FR2602792A1 - Process for stabilising a plasmid contained in a bacterial strain and strain obtained - Google Patents

Abstract

The present invention relates to a process for stabilising a plasmid vector contained in a bacterium, characterised in that the bacterium contains a dap<-> chromosomal mutation and in that the plasmid vector carries a dap<+> gene.

Description

The practical application of recombinant DNA technology to the production of molecules of interest by microorganisms generally comes up against the problem of the instability of recombinant plasmids.

The cloning and expression vectors commonly used in the laboratory are generally multicopy plasmids and their stable transmission to the offspring is ensured by the large number of plasmids per cell genome (Jones et al. 1980). However, the introduction of foreign genes into these plasmids results in varying degrees of instability during bacterial multiplication cycles. However, industrial productions may require cultures of 1000 liters, resulting in more than 1016 cells, after more than 50 generations. It is therefore essential to stabilize the plasmids in the bacteria to ensure their presence and therefore the expression of the foreign molecule, until the end of the culture in a fermenter.

Stabilization of plasmids by integration of a gene encoding resistance to an antibiotic is a standard laboratory practice but which cannot be applied for industrial use for various reasons.
- the use of a strain of bacteria resistant to an antibiotic may represent a risk for the environment
- the amount of antibiotic needed during cultivation considerably increases the production short
- the use of antibiotics cannot be considered for the production of substances for human or veterinary therapeutic use.

It is therefore essential to develop other methods of selecting bacteria carrying a recombinant plasmid. The few models that have already been applied are based on the same principle: the host cell is mutated (auxotrophic mutation or introduction of a lethal gene for the bacteria) so as to prevent its multiplication in the absence of a plasmid encoding a trait that complements the host's deficiency.

For example, Skogman and Nilson (1984 and 1985) used the
complementation of a thermosensitive val S mutation by the val S gene
carried by a plasmid; in this model, the stability of the plasmid, which carries
between the tryptophan operon, is complete after 200 generations at temperature
non-permissive, while 1.2% loss of the plasmid is observed for each
generation under non-selective conditions (300C).

Miwa et al. (1984) used a mutant of
Streptomycin-dependent E. coli (Smd) and a plasmid carrying a gene (rpsl of a SmR strain) which masks the Sm d phenotype and makes the strain independent of streptomycin, ensuring a stability of the plasmid greater than 99%.

Herthberger and Rosteck (1984) made a bacterium lysogenic for a lambda prophage deleted from its repressor; the cell can escape lysis only in the presence of a plasmid carrying the lambda repressor ci.

We have developed a new selection model based on the complementation of a dap chromosomal mutation by a dap + plasmid gene, which ensures the survival of only cells that carry the plasmid.

Diaminopimelic acid (DAP) is a component of the lining of bacteria; it is also an intermediate in the biosynthesis of lysine from aspartate. A strain deficient in an enzyme for the biosynthesis of DAP will not be able to multiply in minimum medium; the addition of lysine to the medium will allow growth but will rapidly lead to lysis of the cell which does not incorporate DAP into its membrane (Work 1950 - Davis et al., 1973).

The deficiency in an enzyme in the biosynthesis of DAP can be complemented by the introduction of the corresponding gene on a plasmid and, in particular, on the vector for expressing the foreign protein which is to be produced. If the bacterium loses the plasmid, it reverts to dap and cannot multiply. This model has the advantage of being able to be applied to any culture medium devoid of DAP, that is to say any industrial production medium and any rich or minimum medium with added lysine.

The system also has the advantage of not disrupting the synthesis of DNA, RNA or proteins.

We constructed a dapD- strain (deleted of one of the 9 genes of the lysine biosynthetic pathway, the D gene, corresponding to tetrahydropicolinate-N-succinyl transferase) and introduced the dapD gene on a commonly used expression plasmid. (carrying the gene to be expressed under the control of the PL promoter). Under selection condition, it can be shown that the plasmid is stable for at least 150 generations.

