of from 5000 to about 50 000 molecules of antigen per bacterium. The affinity of the antigen produced by the bacterium with respect to the anti-ovalbumin antibodies does not appear very different from that of the natural ovalbumin.

The size of the antigen has been measured by electrophoresis of crude extracts labelled with S35 and immuno-precipitated.

The hybrid molecule has the same apparent molecular weight as ovalbumin (about 43000 daltons).

It differs from the ovalbumin formed in chicken egg by a small number of modifications carried out after synthesis of the protein. In fact, natural ovalbu - min is phosphorylated in two places and glycosylated at one place. The absence of glycosylated groups is not of a nature to modify the nutrient properties of the protein obtained.

The theoretical sequence of the modified ovalbumin is the following : formyl-methionine - threnonine - methionine isoleucine - threonine - aspartic acid - serine - leucine - alanine - alanine-serine - methionine - glutamic acid...., the 8 first amino-acids coming from the -galactosidase and the 5 following ones forming the first amino-acids of the ovalbumin fragment Hha ov (in which the 5 first natural amino-acids of the ovalbumin are also lacking).

The plasmid P1397 strainwas deposited on June 2, 1978 in the National Collection of Cultures-of Microorganisms (C.N.C.M.) at the PASTEUR INSTI TUTE under No. 1-064.

The plasmid so obtained,more particularly the portion of this plasmid which contains the sequence modified with DNA coding for ovalbumin, may be incorporated into a plasmid which itself is liable of transforming yeast strains, notably Saccharomyces cerevisiae. The modified protein is then also capable of being expressed in these yeasts. Thus in contrast with what one could have expected, the fragment carries no marker liable of inducing in said yeasts a restriction mechanism with respect to the fragment of bacterium origin.

The construction of a modified plasmid capable of transforming a yeast, its amplification, the transformation of a yeast in the modified plasmid and the detection of the expression of this modified plasmid in the yeast are briefly set forth herebelow.

1 ) Extraction from the plasmid pOMP2 of the lac-ovalbumin sequence pOMP2 is opened by the restriction enzyme Hhal.

Two cleavage sites liable of being recognized by that enzyme, on the two opposite sides of the Hha ov fragments are shown on figure 4e. After re-insertion of this fragment into another vector, it is detectable owing to its capability of rendering the bacteria transformed by it lac-constitutive (the latter transformed bacteria then being liable of producing large amounts of -galactosidase and being detected in that they form colonies in the presence of the X-gal dye (5-bromo-4-chloro-indolylgalactoside-).

Particularly this operation has been carried out as follows: 20 F of pOMP? DNA were digested to completion by Hhal (Biolabs; 25 units ; 30 m. ; 37"C) and the reaction was stopped by heating to 65"C for 10 m. in the presence of 10 mM EDTA. The reaction mixture was loaded on a 5-20 per cent sucrose gradient (w/v in 25 mM Na Acetate pH 6.0, 10 mM EDTA, 0.5 per cent SDS) and centrifuged at 31 000 rpm for 16 h at -20 C in a BECKMAN rotor ultracentrifuge of the SW 41 type. The 2.67 kb (kilobases) fragment carrying the fused lac-ovalbumin sequence could thus be purified from the other much smaller fragments of pOMP2.

2") Construction of the 'plasmid 8" containing the "Eco Rl-D"fragment This plasmid is obtained by recombination in vitro of a plasmid pBR322 comprising a genetic marker Ura+ (described by M.L. BACH, F. LACROUTE and D.

BOTSTEIN P NA S, 1979, vol. 176, n" 1 pp.386 - 390 which had previously been opened by Eco RI, and of thefragment"Eco RI-D" (described by CAMERON et al. Nucl. Ac. Research, 1977, vol. 4, no 5, pp.1429- 1448, originating from "plasmid 2 " which is known to have a capability of trans forming yeast such as Saccharom yces cerevisiae.

