CA1172584A - Vector enabling the insertion of prokaryot or eukaryot gene and the excretion of the expressed protein - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A vector carrier of the promoter of the lactose operon and of at least a fragment of the Z gene of this same operon, wherein at least one sequence, more particularly a sequence including the proximal portion of a gene coding extracytoplasmic protein, notably a bacterial receptor for a phage, or at least of its precursor, is inserted in this vector, more part-icularly in this Z gene or fragment of Z gene, or immediately downstream of the latter. The length of this extracytoplasmic gene sequence is sufficient to permit the excretion, under the control of the promotor of the lactose operon, of the hybrid protein expressible in a bacterial strain, into which said vector has previously been introduced.

Description

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BACKGROUND OF TilE INVENTION
~ The invention relates to novel vectors into the genome of which - can be inserted by genetic fusion, in vivo or in vitro, a DNA fragment com-prising a foreign gene capable of coding the production of a predetermined prokaryot or eukaryot protein, these vectors being constructed so that they permit not only the expression of this gene in a cell, notably a bacterium, but also the excretion of the protein produced.
- The invention also relates, besides the thus modified vectors, to the corresponding DNAs or RNAs (desoxyribonucleic or ribonucleic acids).
It will be said below that a protein is "excreted" by a bacterium when after synthesis it occurs localized outside of the external cell-mem-brane or is released ~or releasable) in the culture medium.
The invention takes more particular advantage of certain of the pro-perties recently elucidated of certain genes which have already been much investigated. In this respect, it is known that the translation product of the lamB gene in E. coli K-12 is a protein of the outer membrane which plays particularly the role of bacterial receptor for the ~ phage. Study of its positioning in the outer membrane could provide information on the transfer of proteins through the internal membrane receptor. In the wild strain, the lamB gene forms part of one of the operons of the malB region.
More particularly, SI~IAVY et Coll., Proc. Natl. Acad, Sci. U.S.A., ~: vol. 74, n 12, pp. 5415 (1977) have constructed strains which have enabled them to show that a recombinant DNA including a gene obtained by in vivo genetic fusion of the lamB gene (which codes the bacterial receptor of the phage~ and of a gene fragment which codes an important part of the COOH--terminal sequence of ~-galactosidase (cytoplasmic protein) could be ex-pressed in these strains in the form of a hybrid protein detectable outside the external membranes of their cells. These recombinant DNAs did not in-clude the promotor of the lactose operon.
It is an object of the invention to provide vectors applying the ~4,~