The cloning of another gene (dapA) encoding an enzyme for the biosynthesis of lysine on a plasmid has already been carried out, with the aim of increasing the production of lysine (Dauce-Le Reverend et al. 1982) but these The authors do not control the stability of their plasmid.

The E. coli dapD gene has already been cloned into the pBR322 plasmid and its nucleotide sequence has been published by Richaud et al.

(1984) but the aim of this work is only the study of the regulation of this gene.

This is why the present invention relates to a method for stabilizing a plasmid vector contained in a bacterium, characterized in that the bacterium comprises a chromosomal dap mutation and in that the plasmid vector carries a dap + gene.

Among the dap chromosomal mutations, use will preferably be made of dapD but other genes encoding an enzyme for the biosynthesis of DAP could be used on condition that a vector plasmid carrying the corresponding gene is provided; in the case of a dapD strain, the gene to be inserted into the plasmid will be dapD.

The plasmids can be used more particularly for the expression of proteins of industrial interest, for example hirudin or gamma interferon as will be described below, but the dap stabilization system does not interfere with the expression of protein, it can therefore be used with any expression vector.

In the following, the expression system comprises the left promoter of phage lambda, but it could be another promoter active in the bacterium in question. This explains why a complete description of the elements of expression of the heterologous protein will not be given, this type of plasmid now being widely known. it will simply be necessary to insert the dapD or other gene into a non-essential site of the plasmid vector.

The offending plasmids can be used to transform any E. coli strain rendered dapD.

Thanks to the process according to the invention, bacterial strains are obtained in which the expression plasmids are stable in complete medium without the need for selective pressure by an antibiotic or a particular medium devoid of an amino acid, for example. In addition, the method according to the invention exerts a counter-selection against bacteria which have lost the plasmid.

The invention therefore also relates to the bacterial strains, in particular of E. coli dap, transformed with a plasmid vector carrying a dap + gene and the elements ensuring the expression of a protein of industrial interest carried by this plasmid.

The invention also relates to a process for the preparation of a protein of industrial interest from a bacterium, in which a bacterium transformed by a plasmid according to the invention is cultured on a complete medium, said plasmid further comprising the. gene for said protein and the signals controlling the expression of this protein in the host bacterium.

The aim of the examples below is to demonstrate the stability of plasmids carrying the dapD gene in dap bacteria in comparison with plasmids carrying the Ampr gene.

These plasmids carrying the dapD gene were also used
to express the genes encoding hirudin and interferon gamma.

These two genes are placed under the control of the PL promoter of
lambda phage and host strains of E. coli contain the repressor
thermosensitive C1857; this system makes it possible to induce the expression of the gene controlled by PL by raising the temperature.

The strain of E. coli TGE 900 which comprises the repressor C1857 was modified to become dapD giving the strain TGE 7615.

The following plasmids were then prepared according to the attached diagrams
Amp r dapD Foreign protein
Figure 1 - pTG764 + + 0
Figure 2 - pTG720 + 0 Hirudin
Figure 2 - pTG771 + + Hirudin
Figure 3 - pTG775 0 + Hirudin
Figure 3 - pTG776 0 + Hirudin
Figure 4 - pTG766 0 + 0
Figure 4 - pTG767 0 + 0
Figure 5 - pTG7671 0 + IFN gamma
Figure 5 - pTG7672 0 + IFN gamma
METHODS
The various enzymes are, unless otherwise indicated, used according to known techniques.

Transformation
Competent cells were prepared according to the method of
Hanahan (1983). Their competence amounts to 104 to 105 transformants / g 6
nN for the RL58 strain (Bollen et al. 1979) and 105 to 106 transformantstpg
DN for strain TGE 7615.

In general, to 0.2 ml of a stock of competent cells are added 1 to 10 μl of a solution having the appropriate dilution, of a DNA preparation containing the plasmid which is to be introduced into the strain. . The cells are left to react with the DNA for 15 minutes at 0 ° C. and then incubated at 370 ° C. for 90 seconds. They are returned to OOC for 5 minutes and then 0.8 ml of LB medium is added thereto. For strain RL58 or for strain TGE 7615, this medium must contain DAP at a rate of 8 gamma / ml. This is necessary for the growth of dapD cells, even those which contain the plasmid carrying the dapD gene. Cells are generally incubated at 30 "C with strong shaking (however, they can be set at 37" C if the plasmid does not contain the PL promoter). The incubation time is 60 minutes, but it can be extended to 90 minutes for growth at 300C for the TGE 7615 strain.

