EP0594776A1

EP0594776A1 - Plasmid replication origin increasing the number of copies of the plasmid containing said origin

Info

Publication number: EP0594776A1
Application number: EP92916263A
Authority: EP
Inventors: Gérard Devauchelle; Laurence Garnier; Martine Cerutti; Viviane Valverde; Jean-Michel Masson
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Recherche Agronomique INRA
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Recherche Agronomique INRA
Priority date: 1991-07-12
Filing date: 1992-07-10
Publication date: 1994-05-04
Also published as: FR2678947A1; WO1993001293A1; US5565333A; FR2678947B1

Abstract

L'invention est relative à une séquence d'ADN, dérivée par mutation d'une origine de réplication de type Co1E1, laquelle séquence est caractérisée en ce qu'elle comprend au moins l'une des mutations suivantes, définies par rapport à l'origine Co1E1 sauvage: une transversion Guanine -> Adénine en position 2976; une délétion d'une Guanine en position 3057. Cette séquence augmente le nombre de copies des plasmides qui la comportent. L'invention concerne également des plasmides comprenant ladite origine de réplication, et leurs applications à l'amplification de séquences d'ADN et à la production de polypeptides par génie génétique.The invention relates to a DNA sequence, derived by mutation from a Co1E1 type origin of replication, which sequence is characterized in that it comprises at least one of the following mutations, defined with respect to the wild Co1E1 origin: a Guanine -> Adenine transversion at position 2976; a deletion of a Guanine at position 3057. This sequence increases the number of copies of the plasmids which contain it. The invention also relates to plasmids comprising said origin of replication, and their applications to the amplification of DNA sequences and to the production of polypeptides by genetic engineering.

Description

ORIGIN OF PLASMIDIC REPLICATION INCREASING THE NUMBER OF COPIES OF PLASMID INCLUDING SAID ORIGIN

The invention relates to an origin of plasmid replication, and to plasmids comprising said origin.

Plasmids are fragments of extrachromosomal circular DNA, transferable from one bacterium to another, and whose replication takes place independently of that of the bacterial chromosome. A given plasmid can be present in a large number of copies inside a bacterial cell. The copy number is a genetic characteristic of each plasmid. For example, in ColE1 type plasmids (such as plasmids of the pBR, pUC families, etc.), the number of copies is under control of a region of DNA corresponding to the origin of replication of the plasmid ( ORI) which extends approximately between bases 2940 and 3130 (numbering of the bases of PBR322 proposed by PEDEN [Gene, 22 (1983) 277-280]. A part of this region, located between bases 2970 and 3089 is transcribed in RNAs called ARNI and RNAII, RNAI, in particular, would play a role in regulating the copy number of the plasmid.

Plasmids are commonly used in genetic engineering as vectors for the cloning and expression of foreign genes in bacteria.

It is particularly desirable, for this purpose, to have plasmids present in a large number of copies, either to obtain the foreign DNA in a large quantity (for example to allow its sequencing), or to increase the quantity of the expression product. of said foreign gene.

A conventional technique for obtaining a large quantity of plasmids is to add chloramphenicol to bacterial cultures, which prevents the cell multiplication by blocking translation, without affecting plasmid replication. However, since chloramphenicol also interferes with the translation of the foreign gene, this method is not optimal when an increase in the expression of said gene is sought.

Plasmids carrying a mutation affecting the number of copies have been described in the literature. BOROS et al. [Gene, 30, 257-260 (1984)] thus describe a mutant plasmid derived from pBR322. The number of copies of this plasmid per cell is increased by approximately 200 times compared to the number of copies of pBR322. This increase in the number of copies results from a G → T transversal located at position 3075 on the HinfI fragment 2846-3363, near the 2 'end of the sequence transcribed into RNA I. MUESINO et al. [Cell, 24, 235-242, (1981)] had previously demonstrated the same mutation in the plasmid ColEl (whose origin of replication is similar in sequence to that of pBR322), with also the effect of an increase in number of copies of said plasmid (up to 300 per cell).