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properties of genes which, such as the lamB gene, are capable of coding extracytoplasmic proteins which either occur in the cell-host in the exter-nal membrane, or are liable of being excreted in the culture medium, for causing the expression in cells of gene fragments coding a predetermined pro-tein and the direct excretion of this protein outside of said membrane or in the culture medium.
The vector according to the invention, which is a bearer of the promotor of the lactose operon and of at least a fragment of the Z gene of - this same operon, is characterized by the fact that at least one sequence, more particularly a sequence including the proximal portion of a gene coding an extracytoplasmic protein, notably of a bacterial receptor for a phage, or at least of its precursor, is inserted into this vector, more particularly in this Z gene or fragment of Z gene, or immediately downstream of the latter, the length of this sequence of extracytoplasmic gene being suffi-cient to permit the excretion, under the control of the promotor of the lactose operon, of the hybrid protein capable of being ~1725~34 expressed in a bac~erial strain, in which this vector would have previously been introduoe d.
The VeCtQr may also include a fragment comprising itself the gene which codes a predetermined foreign protein.
Advantageouslyr the gene ooding an extracytoplasmic protein is constituted by the lamB gene, said sequence oomprising then also, preferably the distal portion of the K gene which, in maltose B operons, is associated with the lamB gene.
me invention also relates to a method for the manufacture and excretion of such a predetermined protein which co~prises the intrcduction of the vector described above into a host-cell, inducing the expression of this vector, and recovering the hybrid protein excreted in the culture medium.
The host- oe ll is, for example, a bacterium.
The method is suitable for producing vaccines with respect to antigen proteins, preferably viral antigen proteins, such as the extra-cytoplasmic protein coated by a fragment with tw~ EcoRI cites comprising the lamB gene, normally ccntained in the maltose B operon. The method ocm-prises introducing a vector ccntaining such a fragment into an innocucus oe ll-host, inducing the expression of this vector, and reoovering the oe ll strain carrying the extracytoplasmic hybrid protein ocmprising said antigen protein.
The obtaining of the excre~ion of a protein derived from a cloned gene is interesting from several points of view. Ihere is thereby avail-able a novel means of studying genetic determinates responsible for the excretion. It permits the study and the easier detection of the expressed protein. The excreted portein is, especially where it relates to cytoplasmic pr~tein, relatively protected from bacterial proteases. In addition, the exposure at the surfa oe can facilitate the perfection of vaccine5 against viral proteins and this mD~e particul æ ly in the case of viral proteins and in the case where the bacteriumrhost is innocuous, so that tne latter can be directly used as a vehicle carrying vaccinating antigens that these viral proteins oonstitute. Lastly, this protein - for example somatostatin 1:~72S84 ~ay be separated and found in the medium, so that its preparation in large am~unt5 is facilitated.
It seems well-established now that the excreted proteins are synthesized in the fqrm of a precursor. Ihe precursor includes an N
terminal peptide of about 20 amino acids or signal peptide. The peptide seems to c~ntain sufficient instructions for the protein to be excreted;
it is detached in the course of the excretion.