Then fixed volumes (0.1 ml) are plated out on solid LB medium. For the plasmids containing the gene for resistance to ampicillin, the clones are selected by adding 100 gamma / ml of ampicillin; clones containing the dapD gene are selected, from a culture which is dapD as a whole, is dapD as a whole, on LB medium without further additions. The dishes are incubated for 24 hours at 30 ° C. The colonies are then subcultured with a toothpick in 3 ml of LB medium and after growth overnight at 30 ° C. the plasmid is isolated and its structure confirmed on agarose gel. , after digestion with the appropriate restriction enzymes.

Plasmids containing the dapD gene can be transformed into any E. coli which carries the dapD mutation (e.g. RL58,
TGE7615); if, in addition, they contain the PL promoter of bacteriophage lambda, they must necessarily be transformed into strains of E.

coli dapD which express the bacteriophage lambda C1857 repressor, either on a plasmid2 or in the chromosome, or the C1 and a Rec mutation (eg RecA441).

EXAMPLE I
Introduction of a mutated dapD Rene in the E. coli strain TGE900
The E. coli TGE900 host strain was rendered dap by conjugation with a known dapD mutant, RL58.

Strain TGE900 is a derivative of strain N4830 described by
Gottesman et al. (1980) whose characteristics are: su F- his ilv bio (acul857 a Bam ÔHI) Smr. It is commonly used as a host for expression vectors in which the foreign gene is placed under the control of the lambda PL promoter because it makes it possible to induce expression at will by an increase in temperature (greater than 370C ) which inactivates the heat-sensitive repressor (lambda c1857) carried by the bacteria.

The dap RL58 strain (met B dapD2 met D279 Hfr P4 X) was described by Bollen et al. (1979).

From this strain, a spontaneous mutant resistant to trimethoprim was selected (after spreading the strain on a dish containing 2 gamma / ml of trimethoprim and confirmation of the resistant character of a colony chosen in medium containing 4 gamma / ml of trimethoprim). In fact, the gene encoding this resistance is sufficiently close to the dapD mutation so that if the recipient strain, after conjugation, becomes resistant to trimethoprim, it has also become dapD.

The resistant strain was named TGE755; its useful characteristics are Hfr dapD Time.

After conjugation of the strains TGE900 oe SmreS dap +) and
TGE755, interrupted after 15 minutes, by spreading on minimum medium containing DAP, streptomycin and trimethoprim, Sm r Tmp r colonies are selected, the dap character of the strains selected on LB medium is then checked for the presence of auxotrophic mutations of the parental strain (his ilv val) on minimum medium + DAP.

One strain (his ilv dap-) was selected: Tue7615.

EXAMPLE 2
Construction of a vector plasmid carrying the dapD gene of E. coli (Figure 1)
a) Construction of a multiple-use cloning plasmid
comprising a 12 restriction site polylinker
The construction was initiated from the plasmid pML2 (Lusky and Botchan, 1981), derived from pBR322 by deletion of nucleotides 1089 to 2491. This plasmid retained the origin of replication of pBR322 and the gene for beta lactamase (resistance to ampicillin).

The recognition sequence of the psti enzyme was eliminated by insertion of an Ahalll-Ahalll fragment of pUC8 which carries a Pstl mutation induced by ethanesulfonate (Vieira and Messing, 1982), between two AhalII sites of pML2, which deletes 19 base pairs from pML2. The resulting plasmid, pTGr90, carries the PstI mutation of pUC8.

A BglII linker (5'-dCAGATCTG-3 '; Collaborative Research) was inserted into the unique Nrul site, by the “linker tailing” technique described by Lathe et al., 1984. The resulting construct is pTG191.