However, such an amplification of the number of copies has certain disadvantages which limit its practical applications. This results in fact:

an increased risk of toxicity for the host bacterium, by too strong expression of the recombinant genes carried by the plasmid, which is also generally accompanied by the production of precipitation granules and moreover prevents the expression from being considered perimasmic proteins;

- an increased probability of uncontrolled recombinations between plasmids, in particular for plasmids carrying certain specific sequences of phage origin, for example). Now, during mutagenesis experiments on the plasmid pUC9, the inventors obtained plasmids carrying mutations in the ORI sequence of the origin of replication, and found that, surprisingly, the number of copies of these plasmids is 10 to 25 times greater than that of the original plasmid.

The inventors have sequenced the origins of replication of the mutated plasmids, and have located the corresponding mutations.

The subject of the present invention is a DNA sequence, derived by mutation from an origin of replication of the ColE1 type, which sequence is characterized in that it comprises at least one of the following mutations, defined with respect to wild type ColE1 origin:

- a Guanine → Adenine transversion at position 2976

- a deletion of a Guanine in position 3057. The position of the mutations is indicated according to the numbering of the bases of pBR322 proposed by PEDEN

[Gene 22 (1983) 277-280].

According to a preferred embodiment of a DNA sequence in accordance with the invention, said sequence carries the two mutations defined above.

FIG. 1 shows in (la) the sequence that region 2970-3089 of an origin of wild replication of pBR 322, and in (lb) a sequence in accordance with the invention, carrying the two mutations defined above.

The inventors have further observed that a mutated sequence in accordance with the invention could be inserted into other plasmids in place of the corresponding sequence of the origin of initial replication, and that this resulted in an increase in the number copies of the recombinant plasmids thus obtained.

The present invention therefore includes plasmids characterized in that their origin of replication, ColE1 type, includes a mutated sequence as defined above.

Figures 2, 2 and 4 show plasmids according to the invention respectively called pLMl, pLM2 and pLM3. The plasmids pLM1, pLM2 and pLM3 derive from the plasmid pUC9 by mutation, as defined above, from the origin of replication, and also contain the complete gene for β-galactosidase, framed by a polylinker, and placed under the control of the promoter of the LacZ gene.

Other plasmids in accordance with the invention can be easily obtained, for example, from plasmids carrying an origin of replication of the ColE1 type, by a directed mutagenesis technique, indeed from a plasmid such as pML1 by excising from the latter the mutated DNA segment and inserting it in place of the corresponding wild-type DNA segment of the recipient plasmid. It will appear to a person skilled in the art that it is also possible to obtain a DNA segment in accordance with the invention by oligonucleotide synthesis, according to techniques known in themselves and to insert it into the recipient plasmid.

The techniques allowing this insertion also feel known in itself. It is possible to cite for example the techniques using homologous recombination, or else the excision of a restriction fragment carrying the non-mutated origin and its replacement by a fragment carrying the mutated origin.

FIG. 5b represents the plasmid pARA 15 thus obtained from a plasmid called pARA 14

(FIG. 5a / which is an expression vector comprising the origin of replication of pBR322, a gene for resistance to ampicilin and a promoter inducible by arabinose.

Receptor plasmids which can be used for the construction of plasmids conforming to the invention are generally, all the plasmids compatible with an origin of replication of the ColE1 type

The number of copies of the plasmids thus obtained is at least multiplied by 10 with respect to the corresponding "wild" plasmids. The expression of foreign genes placed in these plasmids increases in the same proportions. The promoters that control the expression of these genes remain inducible.

In addition, this relatively moderate increase in the number of copies is optimal for obtaining a satisfactory expression of an exogenous gene inserted into the plasmid, insofar as it makes it possible to avoid the drawbacks (mentioned above with regard to the plasmids obtained by BOROS et al.) resulting from too many copies.

The host strains suitable for the multiplication of the plasmids in accordance with the invention and for the expression of the genes carried by these plasmids are the same as those allowing the multiplication of the corresponding wild plasmids and the expression of the genes which they carry, and the behavior and growth of the strains transformed by the plasmids in accordance with the invention are identical to those of the strains carrying the wild plasmids.

