CA1203492A - Process for producing proteins by the expression of the corresponding dna in bacteria, modified adn and vectors applicable in such processes - Google Patents

Abstract

Abstract of the Disclosure 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 microorganism.

Description

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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 modi-fied 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 al. (NUCLEIC ACIDS RESEARC~ 77, Vol. 4, p. 2389) have described the insertion into a plasmid (pCRl~ of a double s~rand DNA derived from the transcription of a puri-fied RNA messenger of chicken ovalbumin. The thus purified plasmid pCRl ov 2-1 could be introduced into strains o~
Escherichia coli K12 by gene construction methods which have become conventional. No ovalbumin synthesis 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. I-t must however be stressed that there is one exception to this still negative general si-tuation. 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

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in Escherichia coli of a chemieally synthesized gene for the hormone somatostatin", published by Keiichi I-takura et al. 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 conclitions particularly favorable for inducing the production by E. coli of a hybrid protein ineluding a fragment formed from the 14 amino aeids from which somatostatin is normally constituted.
They resorted to a synthetie gene, constructed chemieally 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 indieated 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 ~-galactosidase of the lactose operon. If the gene of the somatostatin is only linked to the portion corresponding to the fragment of the lac-tose operon (eomprising the "p", "o" genes and the 8 coding base triplets for the first amino acids of the bacterial ~-galactosidase), hereafter designated as "lac", no somatosynthesis is deteeted.
As a result, the fragment corresponding to the somatostatin only representecl, under the most favorable experimental conditions described by these authors, a small part of the expressed hybrid protein, the largest part of the ~2~3~9~ ~
~ ter consistin~ of a fragment having the amino acid se~ ence of ~-galactosidase. It is possible to consid~r that the somatostatin fragments occured~ with respect to the whole of the hybrid protein formed, in a ratio of the order of 14 amino ~cids tthose of somatostatln) to a figure of the order of 1000 amino ~cids.
It is an object of the invention to provide a process for the production of a prokaryot or eukaryot proteln, bringing lnto 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 typP, 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.

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In one aspect, the invention relates to an improved method of manufaeturing a hybrid protein of which a part belongs to a protein normally coded by a gene contained in the genome of a foreign proearyot or euearyot natural host cell, which comprises transforming a suitable transform-able foreign host eell with a recombinant plasmid obtained by genetie fusion of a DNA fragment eorresponding to said gene and of a plasmid ineluding a bacterial laetose operon fragment whieh itself eomprises at least the eorresponding baeterial promotor and a nucleotide se~uence eonsisting of at least the first nueleotide triplet of the gene normally eonneeted to said promotor and eoding for at least the amino-aeid of the eorresponding protein, betagalactosidase, the improvement wherein said DNA fragment eonsists of a foreign DNA transeript of a messenger RNA linked to and in reading phase with said nueleotide triplets, and which comprises col-leeting said hybrid protein with the translation products of said host cell.
The plasmid according to the invention comprises a non-essential part of its genome, a DNA fragment derived itself - 4a -~Z~3fl~9;~
from a ~ phage fragment, linked to a bacterial operon fragment, no~ably of a lactose operon, comprising at least the bacterial promotor and a fragment of the yene linked to thls promotor, such as the Z gene coding the production of ~ -galactosldase 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 ccrresponding protein,/g-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 ( ~ Z), with a cleavage site by a pre-determined endonuclease, preferably to the exclusion of an~
other cleavage site b~ 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 ~ -galacto-sidase (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 consti-tuted 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 b~ the action of EcoRI and Hind III enz~mes, 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-acidsj as defined above. Preferably the operation is not sensitlve to catabolic repression.