The pTG191 was opened by EcoRI and BglII (which deletes the tetracycline resistance gene) and religated with the segment
EcoRI-Bglll from phage M13TG131 (Kieny et al., 1983) which comprises a polylinker with 12 restriction enzyme recognition sequences.

the resulting plasmid is pTG192.

b) Cloning of a dapD gene in the plasmid pTG192
The E. coli chromosomal dapD gene was inserted into the plasmid pACYC184 to give pDB6 (Bendiak and Friesen, 1981). This dapD gene was recovered from pDB6 in the form of a 1.3 kb Alul fragment and inserted into the EcoRI site of pTG192 (the EcoRI ends having previously been repaired by treatment with the Klenow fragment of DNA polymerase 1 ).

Among the resulting plasmids, pTG764 is chosen in which a single EcoRI site is reconstituted. pTG764 therefore carries the dapD gene and the ampicillin resistance gene of pTG192 (originally from pUC8).

EXAMPLE 3
Hirudin expression plasmid containing the dapD and Amp r genes (Figure 2)
Plasmid pTG720 described in patent No. 84 04755 essentially comprises the hirudin gene under the control of the lambda PL promoter.

Plasmid pTG720 digested with Bglll and Bgll gives a 2.74 kb fragment which carries the hirudin gene and part of the bitter gene. This fragment, treated with phosphatase, is religated to the BglII-BglI fragment of the plasmid pTG764 which carries the dapD gene and another fragment of the ampr gene. The resulting plasmid, pTG771, carries the reconstituted complete amp gene, the dapD gene and the gene encoding hirudin.

The TGE7615 colonies transformed with this plasmid can be selected either on LB medium (selection for the dap + character) or on LB + ampicillin medium.
After induction, the transformed strain produces Hirudin.

EXAMPLE 4
Hirudin expression plasmid containing the dapD gene and no longer containing Vamp '(figure 3)
The starting plasmid is pTG771.

Digestion is carried out with Ahalil to remove the small fragment comprising the 3 'end of the gene encoding Amp and the plasmid is closed by ligation in the presence of an EcoRI "linker":
CCGAATTCGG
The plasmid pTG775 is obtained.

EcoRI digestion followed by ligation completely eliminates the gene encoding Ampr.

The plasmid pTG776 is obtained.

The TGE7615 bacteria transformed with this plasmid can be selected in LB medium, without addition (selection for the dap + character).

After induction, the transformed strain produces hirudin.

EXAMPLE 5
Construction of plasmids carrying the dapD gene and not including the Vamp 'gene (Figure 4)
The starting plasmid is the plasmid pTG192.

After digestion with Semai and Ahalil, then treatment with phosphatase, the 0.95 kb vector fragment is isolated and ligated with the 1.3 kb AluI fragment of pDB6 which carries the dapD gene. The amp r gene has therefore been completely deleted. The strain TGE7615 is transformed with this new plasmid and it is selected on LB medium to obtain clones which carry the dapD gene.

Plasmids are obtained which carry the dap gene in both orientations; The orientation of the insert can be determined by digestion with Pstl. For two selected candidates. pTG766 releases fragments of 1.34 kb and 0.9 kb. pTG767 releases fragments of 1.85 kb and 0.4 kb.

EXAMPLE 6
Interferon gamma expression plasmid carrying the dapD gene (Figure 5)
Plasmid pTG40 (PED) carries the gamma interferon gene on a 1.2 kb Hindi1 fragment under the control of the PL promoter.

After digestion with HindIII, this fragment is recovered and inserted into the HindIII site of pTG767 previously treated with phosphatase. After ligation, TGE7615 is transformed and selected for the presence of the dapD gene on LB medium. Two plasmids are retained which carry the insert in opposite orientations. pTG7671 (which has the PL proximal to the origin of replication and in the same
orientation) and. pTG7672.

The TGE7615 bacteria transformed with these plasmids can be selected in LB medium, without addition (selection for the dap + character).

After induction, the transformed strain produces gamma interferon.

The following examples are intended to demonstrate the characteristics of the strains transformed according to the present invention.