This recombination is carried out by any known method, for instance such as that described by HINNEN etal, P NAS 1978, vol. 75, n 4, pp.1929 1933.

The "plasmid 8" strain was deposited at the C.N.C.M. under Nr. I - 093 on May 1979.

3 ) Construction ofa recombinant of "plasmid" ~ and of the pOMP2 fragment containing the "/ac ova/bumin" 30 clog of plasmid 8 DNA were cut by 1.5 unit of Hha during 5 minutes at 37"C. The reaction was stopped by heating 10 min. at 65"C in the presence of 10-2 M EDTA. The linear molecules were purified by centrifugation on a 5-20 per cent saccharose gradient (10-2M tris HCI, 10-3M EDTA, 10-1M NaCI) for4 hours under 40 rpm at 20"C in a BECKMAN rotor of the SW 41 type.

200 ng of the 2.67 kb lac-ovalbumin Hha/fragment and 600 ng of linearized plasmid 8 were ligated with T4 DNA ligase at 4"C in a final volume of 5 Fl in 40 mM Tris pH 7.5, 10 mM MgCl2, 10 mM DTT, 0.1 mM ATP, 0.05 mg/ml BSA. After transformation of CaCI2 treated lysogenic cells of strain 1398 (CNCM n" 1-094 deposited on May 27, 1979) transformants were selected on ampicillin containing plates (20Fg/ml), and screened by in situ hybridization with a labelled DNA probe specific for the ovalbumin sequence (the Hha ov probe). Positive colonies were then tested for ovalbumin production in the same radio-immuno assay as before.All the clones positive in in situ hybridization were also positive in this assay.

4 ) Amplification and recovery of theplasmid A colony of bacteria E.coli C 600 rk~mk+ inductible into lac-constitutive bacteria is transformed with the DNA fragments isolated during the preceding operation. After recovery of the colonies and identification of lac-constitutive bacteria formed, the latter are cultivated and the plasmids are recovered from the grown bacteria on making use of classical techniques for rupturing the bacteria, recovering the DNA contained therein and separating the band of circular plasmid in a gradient of coesium chloride in the presence of ethidium bromide.

5 ) Transformation of the yeast Saccharomicae cerevisiae {CNCM Nr 1-095 deposited on May 27, 1979.

The abovesaid strain which is Ura- is transformed by the abovesaid plasmid and cultivated on a medium free of uracil. Only those of the cells which have been rendered Ura+ by the said plasmid will develop into that medium.

The production of a hybrid protein containing most of the ovalbumin sequence is detected by means of the radio-immunological assay of the type already disclosed hereabove in the yeasts containing said plasmids.

The vector according to the invention containing the gene of ovalbumin can, in turn, serve as a vector for manufacturing other hybrid proteins including a separate protein; this can be carried out by insertion in the ovalbumin gene fragment, replacement thereof by- or addition thereto of an RNA transcript fragment corresponding to the desired protein and expressed in the form of this protein in its natural host-cell. By way of example, the gene of somatostatin (or of another protein) can be inserted therein.

This application is all the more interesting as it has been found that most of the hybrid protein containing ovalbumin as expressed in the bacteria by the above-defined vector, is freed in the periplasmic space, that is between the cytoplasmic membrane 13Q and the bacterial wall.

Particularly it has been found that upon expression of pOMP2 in a strain of E.coli K 12 88% of the synthetized ovalbumin is found in said periplasmic space,12 only remaining within the cytoplasm.

These dosages have been carried out by the abovesaid radio-immunologic method applied to the growth medium of the bacteria, after selective splitting of the bacterial walls according to the method of NEU and HEPPEL, described in"J. Biol.

Chem." ,1965, vol.240, pp.3685 - 3692. Intracellular ovalbumin has been dosed too, after freeing from the spheroplastes after splitting thereof.

The vector containing the DNA fragment corresponding to ovalbumin is thus appropriate for insertion in its genome of a DNA corresponding to the desired protein (somatostatin or another protein).