- 4a -1~72584 -~ By fusing the proximal end of a gene of structure (X) coding for a protein ~x), to a DNA (S) corresponding to at least one signal peptide (s), a recombinant is obtained which can be used for excretion experiments.
A preferred embodiment of the invention consists of inserting, by genetic fusion, the X gene in a vector comprising the S sequence of the lamB gene determining the bacterial receptor for the ~ phage, more particularly a fragment proximal the lamB gene and itself fused to a fragment of the structural lacZ of ~-galactosidase, including here the promotor of the lactose operon. The hybrid protein obtained possesses the N
terminal end of the receptor of the ~ phage. It must be noted `! that the synthesis of the hybrid protein is then subjected to :
the regulation by the lactose system. This property permits the triggering of excretion by the addition of isopropyl-thiogalcatoside (IPTG) to the culture medium or the constitut-ive excretion of the hybrid protein sought after the introduct-ion of the so modified vector in a suitably selected - in a manner known per se - bacterial strain.
The protein is capable of being excreted as soon as the size of the receptor fragment is sufficient, corresponding notably to a protein fragment having a molecular weight reach-ing preferably, of the order of 2000, (preferably 2500) in the case of the receptor of the ~ phage (the molecular weight of the purified ~ receptor being of the order of 55,000). Said : receptor fragment can naturally have a greater size, correspond-ing particularly to a protein of higher molecular weight, advantageously above 20,000 or even above 30,000. By way of example this receptor fragment corresponds to a protein fragment having a molecular weight from about 35,000 to about 40,000.
Advantageously, this vector is constituted by a ~72S84 phage whose one or more corresponding bacterial receptors are distinct from that for those corresponding to the phage from which the sequence of the extracytoplasmic gene inserted therein originates. For example, when the in-serted sequence corresponds to the lamB gene, recourse may be had to a vector derived from the h434 phage.
Such a type of vector enables:
a) the insertion in its genome, downstream of the promotor of the lactose operon, of the X DNA fragment, preferably provided previously with EcoRI
- cohesive ends;
b) the introduction of the whole so modified vector in bacteria resistant to the ~ phage due to the specificity of the h434 host. This property en-ables the possible expression of the lamB gene inserted in the vector to be examined directly. It suffices to check if the lysogen bacteria for the lamB carrier have recorded sensitivity to the ~ phage.
The above-said insertion of the X DNA fragment - corresponding for example, to the gene fragment of somatostatin described in the article en-titled "Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin", published by Keiichi ITAKURA et Coll. in SCIENCE, vol. 198, pp. 1056-1063 of December 9, 1977 - occurs downstream of the gene sequence corresponding to the minimum receptor fragment required for the excretion, but upstream of the end of either the end of the Z gene or frag-ment of Z gene which is the remotest of the lactose promotor or of the end of the gene sequence corresponding to the extracytoplasmic protein, notably lamB, in the case where this sequence would not be followed by a distal frag-ment of Z gene in the direction of the translation.
BRIEF DESCRIPTION OF THE DRAWINGS
Other features of the invention will also appear in the course of the description which follows of certain preferred embodiments thereof, not-ably with reference to the drawings in which:
Figures 1 to 12 are representations ~diagrammatically) respec-58~
tively of different forms of phages which have been brought into play; the promotor of the maltose operon (between the K and E genes) not being shown in the vectors which carry the two genes concerned.
In these figures J the symbols:
- K, E, F denote the K, E, F genes of the B maltose operons;
- QB represents the lamB gene;
- L is the promotor of the lactose operon;
- Z, Z', `Z, Z'' and ``Z denote the Z gene or fragments of the Z gene of the lactose operon, the position of the signs ', `, '', ``, being indica-~- 10 tive of the cleavages (fragments respectively proximal or distal), in the , direction of translation of the vectors concerned (from left to right in Figures 1 to 5 and from right to left in the others);
- X corresponds to the inserted gene and whose expression is sought;
- Y and A correspond respectively to the genes of permease and of acetylase or to fragments of the latter;
- b, x, z correspond to proteins coded by the genes QB, X and Z respectively, these letters being also modifiable with the same signs ', `, '', ``, in conformity with the annotation of the corresponding genes in the figures concerned.
The other indications are conventional either of mutations, or of deletions introduced into tlle diagrammatically shown vectors.
DESCRIPTION OF PREFERRED EMBODIMENTS
1) Manufacture of a lamB fragment (EcoRI) It was obtained by EcoRI hydrolysis of the DNA of the ~pmalB21 phage (deposited in the National Collection of Micro-organism Cultures of the PASTEUR INSTITUTE (C.N.C.M.) under No. I-072) and separation of the frag-ments by ultracentrafugation on a saccharose gradient. The ~pmalB21 phage is shown diagrammatically in Figure 1. The arrows ey denote the sites of cleavage by the EcoRI enzyme. There will also be seen the respective loca-tions of the lamB fragment (EcoRI) and of a fragment (a), whereby the latter .

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will be used for the preparation of a phage enabling the cloning of the lamB
fragment (EcoRI). It has been noted that the lamB gene was in the B maltose operons surrounded by two close EcoRI sites, of which one is in the K gene.

2) Cloning of the lamB fragment (EcoRI) .. . _ ............. _ _ _ By _n vivo recombination of the ~pmalB21 phage and of the ~plac5-1 phage (C.N.C.M. ~o. I-074) shown diagrammatically in Figure 2, the ~pmalB20 phage is obtained, shown in Figure 4 which forms plates and bears malE . It results from the transformation of the unstable intermediate phage shown in Figure 3, as it results from the recombination in vivo of ~lac 5-, and ~apmalB12, by in vivo internal deletion of the _ portion. The ~pmalB20 phage is recognized in that it forms transducing particles of malK . The arrows fl~ f2 and f3 show the nature of the transformation carried out.
The lamB fragment (EcoRI) is inserted in the ~pmalB20 phage by in vi recombination (insertion of the lamB fragment (EcoRI) in the EcoRI site of ~pmalB20) and the ~pmalB21 phage (Figure 5) enabling the cloning of the lamB (EcoRI fragment) is detected owing to its being transducer particles of malK .
The lengths 1 (orders magnitude expressed by the ~ sign) of the phages of Figures 3, 4 and 5 are expressed in % of the length of the ~ phage.
The sign ~ indicates the locations of fusion of the genes of the type con-cerned.