All the plasmids carrying the dapD gene are found in the Tue7615 strain, the others are in the TGE900 strain.

The stability of the various plasmids was tested on the appropriate media - the LB medium will be called the selective medium for the plasmids according to the invention and the LB + ampicillin medium for the plasmids pTG720 and pTG740 - the non-selective medium will be called the LB + DAP medium. for the plasmids according to the invention and the LB medium for the other plasmids.

A stability study is carried out over 110 generations, by several successive cycles of dilution and multiplication of the bacteria. At the end of this multiplication phase, the stability of the plasmid is measured as follows - the number of viable cells in the culture is measured, by appropriate dilution and counting of the cells in non-selective agar medium; a significant sampling of these colonies is subcultured in parallel on selective and non-selective agar media and the percentage of colonies having lost the plasmid and therefore the capacity to grow on selective medium is determined.

The results obtained with the plasmids carrying the interferon gene are presented in Table 1:
TABLE 1
Evaluation of the loss of the plasmid by bacteria
after 110 generations
Strain TGE900 TGE7615 TGE7615 TGE7615
Plasmid pTG40 pTG766 pTG767 pTG7671
Amp selection dapD dapD dapD
LB medium 2.10-3 2.10-3 <5.10-5 <5.10-5 3.10-5 p- / c / generation *
LB + DAP medium 2.1o-3 C <5.10-5 <5.10-5 (<5.10-5 3.10-5 3.10-5 8 * c = number of viable cells
p- = cells that have lost the plasmid
p- / c / generation = (number of p- / number of c tested) x number of
generations
Equivalent tests carried out with the plasmids carrying the hirudin gene and with the cloning vector pTG766, after multiplication of the bacteria for 170 generations in LB medium, give the following results
TABLE 2
Evaluation of the loss of the plasmid by bacteria
after 170 generations in LB environment
Strain TGE900 TGE7615 TGE7615 TGE7615
Plasmid pTG720 pTG771 pTG776 pTG766 p + (%) 96 98 100 100 p- / c / generation 2.1 2.10-4 1.5.10-4 <1.1.10-4 <1.1.10-4 <
The results shown in Tables I and 2 show
10) that the stability of the dap vectors is improved by a factor of 10 compared to the plasmid carrying the ampr gene
20) that the difference between selective and non-selective medium is minimal
30) that the pTG766 and pTG767 cloning vectors have a loss of less than 5.10-5 p- / c / generation ;;
4) that the stability of pTG40 is markedly lower than that of the dap + vectors, but it should be noted that the phenomenon is especially marked after 50 generations; at the start of culture, the loss is only 2.5.10-4 p- / c / generation
5) that the loss of the control plasmids ampr, pTG40 and pTG720, carrying the interferon and hirudin genes respectively, is of the same order of magnitude; this loss of plasmids over time is reduced when they carry the dapD gene (pTG766, 767, 7671 and 776).

However, it should be noted that the stability of the plasmid carrying the dapD gene and the ampicillin resistance gene (pTC771) is less good and remains comparable to that of pTC720. This result is interpreted by the analysis of the plasmid content on agarose gel; this shows that pTC771 tetramerizes, a phenomenon which has repercussions on the partition of the plasmids and which makes their loss higher (Summers and Sherratt, 1984). It should be added that over a reduced number of generations (30) this multimerization phenomenon does not occur and that over a total of 70 generations the loss of pTG771 remains less than 2.8.10 4 p- / c / generation.

Therefore the plasmids which contain the dapD gene see their stability increased compared to the plasmids carrying the amp r gene and their loss becomes minimal.

EXAMPLE 8
Stability of plasmids at 37 or 42 "C
The influence of temperature on the stability of plasmids cannot be studied, for those which contain a gene encoding a foreign protein placed under the control of the PL promoter without inducing the expression of this foreign protein. Nevertheless, it should be noted that under the conditions in which the induction is being worked on, it is followed by an increase in OD from 0.3 to 3.6, which corresponds to 3.6 generations.

So, in fact, the bacteria make the greatest number of generations at 30 ° C, before induction, with a loss that was already determined in the previous example.