3~2~3~92 It is possible to incorporate into such a vector 1~
an-~ fragment corresponding to the protein whose sxpression is sought, which genefraqment ma~ be either s~nthesized chemically, or synthesiæed by an enz~matic rcute, notably from an R~A 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 ~ragment 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 here-after described and aiming at the production of a hybrid protein comprising most of the sequence of the ovalbumin D~
amino-acids, use is made preferably of an ~* fragment synthesized by the enzymatic route starting from the corres-pcnding messenqer RNA. The latter may be obtained for instance through enzymatic transcription according the method des-cribed b~ HUMPHRIES et al, referred to hereabove. The possibilit~ of causing said DNA fragment to be expressed in a bacterilm, even in a ~east, 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 alread~ been indicated that the D~
genes coding for ovalbumin are formed of ~ sequences separa-ted from one another by non-coding 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, notabl~
E.coli so that it is possible to obtain the production of a hybrid protein of which the major part corresponds to the amino-aci setl~ence of ovalbumin~
Tlle 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 abovesaid Z gene fragment .
More preferably the invention concerns the so modiEied 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 r~sulting 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,form~l-methionine, of the bacterial ~ galactosidase, on the one hand, and a sequence capable of coding the major portion of ovalbumin, possibly except for the first amino-acids thereof (partlcu-larly the first 5 amino-acids), linked to the preceding sequence, either directly, or by means of a whole number of triplets selscted among those which are not liable o-f .
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 necessarlly 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 3~92 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 correspond~ng to the first amino-acids of ~ -galacto-sidase.
In particular, it will be noted that in the case of ovalbumin which is formed from about 385 amino-acids, 380/387 (expressed in number of amino-acids) of the hybrid protein finall~ obtained will be constituted by the protein sought.
Thus invention concerns in a general manner plasm~ds 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 forat least ~art of the amino-acids of ~ -galactosidase and, on the other hand, the DNA
sequence coding for the production of the nutritional protein, for the sake of causing ~le2 fragments to be in proper phase when later translated.
The invention therefore also provides a process for manufacturing a h~brid 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, lZ~92 noLably E~ coli, or as a yeast, such as 6accharomyces cerevisiae, a reccmbinant plasmid resulting from the insertion by genetic fusion of the above-menticned 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 contiguously to 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 cf the desired protein has a size, expresséd in number o~
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-aclds as contained in ovalbumin, however essentially free of glycosylated groups cf the type contained in the natural protein;
-the corresponding ~ fragments which comprise, on the one hand - a DNA sequence corresponding to at least part of the -galacto-sidase protein, starting with the first amino-acid thereof (formyl-methionine , more particularly those comprising the triplets corresponding to the 8 first mentioned amino-acids of -galactosidase and, on the other hand, the whole or the malnr part of the DNA sequence such as the whole ovalbumin except for the first amino-acids, notably the S 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 fragment in which are associated, on the one hand, A DNA fragment co~-respondin~ to a portion of the lactose operon including the ' F)romctor and a Eragment of the associated gene Z and, on the other hand, a DNA fragment capable of coding a nutritional protein in phase with the preceding fragment.
Preferably the DN~ fragment which codes for the major part of the nutritional protein, particularl~ 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 prctein, in current radio-immunological assays.
The invention also relates again to the strains o~ micro-organisms, particularly bacteria, notably ~. coli1 or ~easts, notably Saccharomyces cerevisiae~ containing the plasmids according to the invention and permitting their replication simultaneously with~their own division.

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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, essential-ly 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 descrip-tion to the drawings in which the transformations to which the initially treated plasmid gives rise are represented diagram-matically.
EXAMPLE:
1) Construction of the vector.
It is proposed to incorporate in a non-essential part of the plasmid pBR322 (Figure 4a) already mentioned, a DNA including a ~ 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 ~-galactosidase (formyl-methionine not included).
The latter DNA was obtained in the following manner:
As a start there is used ~ phag~ in which there has been incorporated by genetic fusion, a lactose operon segment such às has been described by BACKMAN et al~ in PROC. NATL.
ACAD. SCl. U.S.A. (Vol. 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.

lZir)3492 In figure 1 is shown a diagrammatic map of the ~ 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 ~ -galactosidase (with ~8 and W 5 mutations). This fragment had been inserted in vitro into the EcoRI site situated at the end of the Z gene of a ~ 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 III 203 fragment also described by these authors (Flgure 3a). The latter fragment includes, on opposite sides of a central portion OP, on the one handl a fragment Z' coming from the start of the Z gene and corresponding to the seven first amino-acids capable of being coded b~ the Z gene9 and on the other hand, a terminal I" fragment of the lactose operon repressor. When the OP Hind III 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"

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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), intra-molecular recombination creates a dele~ion (Figure 3c) of almost the whole of the Z gene. This permits, also, the creation of an ~coRI site (EcoRI- ~ Zsite) in the place of the Hae III site, very close to the origin of the Z gene at a site corresponding to the 7th amino-acid of the ~ -galactosidase.
~f the initial Z gene there only subsist inally the fragments Z"
and Z', on both sides of the EcoRI ~ Z site.
In ~igure 2, there is also shown the site of cleava ge by the Hind III enzyme closest to the lactose operon segment on the DNA of bacteriophage~ ~. This site is called s Hind III~ 2.
The vector thus obtained has been cut according to techniques known in themselves by the enzymes ~coRI 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 of Z'), corresponding to the EcoRI- ~2 cleavage site and whose opposite end corresponds to the sHind III cleavage siteO
This identification can be carried out for example by taking advantage of the capacity of this fragmen-t 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- ~-galactoside.
The DNA of the plasmid pBR322 is itselF cleaved by the enzymes ~coRI and Hind III at the level o its corresponding cleavage sites.