3) Preparation of a vector derived from a phage other than the ~ phage and containing the lamB_gene under the control of the promotor of the lact _ operon.
The lamB fragment (EcoRI) was inserted in a vector carrying the ; operator and of the promotor (affected by the mutation L8W 5) of the lactose operon: ~Yh434 (Figure 6). The insertion was carried out by complete hydrolysis of the vector by EcoRI and ligation in the presence of the lamB
fragment (EcoRI) (Figure 7) by the ligase of the T4 phage. By transfection of a lac indicator, lysogen bacteria for the phages comprising the insert could be isolated.

-~r ~L3L7~S~34 o The ~Yh434 vector, which includes the late functions giving the host 434 specificity, was obtained by crossing of a ~oh434 phage (C.N.C.M.
No. I-070) devoid of EcoRI cleavage site (resulting itself from a hybrida-tion between a ~ phage and a 434 phage), on the one hand, and of a ~Y
(C.N.C.M. No. I-701), on the other hand, previously prepared and including in its genome a portion of the lactose operon, notably its promotor I., a fragment of the Z gene (Z, Z') including a single EcoRI site and a fragment A' of the gene of acetylase. The ~Yh434 phage is in addition provided with sites corresponding to the restriction enzyme BgIII (shown by double arrows) which then enable the verification, by hydrolysis of these sites, of the cor-rect orientation of the lamB fragment (EcoRI), when the latter is inserted in this vector.
a: The presence of the fragment and the possible expression of the lamB gene have been examined by recombination and complementation in lysogen strains for vectors in which the previously constructed fragment lamB' (EcoRI) were inserted.
-al) "Maltose plus" recombinants have been obtained in two strains carrying point mutations in the distil portion of the malK gene. It is the proof of the presence of this portion of the gene on the fragment carried by the vector.
-a2) Four strains initially resistant to the ~ phage for different reasons become sensitive to it. I`he genetic properties of these strains en-able the conclusion that this sensitivity is then:
- due to the lamB gene carried by the vector;
- independent of the expression of the maltose system; it is observed in the absence of the product of the malT positive regulator gene;
- insensitive to catabolic repression: this is in accordance with the fact that it is dependent on the L8UV5 promotor;
-a3) A mal strain, but incapable of growth on dextrins following a mutation, becomes capable of using dextrins but only in the presence of ' ~

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IPTG. This indicates that the expression of the lamB gene of the vector is inducible by IPTG.
b: The presence of the lamB (EcoRI) fragment and its direction of integration in the ~Yh434 vector have been established on agarose gel by using DNA preparations of two phages expressing the lamB gene and a phage not expressing it.
Analysis of the results enables the conclusion that the phage con-cerned has integrated the lamB fragment (EcoRI) in the direction enabling - the expression of the lamB from the promotor of the operon lac. This could not be done by recombination phages which would have integrated this frag-ment in the reverse direction, (Figure 9).

4) Preparation of a ~LBZ vector, derived from a ~ phage and containing the lamB gene under the control of the promotor of the lactose operon.
The phage ~i ltSSam7 3221 (spij (C.N.C.M. No. I-075), was first pre-pared, the fusion of genes 42-1 between lamB and lacZ, by recombination of ; the DNA obtained from the pop 3221 strain, described in the above mentioned SILHAVY et al publication, on the one hand, and of a ~ phage, on the other hand (Figure lOa).
By in vivo recombination between the ~i Sam7 3221 (~E~ phage :. o and the ~pYh434(1B)10 Phage (Figure 8, also reproduced in Figure lOb), the ~LBZ phage (Figure lOc) is obtained, in which the lamB gene is thenceforth under the control of the promotor of the lactose operon. The broken line of `- Figure 10, between Figures lOa and lOb, shows the portions of the ~LBZ phage ` which arise from the phages of Figures lOa and lOb respectively. The o I ~pYh434(1B)10 phage carries the malK5 mutation. The ALBZ phage also. The ` expression of the fusion of genes 42-1 hence becomes under the control of the promotor of the lactose operon.