When the expression of a foreign protein (for example hirudin for pTG720 and interferon gamma for pTG40) is induced, we observe, after a certain time (2 to 4 hours), a mortality of the culture of E. coli at 90% to 95%. These are cells containing the plasmid (since the p- host cell is capable of growing at 37 and 42 ° C).

This mortality necessarily increases the proportion of p- cells relative to living cells by a factor of 10 to 20. When all of the living cells have greatly diminished and the p- cells form a significant fraction of the population, the multiplication of the p- cells is measured, which greatly reduces the p + / p- ratio (see, for example, Table 3, the results obtained after 5.30 hours and 7 hours).

It is obvious that the parameter p- / cell / generation has lost its meaning under such conditions since the p + no longer multiply.

The following parameter is proposed: p- / ml / unit of optical density, which measures only the p- cells at each time of culture. In addition, it makes it possible to compare two different cultures for their loss of plasmid.

This parameter is calculated according to the following formula
p- / ml / OD = C1 - (% p + / 100) 1 x c. viable / ml / OD.

Finally, if we relate this parameter to the total number of cells (which is 4,108 under the present conditions) we can obtain the percentage of cells (Fp-) in the culture and estimate the degree of contamination of the culture by cells. p- at every moment. The Fp is calculated as follows
Fp- = 4.108 c / ml / OD x 100 / (p- / ml / OD).

Fp- is an objective parameter which makes it possible to decide whether a culture intended for the production of a molecule is sufficiently pure to warrant further development and which makes it possible to compare different plasmids with each other under the conditions of induction.

We will, in the inductions described in the following examples, measure the viable cells, the percentage of p + and then calculate the p- / ml / OD and the Fp-. These data will be presented in tabular form.

EXAMPLE 9 induction of hirudin
We will present the results relating to the induction of hirudin, first with a vector containing the gene for resistance to ampicillin, then with the vector also containing the dapD gene.

1) Stability of plasmids
a) Induction of the expression of hirudin in a vector which
contains the ampicillin resistance gene (TGE900 / pTG720)
in LB at 37 "C
Typical results for TGE900 / pTG720 are shown in Table 3.

TABLE 3
Stability Parameter of Plasmids During Expression
at 42 "C of hirudin in TGE900 / pTG720
Time in hours c / ml / OD% p + p- / ml / OD Fp- (%) 0 h 8.3.107 100 C1.7.106 <0.4 1 h 30 2.4.108 100 <4.8.106 <1.2 3 h 2.1.108 96 8.4.106 2.1 4.30 h 4.7.107 82 8.5.106 2.1 5.30 h 1.8.107 48 9.4.106 2.35 7 h 2.8.107 20 2.2.107 5.5
It is seen that after 3 hours the number of viable cells per unit of OD decreases at the same time as the proportion of p +. After 5:30 a.m.
Fp- (% of p- relative to all of the cells) is of the order of 2.35%.

After 7 hours, this number will have doubled and it is likely that this is due to the growth of p- cells only.

b) induction of the expression of hirudin in a vector which
contains the dapD gene (TGE7615 / pTG771) in LB at 37 "C
The results shown in Table 4 are comparable to those obtained for pTG720.

TABLE 4
Stability Parameters of Plasmids During Expression
at 42 "C of hirudin in TGE7615 / pTG771
Time in hours c / ml / OD% p + - p- / ml / OD Fp- (%) 0 h 3.4.106 99.8 7.7.105 0.2 1 h 30 2.1.107 99.2 lJ.l05 0, 04 3 h 9.7.106 100 (1.0.105 <0.025 4 h 9.3.106 100 <8.0.104 (0.005 5 h 7.5.106 98 1.5.105 0.04
It can be seen in Table 4 that despite a mortality comparable to that recorded for TGE900 / pTG771, the loss of plasmid (% p +) is low (after 5 hours 98% p +) as is the absolute quantity of p- cells (p- / ml / OD). After 5 hours the Fp- is quantified at 0.04%, which is 10 times less than for pTG720 at the same time. This shows the better stability of pTG771 compared to pTG720. It should be remembered that in an induction experiment pTG771 does not tetramerize and that its loss is assumed to be less than that measured over 170 generations at 30 ° C. (see Table 2).