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The recombination in vitro follows of the Eragments of DNA of the plasmid pBR322 and of the A 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 al. already cited, the desired vector shown diagrammatically by Figure 4b. This vector com-prises the essential part A of the DNA of the initial plasmid and the inserted part B of the DNA comprising the fragment of ~A phage and the fragment of lactose operon (~Z, o,p(i)) ((i) denoting a fragment only of the gene (i)).
The novel plasmid thus obtained, named pl397, 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 Bth if counting formyl-methionine as such) of the ~-galactosidase. The novel plasmid will also hereafter be referred to as "p OMPO".
2) Construction of the 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 enzymè Hha I from the pCRI ov 201 plasmid and isolated by sedi-mentation 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 diagra~lmatically in Figure 4c in the ; drawing (reference Hha ov).
The plasmid pl397 is opened by EcoRI and treated by the specific nuclease of single strand Sl DNA, so as to trim the cohesive short ends produced by EcoRI (Humphries et al, `'~i' `" 12~3492 1977).
The DNA obtained shown diagrammatically at 4d, is treated with DNA polymerase 1 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 1 of E. coli in the presence of the 4 deoxy-triphosphates.
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 Ko~ Hirose T., Crea R~, Riggs A.D., Heynecker H.L., Bolivar F., Boyer EI.W. (1977) SCIENCE, 198, 1056).
Among the recombinant molecules 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 ~-galactosidase (or the 8 first amino acids if one counts formyl-methionine (Fiqure 4e) ). The strain which has finally been retained for the further experimentation herein-after 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 lysogene strain of E.coli K12, bearing a thermo-inducible 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 ~L2Q3~92 detected in s u by hybridation with a DNA radioactive probe, at a spot whlch 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 to the colonies responding positively 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 rk mk+), according to the technique described by Rougeon F., Kourilsky P., Mach B. (1975), NUCLEIC ACIDS RE-SEARCH, 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 sub]ected at 0C 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 to 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-immunological assay indicates the presence of from 5000 to about 50 000 molecules of antigen per bacterium.
The ~ ,~

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affinity of the antigen produced by the bacterium with respect to the anti-ovalbumin antlbodies does not appear ver~ different from that of the natural ovalbumin.
The size of the antigen has been measured by electro-phoresis of crude extracts labelled with S and immuno-precipitated.
The hybrid molecule has the same apparent molecular weight as ovalbumin (about 43 000 daltons)~
It differs from the ovalbumin formed in chicken egg by a small number of modlfications carried out after synthesls of the protein. In fact, natural ovalbumln is phosphorylated in two places and glycosylated at one place. The absence of glycosylated groups is not of a nature to modify the nutrlent properties of the protein obtained.
The theoretical seqllenCe of the modified ovalbumin ls the following :
formyl-methionine - threnonine - methionine - isoleucine -threonine - aspartic acid - seriné - leuclne - alanine -~lanine-serine - methionine - glutamic acid...., the 8 first amino-acids coming from the ~ -galactosidase and the 5 following ones forming the first amino-acids o~ the ovalbumin fragment Hha ov (in which the 5 first natural amino-acids of the ovalbumin are also lacking).
The plasmid P1397 strain was deposited on June 2,1978 in the National Collection ofCultures of micr~rgan:i.ms (CoN~C~M~ ) at the PASTEUR INSTITUTE under No. 1-064.
The plasmid so obtained, more particularly the portion of this plasmid which contains the sequence modified with coding for ovalbumin, may be incorporated into a plasmid which itself is liable of transforming yeast strains,notably ~21?3gL9~
Saccharomyc~-; cerevisiae. The modified protein is then also _ _ _ _ capable of belng expressed in these yeasts. Then in contrast with what one could have expected, the fragment carries no marker liable of inducing in said yeas~ a restriction mechanism with respect to the fragment of bacterium origin.
The construction of a modified plasmid capable of trans~orming a yeast, its amplification, the transformation of a yeast in the modified plasmid and the detection of the expression of this modified plasmid ~n the ~east are briefly set forth hereabove.
1) Extraction from the elasmid eOMP2 of the lac-ovalbumin ____________________ _______ ___________. _ ___________ se~uence _ _ _ _ _ _ _ poMP2 is opened by therestriction enz~me HhaI. Two cleavage sites liable of being recognized b~ that enzyme, on the two opposite sides of the Hha ov~fragments are shown on figure ~e. After re-insertion of this ~ragment into another vector, i~ is detectable owing to its capability of rendering the bacteria transformed b~ ~t lac-constitutive (the latter ~ransformed bacteria then being liable of producing large amounts of ~ -galactosidase and being detected i~ -that the~ f~rm ~olonies in the presence of -the X-gal dye (5-bromo-4-chloro-indolylgalactos~de).
Particularly this operation has been carried out as follows :
20 ~g of pOMP2 DNA were digested to completion by HhaI
(Biolabs ; 25 units ; 30 m ; 37C) and the react~on was stopped by heating to 65C 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 EDTA1 0-5 per cent SDS) and centrifUged at 31 000 rpm for 16 h at 20C