In Figure 10 are hence to be found:

~ = fusion of genes giving a hybrid protein, L_J= hybrid protein, L = the presence of the mutation of the L8UV5 promotor of the lactose operon, Q = approximate length in percentage of that of the ~ phage.

5) Preparation of a modified h434 phage comprising both a fragment of the lactose operon and the X gene .

The EcoRI fragment containing the X gene- such as that of somato-statin - is inserted in the EcoRI site of a ~plac5-1 L8UV5 i n 5 h434 vector by in vitro recombination. This vector (Figure 11) possesses the o host specificity h434. The insertion of the [X] EcoRI fragment leads to the formation of a hybrid protein z'-x. The ~plac5-1 L8UV5 i21tSn 5 h434 vector is derived from a crossing between the ~apmalB21 h434 (recombinant be-tween ~pmalB21 and ~oh434) and ~p~lac5-1 L8UV5 n 5 (C.N.C.M. No. I-073).
The phage obtained, also named ~ZX, is shown in Figure 12a.

6) Preparation of the phage containing both the X gene and the lamB gene .

The phage containing the insertion of the X gene (Figures 12a) is crossed with a ~LBZ phage (shown both in Figure lOc and Figure 12b). The phages produced by in vivo recombination in the gene lacZ are located. The broken line of Figure 12 establishes the constituent parts of the recombin-ants obtained (Figure 12c). They are carriers of a double gene fusion which leads to the formation of a hybrid protein b' - 'z - y, which can be excreted, when these phages are expressed in a bacterium devoid of receptors for the phage, in which the abovesaid phage has previously been introduced.
When x is human somatostatin, it can then be recovered by treatment of the carrier bacteria, under conditions similar to those described in the ITAKURA and Coll. article mentioned above as regards the isolation of human somatostatin from the hybrid protein that these authors had obtained.
By constituting the X gene by an adenovirus DNA fragment 5 bearing EcoRI* ends (EcoRI star), the modified vector is available whose expression is liable of being manifested by the production of an extracytoplasmic hybrid protein comprising a protein with an antigen character corresponding to the adenovirus 5.

1~'725~34 Modifications permit the vector system described to accept a DNA
fragment of greater size. They consist for example of changing by in vitro recombination the early and late functions of the ~Z and the ~LBZ to obtain finally a derivative ~i tSninS LBZXh~ of which the DNA has a size less than that of the ~LBZX by more than 10%. In addition, it is possible to intro-duce deletions into the lacZ gene so as to reduce the size of the double hybrid. The deletions could also result in structural modifications of the double hybrid.
A second vector system can also be developed from phages li~e ~LBZ.
As a matter of fact, as soon as the DNA sequence corresponding to the start of the lamB gene (and hence to the signal peptide) is known, it is possible to contemplate the possibility of inserting an X gene directly into a good position in the lamB gene so that the hybrid proteins resulting from this fusion may be excreted.
The thus modified phages obtained or a portion of these phages com-prising notably the promotor of the lactose operon, the Z gene or the Z gene fragment and the DNA fragment including the lamB gene or a sequence of lamB
gene (or any other gene or sequence of gene coding the preparation of an extracytoplasmic protein) could be inserted into another vector system, for example, a plasmid or a bacterial chromosome.
Thus it would be possible for example to insert the vector phage or the portion of vector phage mentioned above, for example into a plasmid such as the pBR322 plasmid. Such modified vectors would be suitable for the expression of proteins of high molecular weight, such as for example chicken ovalbumin.
As is self-evident and as emerges already from the foregoing, the invention is in no way limited to those of its types of application and embodiments which have been more especially contemplated; it encompasses on the contrary all modifications, notably those where recourse would be had to cleavages at the level of other sites, for example those corresponding to other restriction enzymes, for example Sst enzyme.