The results show greater stability of the plasmid pTG771 than that of pTG720.

2) Plasmid content
Plasmids pTG1'20 and pTG771 were isolated at different times during induction. In general, at the start of induction, in the exponential phase, a low plasmid content per cell is observed, which increases significantly over time. During this same period, there is production of hirudin.

Interestingly, this increase in plasmid content occurs in a population where over 95% of the cells have died.

3) The induced activity
The hirudin activities produced from pTG720 or pTG771 are not significantly different. The values (in antithrombin units, ATU) are 2720 ATU / 1 / OD for pTG720 and 2380 ATU / L / OD for pic771, after 5 hours of induction.

In conclusion, we can say that pTG771 shows increased stability compared to pTG720, while keeping the same production capacity and the same properties of increasing its copy number at the end of the exponential phase as pTG720.

EXAMPLE 10
Induction of gamma interferon
The data concerning the induction of interferon gamma are presented in the same way as those of hirudin.

1) Stability of plasmids
a) Induction of the expression of interferon gamma in
a vector containing the ampicillin resistance gene
(TGE900 / pTG40) in LB at 42 "C
The results shown in Table 5 show that the loss of the plasmids is comparable to that of pTG720- and that after 5 hours the Fp rises to 6 96. However, unlike pTG720, the viability is lost more quickly and after 3 hours already a minimum number of viable cells is reached (to be compared with the 5 hours observed for pTG720); moreover, the p- cells present in the culture remain viable and already divide significantly after 3 hours and contribute to this Fp- of 696 after 5 hours.

TABLE 5
Stability Parameters of Plasmids During Expression
at 42 "C of interferon in TGE900 / pTG40
Time in hours c / ml / OD 96 p + p- / ml / OD Fp- (%)
(No. of colonies
tested)
Oh 1 h 30 1.8.106 100 <î06 (I
(202/202) 3 h 6.6.106 10 6.0.106 1.5
(2/20) 5 h 2.5.107 2.7 2.4.107 6
(10/366) 6 h 4.3.107 1.7 7.3.107 18
(11/645) 8 h 1.3.108 0.9 1.3.108 32
(4/465)
b) Induction of the expression of interferon in a vector
containing the dapD gene (TGE7615 / pTG7671) in LB at 42 C
In table 6, the data relating to an induction of TGE7615 / pTG7671 at 42 C in LB are given. Note that the maximum mortality is only reached after 5 hours and that at this time, although the% p + is comparable to that of pTG40 (10%), the number of p- cells (p- / ml / OD) is 5 times lower than that obtained after 3 hours for pTG40. In fact, after 5 hours the Fp- is 0.3% while the Fp- for pTG40 is then already 6%. There is therefore a difference of a factor of 20, which is a reflection of the increased stability of pTG7671, an observation which confirms the stability data at 300C (see Table 1).

TABLE 6
Stability Parameters of Plasmids During Expression
at 42 C of interferon in TGE7615 / pTG7671
Time in hours c / ml / OD% p + p- / ml / OD Fp- (%)
(No. of colonies
tested) 0 h 1.7.108 100 <.3.8.106 (1
(50/50) 1 h 30 1.35.108 100 <2.7.106 <1.5
(50/50) 3 h 4.2.107 100 <8.4.105 <0.2
(50/50) 5h 1.3.106 12 1.1.106 0.3
(6/50) 6 h 2.2.106 5 2.1.106 0.5
(5/100)
Under conditions of induction of TGE7615 / pTG7671 at 42 ° C, the stability of the plasmid is practically identical in selective and non-selective media, the stability of pTG7671 being already very good in the absence of selection. The loss of the plasmid is therefore reduced to a very satisfactory rate and compatible with industrial production.

2) Plasmid content
Agarose gel analysis of the plasmid content shows that the strains transformed with the plasmids pTG40 and pTG7671 contain equivalent amounts of material and that this plasmid content per cell increases over time during induction.