The expression of the so modified vector therefore entails the freeing of the hybrid protein expressed, containing the desired protein, in part of the bacteria (periplasmic space) wherefrom it is easily recovered.

The invention concerns more generally any recombinant vector comprising in itsgenomea DNA sequence coding for at least the major portion of ovalbumin. Preferably this DNA sequence corresponding to ovalbumin in the vector under consideration, is linked to a DNA sequence corresponding to at least part of the lactose operon including the promotor and part of the gene Z associated thereto, either directly or by means of a whole number of triplets selected among those which are not liable of interrupting the translation.The recombinant vector according to the invention comprises advantageousliy the preferred DNA fragment comprising the sequence corresponding to the 8 first aminoacids of p-galactosidase and the sequence capable of coding the major part of ovalbumin, except for its first, particularly its 5 first, aminoacids, the latter sequ ence being linked directly to the former sequence.

The invention is in no way limited to those of its uses and embodiments which have been more especially envisaged; it encompasses, on the contrary, all modifications, in particular that where recourse is had to other plasmids than those which have been more particularly described in the fore going, in particular the plasmid pSOMI contem plated in the article already mentioned of Keiichi Itakura et coll., notably to the extent that the expressed ovalbumin can be considered as not being sensitive to the action of the endogenic proteolytic enzymes of the ost bacterium; the invention also relates to plasmids modified according to the invention, but in which the host operon fragment instead of originating from the lactose operon comes from another operon, such as a galactose operon or a tryptophane operon; as regards the latter, it is recalled in fact that the construction of a recombinant of a X phage and of a tryptophane operon has been described notably by Anne Moir and W.J. Brammar in the article entitled "The use of specialized transducing phages in the amplification of enzyme production" (MOLEC.GEN.GENET.1 49,87 99 (1976).

Claims

CLAIMS:

1 - Plasmid which comprises in a non-essential part of its genome a DNA fragment derived itself from aX phagefragment, linked to a bacterial operon fragment and at least the starting part of the gene connected to said bacterial promotor, said starting part comprising a site of cleavage by a predetermined endonuclease.

2 - The plasmid of claim 1 wherein the operon fragment is a fragment of the lactose operon, comprising at least the bacterial promotor and a sequence of the gene Z linked to this promotor, said gene Z sequence coding for at least the first amino-acids of p-galactosidase.

3 - The plasmid of claim 2 which comprises no other site of cleavage by the same endonuclease in the other parts of its genome.

4 - The plasmid of claim 1 wherein the abovesaid bacteria I operon fragment is a lactose operon fragment and which comprises at least the sequence of the Z gene adapted to code the 8 first amino-acids, inclusive formyl-methionine, of the bacterial ss- galactosidase.

5 - The plasmid of claim 2 wherein said site of cleavage is an EcoRI site.

6 - The plasmid of claim 1 which forms a genetic recombinant containing a supplementary foreign DNA fragment inserted in its genome, coding for at least a part of a predetermined protein, in the prokaryot or eukaryot host-cell, from which it originates.

7 - The recombinant plasmid of claim 6 wherein the bacterial operon fragment is a lactose operon comprising at least the promotor and linked thereto a gene Z sequence coding for at least the first aminoacids of ss-galactosidase.

8 - The recombinant plasmid of claim 7 wherein supplementary foreign gene fragment has a size, expressed in number of nucleotides, higher than that of the Z gene sequence, preferably at least equal to 50.

9 - The recombinant plasmid of claim 8 wherein the supplementary gene fragment has a size at least 50 times as large as that of the Z gene sequence.

10 - The recombinant plasmid of claim 7 wherein the foreign DNA fragment is formed of a DNA transcript of the messenger RNA corresponding to said predetermined protein.

11 r The recombinant plasmid of claim 7 or 8 wherein the foreign DNA fragment is a fragment coding a nutritive protein.

12 - The recombinant plasmid of claim 7 or 8 wherein the foreign DNA fragment codes for at least part of ovalbumin.