In addition, there is no reversion of the mutation, and the phenotype characterized by the increase in the number of copies remains stable over the generations.

The present invention further relates to a process for the multiplication of plasmids in accordance with the invention, which process is characterized in that, in a first step, the transformation of an appropriate host bacterial strain is carried out by at least one of said plasmids, and in a second step, the culture of said bacterial strain is carried out. The invention also includes:

- A method of amplifying a DNA sequence, which method is characterized in that, in a first step, said sequence is inserted into a plasmid according to the invention, and in that, in a second step, one proceeds to the multiplication of said plasmid, as indicated above.

- A process for the production of polypeptides by genetic engineering, which process is characterized in that, in a first step, the gene encoding said polypeptide is inserted into a plasmid according to the invention, in a second step, one proceeds to the transformation of an appropriate bacterial host strain with said plasmid, and in a third step, one proceeds to the culture of said bacterial strain under conditions suitable for the expression of said gene.

The present invention will be better understood with the aid of the additional description which follows, which refers to examples of obtaining plasmids carrying the origin of replication in accordance with the invention.

It goes without saying, however, that these examples are given solely by way of illustration of the object and of the invention, of which they in no way constitute a limitation.

EXAMPLE 1: OBTAINING PLASMIDES CONFORMING TO

THE INVENTION

The plasmid pARA 15 is derived from the plasmid pARA 14 by replacing the origin of initial replication of the latter (which is that of pBR322) by the origin of mutated replication in accordance with the invention. For this, a 2060 bp SstI / BglI fragment of pARA 14, comprising its origin of replication, is excised and replaced by a 1430 bp SstI / BglI fragment from pLM3, comprising the origin of replication in accordance with the invention. FIG. 5 represents the plasmid pARA 14 (5a), carrying an origin of wild-type replication, and the plasmid pARA 15 (5b) carrying an origin of replication in accordance with the invention.

In the plasmids pARA 14 and pARA 15, the foreign genes which it is desired to express are under the control of a promoter inducible by arabinose.

EXAMPLE 2 EVALUATION OF THE NUMBER OF COPIES OF PLASMIDS CONFORMING TO THE INVENTION

a) Measurement of the quantity of plasmid DNA

The DNA was extracted from cultures of E. coli TG1 cells (2 ml), harvested at 2.3 U OD _{6 0 0} . The cells were lysed with a mixture of lysozyme, detergent and sodium hydroxide. After centrifugation, the plasmid DNA contained in the supernatant was separated from the proteins by phenol extraction followed by treatment with chloroform. The pure plasmid DNA is then precipitated with ethanol, the precipitate is centrifuged and dried under vacuum, then redissolved in 50 μl of buffer (Tris 10 mM, EDTA ImM pH8) for each sample. Extracts of E. coli TG1 cells containing either the plasmid pARA 14 (control strains) or the plasmid pARA 15 were prepared in this way.

5 μl of each preparation were incubated for 2 hours at 37 ºC with 5 units of XhoI restriction enzyme. The hydrolysis product was then deposited on a 1% agarose gel and subjected to electrophoresis in TBE buffer (containing 20 μg of ethidium bromide per liter) for 2 hours at 250 volts. The gel is then subjected to irradiation with ultraviolet rays. The intensity of the fluorescence emitted by the DNA bands in the gel is proportional to the quantity of DNA, for a given fragment size, and therefore reflects the relative number of copies of plasmids. Examination of the gels shows that there are at least 10 times more plasmid DNA with the strains carrying the mutated plasmid than with the control strains.

EXAMPLE 3 MEASUREMENT OF THE EXPRESSION OF EXOGENOUS GENBS INSERTED IN PLASMIDS CONFORMING TO THE INVENTION.

a) Expression of the β-lactamase gene

In another experiment, the β-lactamase (expressed from the bla gene present on each of the plasmids pARA 14 and pARA 15 and conferring resistance to ampiciiline) produced in the exponential growth phase was assayed for an E. coli strain TG1 containing either the plasmid pARA14 or the plasmid pARA15.