-1~72584 ; It should also be mentioned that the word "gene" as used in the claims hereafter also extend to any DNA capable of coding the same protein - as the natural DNA, for instance to the double stranded DNA obtained by enzymatic synthesis starting from the corresponding messenger RNA.

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Claims (18)

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

1. A vector which carries the promotor of the lactose operon and of at least a fragment of the Z gene of this same operon which comprises an insert located in said Z gene or fragment of Z gene, or immediately downstream of the latter, said insert consisting of a sequence including at least the proximal portion of the lamB gene coding an extracytoplasmic protein located outside of the cell in which said vector can then be introduced or releasable in the cell-culture medium, the length of said extracytoplasmic gene sequence being suffi-cient to permit the excretion, under the control of the promotor of the lactose operon, if the hybrid protein liable of being expressed in said cell.

2. The vector of claim 1 wherein said gene coding an extracytoplasmic protein is capable of coding a bacterial receptor for a phage, or at least of a precursor thereof.

3. The vector of claim 2 wherein said sequence also comprises the distal portion of the K gene which, in B maltose operons, is associated with the lamB gene.

4. The vector of claim 2 which contains the S sequence of the lamB gene determining the bacterial receptor for the phage and a fragment close to the lamB gene and itself fused with a fragment of the gene of .beta.-galactosidase lacZ structure.

5. The vector of claim 2, wherein the size of the proximal fragment of said lamB gene corresponds to a protein fragment having a molecular weight reaching at least 2000.

6. The vector of claim 5 wherein the said size corresponds to a protein having a molecular weight reaching at least 20,000.

7. The vector of claim 5 wherein the said size corresponds to a protein having a molecular weight reaching at least 30,000

8. The vector of claim 5 wherein the said size corresponds to a protein having a molecular weight from about 20,000 to about 40,000.

9. The vector of claim 5 wherein the said size corresponds to a protein having a molecular weight from about 35,000 to about 40,000.

10. The vector of claim 1 which is derived of phage whose one or more corresponding bacterial receptors are distinct from that or those which correspond to the extracytoplasmic gene sequence which is inserted therein.

11. The vector of claim 1 which is derived of the h 434 phage and wherein said insert comprises at least a sequence including the proximal portion of the lamB gene.

12. The vector of claim 2 wherein the sequence coding said extracytoplasmic protein is formed from a fragment with two EcoRI sites comprising the lamB gene, normally contained in the maltose B operon.

13. The vector of any one of claims 1, 3 or 4 including also a fragment comprising itself the gene which codes a predetermin-ed foreign protein.

14. The vector of claim 9 wherein said fragment is inserted in said vector downstream of the gene sequence corresponding to a minimum receptor fragment required for the excretion, but upstream of the end which is the remotest from the lactose promotor either of (a) the Z gene or fragment of Z gene, or of (b) the gene sequence corresponding to the extracytoplasmic protein, in the case where this sequence would not he followed by a distal fragment of the Z gene in the direction of the translation.

15. Method for the manufacture and excretion of a predetermined protein which comprises the introduction of the vector of claim 1 into a host-cell, including the expression of the vector and recovering the hybrid protein excreted in the culture medium.

16. The method of claim 15 wherein said host-cell is a bacterium.

17. Method of producing vaccines with respect to antigen proteins, corresponding to that coded by the fragment included in the vector of claim 12 which comprises introducing said vector into an innocuous cell-host, inducing the expression of the vector and recovering the cell-strain carrying the extra-cytoplasmic hybrid protein comprising said antigen protein.

18. The method of claim 17 of producing vaccines with re-spect to viral antigen proteins.