13 - The recombinant plasmid of claim 10 which comprises a DNA fragment resulting from the com bination of the sequence comprising the triplets of those nucleotides which are capable of coding the translation of the 8 first amino acids, inclusive formyl-methionine, of the bacterial ss-galactosidase, on the one hand, and a sequence capable of coding the major portion of ovalbumin linked to the preced ing sequence, either directly, or by means of a whole number of triplets selected among those which are not liable of interrupting the translation, on the other hand.

14 - The recombinant plasmid of claim 13 wherein the sequence capable of coding the major portion of ovalbumine does not code for the first 5 aminoacids of this protein.

15 - The recombinant plasmid of claim 13 which is capable of transforming a yeast.

16 - The recombinant plasmid of claim 15 wherein said yaest is Saccharomyces cerevisiae.

17 - The recombinant plasmid of claim 15 which comprises DNA fragments of the "plasmid 2 w

18 - Method of manufacturing a hybrid protein of which a part belongs to a protein normally coded, in a prokaryot or eukaryot natural host-cell, by the corresponding predetermined DNA consisting of the gene belonging to the genome of the latter cell or of a DNA transcripts of the corresponding RNA, which process consists of introducing into a bacterium, notably E. coli, a recombinant plasmid resulting from the insertion by genetic fusion of the abovesaid predetermined DNA in the plasmid, the hybrid protein containing the desired protein part being then recoverable from the cellular proteins formed by the bacterium.

19 - The method of claim 18 wherein said predetermined DNA codes for a nutrient protein.

20 - The method of claim 19 wherein said predetermined fragment codes for ovalbumin.

21 - The DNA fragment which comprises, on the one hand, a DNA sequence corresponding to a portion of the lactose operon including the promotor and a fragment of the associated gene Z and, on the other hand, a DNA sequence capable of coding a nutritional protein in phase with the preceding fragment in respect of translation.

22 - The DNAfragment of claim 21 which comprises, on the one hand, a DNA sequence corresponding to at least part of the -galactosidase protein, starting with the first amino-acid thereof, inclusive formyl-methionine, and, on the other hand, the whole or the major part of the DNA sequence coding for ovalbumin, the latter DNA sequence being linked to the former DNA sequence, either directly or through a whole number of triplets.

23 - The DNA fragment of claim 22 which comprises, on the one hand, a DNA sequence coding for the 8 first amino-acids of ss-galactosidase and wherein the major part of the DNA sequence coding for ovalbumin codes the whole latter protein except for its first 5 amino-acids.

24 - The DNA fragment of anyone of claims 21 to 23 wherein the DNA sequence coding for the whole orthe major part of the nutritional protein is the DNA transcript of the corresponding RNA messenger.

25 - The hybrid protein which comprises the amino-acid sequence coded by the DNA fragment according to anyone of claims 21 to 23.

26 - Recombinant vector which comprises in its genome a DNA sequence coding for at least the major part of ovalbumin.

27 - The vector of claim 26 wherein said DNA sequence is connected to another DNA sequence corresponding to a fragment of the lactose operon inclusive the promotor and at least the triplets coding for the first amino acids of p-galactosidase.

28 - Vector of claim 27 wherein the DNA sequence coding for the major part of ovalbumin is a transcript of the corresponding RNA messenger.

29 - The vector of anyone of claims 26 to 28 which consists of a plasmid.

30 - A method of manufacturing a hybrid protein of which a part belongs to a protein normally coded in a prokariot or eukariot natural host-celi by a determined DNA which comprises transforming a microorganism with the vector of anyone of claims 26 to 29, previously modified by insertion therein of said determined DNA, growing said bacteria, splitting the walls of the transformed bacteria and recovering said hybrid protein from the cellular proteins previously contained in the periplasmic space of said bacteria and freed in the medium.

31 - The microorganism containing anyone of the vectors of claims 1 to 17 and 26 to 29.

32 - The microorganism of claim 31 which is a bacterium.

33 - The microorganism of claim 31 which is a yeast.