Cultures of 5 and 10 ml, in LB broth supplemented with 100 μg of ampiciilin per ml, were moculated with 250 μl of a saturated culture of E. ccli TG1 containing either pARA 14 or pARA 15. After one hour of culture with stirring at 37 ° C., the cells are harvested by centrifugation and subjected to osmotic shock. The osmotic fluid thus obtained, with a volume of 400 μl, contains all of the β-lactamase produced.

On each of the fluids, a measurement of the total quantity of proteins is carried out, and a determination of the β-lactamase activity. This is operated according to the protocol described by O'CALLAGAN et al. (Antimicrobial Agents and Chemotherapy, 1972, 1, 283-288), using Nitrocefine (Oxoid) as a substrate.

The results are shown in Table I below:

We know that the expression of the bla gene is constitutive. The production of β-lactamase in the exponential growth phase is therefore directly proportional to the number of copies of the gene, therefore of the plasmid which carries it. It can be concluded from this that the E. coli TG1 strain which carries pARA15 hosts 15 to 20 times more copies of plasmid than the E. coli TG1 strain which carries pARA14, under identical culture conditions.

b) Expression of the β-galactosidase gene Two constructs were tested: one with the β-galactosidase gene alone in pLM3, the other with the β-galactosidae gene fused downstream of the protease gene of the HIV 1 virus in the plasmid pARA 15 (pARA15PR107β-gal).

The control plasmids are on the one hand pUC 9 and on the other hand the plasmid pARA14PR107β-gal which differs from pARAl5PR107β-gal only by its origin of replication, which is that of the "wild type", from pBR322).

Β-galactosidase assay protocol: 5 ml of LB medium supplemented with 100 μg of ampiciilin per ml, were inoculated with 250 μl of a saturated culture and then incubated with shaking at 37 ºC. When the DO ₆₀₀ reached 3.4, the induction is carried out with 2 mM of IPTG for the strains hosting pCU9 or pLM3, eu 0.2% of arabinosis for the strains hosting pARA14PR107β-gal or pARA15PR107β-gal. The β-gaiactosidaεe activity is then assayed in culture samples taken at different incubation times (0 min, 40 min, 120 min.)

Under these conditions, and after 2 hours of incubation, the level of β-galactosidase obtained in the cell pellets is approximately 16 to 20 times higher with pLM3 than with pUC9, and approximately 2.5 times higher with pARA 15 that with pARA 14.

For these last two plasmids, measuring the expression of the β-galactosidae gene makes it possible to know the rate of increase in the production of the KIV-1 protease in the E. coli MC1061 strain hosting these plasmids. This protein is difficult to produce in E. coli, being known to be toxic to the bacteria. The 2.5-fold increase in production is therefore very significant.

Claims

1) DNA sequence, derived by mutation from an origin of replication of the ColEl type, which sequence is characterized in that it comprises at least one of the following mutations, defined with respect to the origin

Wild ColEl:

- a Guanine → Adenine transversion at position 2976

- a deletion of a Guanine in position 3057. 2) DNA sequence according to Claim 1, characterized in that it carries both the transversion in position 2976 and the deletion in position 3057.

3) Plasmids characterized in that their origin of replication comprises a mutated sequence according to any one of Claims 1 or 2.

4) Process for the multiplication of plasmids according to Claim 3, which process is characterized in that, in a first step, a suitable host bacterial strain is transformed with at least one of said plasmids, and in a second step, one proceeds to the culture of said bacterial strain.

5) Method for amplifying a DNA sequence, which method is characterized in that, in a first step, said sequence is inserted into a plasmid according to Claim 3, and in that, in a second step , one proceeds to the multiplication of said plasmid, by the method according to Claim 4.

6) Method for producing polypeptides by genetic engineering, which method is characterized in that, in a first step, the gene encoding said polypeptide is inserted into a plasmid according to Claim 3, in a second step , we give way to the transformation of a bacterial host strain appropriate by said plasmid, and in a third step, the culture of said bacterial strain is carried out under conditions suitable for the expression of said gene.