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BECKMAN rotor ultracentrifuge of the SW 41 type9 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-~ containin~ the "Eco RI-D"
fra~ment This plasmid is obtained by recombination in vitro of a plasmid TBr 3Z2 comprising a genetic Ura (déscribed by M.L~ BACH, ~ LACROUTE and D~ E30TSTEIN P N A S, 1979, vol. 176, n 1 pp. 386 - 390 which had previously been opened by Eco RI, and of the fragment "Eco RI-D" (described by CAMERON
et al. Nucl. Aco Research, 1977, vol. 4, n 5, pp. 1429 - 14489 originating from "plasmid 2 /u which is known to have a capabl-lity of forming ~east such as Saccharomyces cerevisiae .
This recombination is carried out by any known method, for instance such as that described by HINNEN et al, P N A 5 1978, vol. 75, n 4, pp~ 1929 - 1933.
The "plasmid 8" straln was deposited at the CoN~CaM~
under Nr. I - 093 on May 27, 19790 3) Construction of a recombinant of "plasmid 8" and of the _______~__________ _______________ __________________~_ poMP2 fragment containi.ng the "lac-ovalbumin"
_____ __ ______________ _ _________________~
30 ug of plasmid 8 DNA were cut by 1.5 unit of Hha I
during 5 minutes at 37C. The reaction was stopped by heating 10 min9 at 65C in the presence of 10 M EDTA. The linear molecules were purified by centrifugation on a 5_20 per cent saccharose gradient (10 M tris HCl, 10 M EDTA, 10 M NaCl) for 4 hours under 40 rpm at 20C in a ~3~CK~N r~to~ of the SW 41 type.

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200 ng of the 2.67 kb lac-ovalbumin HhaI fragment and 600 ng of lineari~ed plasmid ~ were ligated with 1'4 DNA ligase at 4C in a final volume of 5Jul in 40 mM Tris pH 7.5, 10 mM
MgCl2, 10 mM DTT, 0.1 mM ATP, 0.05 mg/ml BSA. After transfo~ a-tion of CaCl12 treated lysogenic cells of strain 1398 (CNCMn~ I-094 deposited on May 27, 1979)transformant.s were selected on ampicillin containing plates ~20 ~g/ml), and screened b~ in situ hybridization with a labelled DNA probe specific for.the ovalbumin sequence (the Hha ov probe)~ Positive colonles were then tested for ovalbumin production in the same radio-lmmuno assay as before. All the clones positlve ln in situ h~bridiza--tion were also positive in this assay .
4) Am~lification and recover~ of the elasmid __ ___________________~__ ________ ______ A colony of bacteria E.coli C 600 rk mk~
induct;ble lnto lac-constitutive bacteria is transformed with the DNA fragments isolated during the preceding operation~
After recovery of the colon~ and identification of lac-consti-tutive bacteria formed, the latter are cultivated and the plasmids are recovered from the grown bacteria on making use of classical techniques for rupturing the b~c t erl a , recovering the DNA contained th~rein and sep~rating the band of c~rc~i~ar pla.sm;.d in a gradient of coesium chloride in the presence of ethid~um bromide~

5) Transformation of the ~east Saccha.romicae cerevisiae (CNC~
______________________ ______._____________________________ Nr l-O~-d~Qsl-t-e---on-Ma~-27l-l979 -The abovesaid strain which is Ura is transformed b~ 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 medlum.