3) Selection of p + cells containing the dapD gene
In order to demonstrate the selective power of the dapD system, taking into account the growth of p- cells, which will no longer be generated as soon as the major part of the cells have lost their viability, an induction is carried out during which the culture is diluted d a factor of 5 three times spaced 2 hours apart, after which this culture is allowed to reach the stationary phase. The results for the 42 ° C induction of TGE7615 / pTG7671 are shown in Table 7 for growth in LB selective medium and in Table 8 for growth in non-selective LB + DAP medium.

TABLE 7
Stability Parameters of Plasmids During Expression
at 42 C of interferon in TGE76l5 / pTC767l in LB selective medium
Time in hours c / ml / OD% p + p- / ml / OD Fp- (%)
(No. of colonies
tested) 0 h 3.8.107 100 <6.105 <0.2
(50/50) 2 h 8.3.107 97 1.4.106 0.4
<74/76) 4 h <106 6 h <106 9 h 3.7.106 100 <3.7.104 C 0.01
(60/60)
TABLE 8
Stability Parameters of Plasmids During Expression at 42 "C
interferon in TGE7615 / pTG7671 in LB + DAP medium
Time in hours c / ml / OD 96 P ± p- / ml / OD Fp- (, ó)
(No. of colonies
tested) O h 5.5.107 100 z 1.2.106 (0.3
(50/50) 2 h 9.9.107 99 1.3.106 0.3
(74/75) 4 h (106 6 h lo6 9 h 1.7.107 5 1.6.107 4
(5/100)
It is noted in Table 8 that after 9 hours of induction the viable cells all contain plasmid, in LB selective medium (the presence of the plasmid is confirmed by minipreparation from 30 colonies out of a total of 60 colonies positive on LB and identification of the plasmid by agarose gel electrophoresis). In non-selective medium, on the other hand, the viability is 5 times higher, but the culture only contains 5% of cells which have retained the plasmid. This higher viability in LB + DAP than in LB shows the growth of the p- cells in medium supplemented with DAP and a fortiori the mortality of the p- cells in the selective medium. Therefore the LB medium allows an efficient counter-selection of the cells. p-.

4) Production of interferon
The pTG7671 keeps the same production capacity of
Gamma interferon than the original plasmid pTG40.

The strains and constructions representative of the invention were deposited in the National Collection of the Institut Pasteur on July 25, 1986:. TGE7615 / pTG7671 under number 1-586
TGE7615 / pTC77l under number 1-585.

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Claims (10)

1) Process for stabilizing a plasmid vector contained in a bacterium, characterized in that the bacterium comprises a dap chromosomal mutation and in that the plasmid vector carries a dap + gene. 2) Method according to claim 1, characterized in that the chromosomal dap mutation is a mutation of the dapD gene and in that the vector plasmid carries an intact dapD gene. 3) Method according to one of claims 1 and 2, characterized in that the vector plasmid carries the gene encoding a protein of industrial interest and the elements ensuring its expression in the host bacterium. 4) Method according to one of claims 1 to 3, characterized in that the host bacterium is a strain of E. coli. 5) Method according to one of claims 3 and 4, characterized in that the protein of industrial interest is chosen from hirudin and gamma interferon. 6) Bacterial strain containing a stabilized expression plasmid vector obtained by carrying out the method according to one of claims 1 to 5. 7) Bacterial strain according to claim 6, characterized in that it is a strain of E. coli exhibiting a dap chromosomal mutation and transformed by a plasmid vector carrying a dap + gene and a gene which codes for expression of a protein of industrial interest. 8) Bacterial strain according to claim 7, characterized in that the protein of industrial interest is chosen from hirudin and gamma interferon. 9) Bacterial strain according to claim 8, characterized in that the gene for hirudin or gamma interferon is under the control of the elements necessary to ensure its expression in the bacteria. 10) Process for preparing a protein of industrial interest, characterized in that a strain according to one of claims 6 to 9 is cultivated on a complete medium and that the protein in question is recovered.