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- The production o~ a hybrid protein containing most of the ovalbumin sequence is detected by means of the radio-immuno-logical assay of the type already disclosed hereabove in -the ~easts containing said plasmids.
The vector according to the invention containing the gene of ovalbumin can, in turn, serve as a vector for manufacturing ot~ler hybrid proteins including a separate protein; this can be carried out by insertion in the ovalbumin gene fragment trepla-cement thereof b~- or addition thereto of an RNA transcrlpt 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 h~brid protein containing ovalbumin as e x pr e ~ sed in the bacteria b~-the above-defined vector, is freed in the periplasmlc space9 that is between the cytoplasmic membrane and the bacterial wall .
Particularl~ it has been found that upon expression of poMP2 in a strain of E.coli K 12 8 % of the synthetized ovalbumin is fo~nd in said perlplasmic space, 12 % onl~ 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 ~EU and HEPPEL, described ~n"J.
Biol. Chem." ,1965, vol. 240, pp. 3685 - 3692 0 Intracellular ovalhumin has been dosed too, after freeing from the sphero-plastes after splitting thereof.

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The vect~r containing the DNA fragment corresponding to ovalbumin is thus approprLate for insertion in its genome of a DNA corresponding to the desired protein (somatostatin or another protein). The expression of the so modifled vector therefore entails the freeing of the hybrid protein expressed, containing the desired protein, in a part of the bacteria (periplasmic space) wherefrom it is easily recovered.
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 foregoing, in particular the plasmid pSOMl contemplated in the article already mentioned of Keiichi Itakura et coll~, notably to the extent that the expressed ovalbumin can be considered as not being sensitiv e to the action of the endogenic proteolytic enzymes of the host bacterium;
the invention also relates to plasmids modified according to the invention, but in which the 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 ~ phage and of a tryptophane operon has been described notably by Anne Moir and W.J. Brammar in the article entitled "The use of specialised transducing phages in the amplification of enzyme production" (MOLEC.GEN.GENET.149,87-99 (1976) ).

Claims (13)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a method of manufacturing a hybrid protein of which a part belongs to a protein normally coded by a gene contained in the genome of a foreign procaryot or eucaryot natural host cell, which comprises transforming a suitable transformable foreign host cell with a recombinant plasmid obtained by genetic fusion of a DNA fragment corresponding to said gene and of a plasmid including a bacterial lactose operon fragment which itself comprises at least the corresponding bacterial promotor and a nucleotide sequence consisting of at least the first nucleotide triplet of the gene normally connected to said promotor and coding for at least the first amino-acid of the corresponding protein, betagalactosidase, the improvement wherein said DNA fragment consists of a foreign DNA transcript of a messenger RNA linked to and in read-ing phase with said nucleotide triplets, and which comprises collecting said hybrid protein with the translation products of said host cell.

2. The method of claim 1 wherein the nucleotide sequence is at least the first triplet of the z gene coding for at least the first amino-acid (methionine) of betagalactosidase.

3. The method of claim 2 wherein the bacterial lactose operon fragment in said plasmid is linked to a lambda phage fragment.

4. The method of claim 2 wherein the said nucleotide sequence of the Z gene codes for the first eight amino-acids of the bacterial betagalactosidase.

5. The recombinant plasmid described in any one of claims 2, 3 or 4 wherein the DNA fragment has a size expressed in number of nucleotides at least 50 times as large as that of the Z gene sequence.

6. The method of any one of claims 2, 3 or 4 wherein the foreign DNA transcript of said recombinant plasmid is a fragment coding for a nutritive protein.

7. The method of any one of claims 2, 3 or 4 wherein the foreign DNA transcript of said recombinant plasmid codes for at least part of ovalbumin.

8. The method of claim 2 wherein said recombinant plasmid contains a nucleotide sequence of the Z gene capable of coding the first eight amino-acids of the bacterial beta-galactosidase, on the one hand, and a sequence capable of cod-ing the major portion of ovalbumin linked to the preceding sequence, either directly, or by means of a whole number of triplets selected from among those which are not liable of interrupting the translation, on the other hand.

9. The method of claim 8 wherein said sequence contain-ing the major portion of ovalbumin does not code for the first five amino-acids of this protein.

10. The method of claim 1 wherein the host cell transform-ed is E. coli.

11. The method of claim 10 which comprises recovering the hybrid protein formed from the periplasmic space of the E. coli.

12. The method of any one of claims 1, 2 or 8 wherein the host cell transformed is a yeast and the recombinant plasmid comprises DNA fragments of the "plasmid 2 µ" capable of causing the transformation of said yeast.

13. The method of any one of claims 1, 2 or 8 wherein the host cell transformed is Saccharomyces cerevisiae and the recombinant plasmid comprises DNA fragments of the "plasmid 2 µ" capable of causing the transformation of said Sacc. cer.