AU616637B2 - Method for the stabilization of a plasmid contained in a bacterial strain and strain obtained - Google Patents

Abstract

Process for stabilising a plasmid vector present in a bacterium, characterised in that the bacterium comprises a dap<-> chromosome mutation and in that the plasmid vector carries a dap<+> gene.

Description

I4V IM I I L'j I^ PI IiJI OR SEAL

(MANAGER

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To: The Commissioner of Patents, Australia P/00/011 61b'6637 FATENTS ACT 1952-1973Fom1 COMPLETE SP~"ECIFICATION

(ORIGINAL)

FOR OFFICE UISE Class Int. CI: Application Number: Lodged: Complete Specification-Lodged: a Accepted: 0 4 Published: Rei,,ted Art: TO BE COMPLETED BY APLCANT a a a a a Name of Applicant: TRANSGENE S.A. a nc h Body Corporate, 16 rub Heori Regnault 92400 COURBEVOTE FRANCE.

,0Address of Applicant:, Actual Inventor'.

Address for Service: ERIC D80RYSE VOW-, THRSOD A CARTER MUTNT A TFIA0n1YiARR;ATIORNtYS 71 QUEENS RlOAD MElDuouNe, 8004. AUSTRA i Complete Specificatioi fni ihe Invention entitled:& METHOD FOR THE STABILIZATION OF A PLASMID CONTAINED IN A BACTERIAL, STRAIN AND STRAIN OBTAINED The following statement is a full d. a ption of this. invention, including the best met'hod of performing It known to me:-,'1 *N t:he description Is to be typed In double spacing, pica type face, In an area not exceeding 250mrm In depth and 160 mm In width, on tough white paper of pod quality and It Is to be Insemtsd Inside this, fotm.

117 100,3- L 117 1O/73-L C. JTigoaw~w~. Cornm~nw~,Ih Oo~rnnient PriaterCInb4tr4 S- 1A The practical application of recombinant DNA technology to the production of molecules of interest by microorganisms generally runs into the problem of the instability of the recombinant plasmids.

Cloning and expression vectors commonly used in the laboratory are generally multicopy plasmids, and the4r stable transmission to the progeny is assured by the Large number of plasmids per cell genome (Jones et al. 1980). However, the introduction of foreign genes into these plasmids gives rise to various degrees of instability during the multiplication cycles of the bacteria. Thus, industrial production operations may require 1,000-Litre cultures, resulting in more than 10 6 cells, after more, than 50 generations. Therefore, it is essential to stabilize the plasmids in the bacteria in order to ensure their presence, and therefore the expression of the foreign molecule, until the end of the culture in the fermenter.

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s The stabilization of plasmids by integrating a gene coding for resistance to an antibiotic is a conventional '.eO Laboratory practice which cannot, however,' be applied to an industrial scale use for several reasons: the use of an antibiotic-resistant bacterial strain may represent a risk to the environment; the quantity of antibiotic required during tha culture significantly increases the cost of proouction; and the use of an antibiotic cannot be envisaged in the production of substances to be used in human or veterinary therapy.

Therefore, it is essential to develop other methods for the selection of bacteria carrying a recombinant plasmid. The few models which have already been applied are based on the same principle: the host cell is made to undergo mutation (auxotrophic mutation or introduction of a gene which is lethal to the bacterium> so as to prevent it from multiplying in the absence of a plasmid coding for a character which makes up for the deficiency of the host.

For example, Skogman and NiLsort (1984 and 1985) have used the complementation of a heat-sensitive val S mutat ior -2by the val S gene carried by a plasmid; in this model the stability of the plasmid, which carries the tryptophan operon, is total after 200 generations at non-permissive temperature, whereas under non-selective conditions (30 0

C),

a plasmid Loss of 1.2% is observed in each ge'eration.

Miwa et aL. (1984) have used, as the host strain, a streptomycin-dependent (Smd) E. c ci mutant and a plasmid carrying a gene (rpsL of an SmR strain) which masks the Smd phenotype and renders the strain independent of streptomycin, thereby ensuring a plasmid stability greater than 99%.

Herthberger and Rosteck (1984) have rendered a bacterium Lysogenic for a prophage Lambda whose repressor has been deleted; the cell can escape lysis only in the presence of a plasmid carrying the Lambda cI repressor.

A new selection model has been developed, based on S a the complementation of a dap- chromosomal muta.tion with a 0 dapr plasmid gene, which ensures the survival of only the S plasmid-carrying cells.

Diaminopimelic acid (DAP) is a component of bacte- L94.

arial cell walls; it is also an intermediate in the biosynthesis of lysine from aspartate. A strain which is deficient in an enzyme for DAP biosynthesis will not be able r 2 to multiply in a minimal medium; the addition of lysine 2 2 to the medium will permit growth, but will soon bring about the lysis of the cell which does not incortorate DAP in its membrane (Work 1950 Davis et al., 1973).

The deficiency of an enzyme for DAP biosynthesis may be compensated for by the introduction of the corresponding gene on a plasmid and, in particular, on the expression vector of the foreign protein required to be produced. If the bacterium loses the plasmid, it becomes dap- and can no longer multiply. This model ha, the advantage that it can be applied to any DAP-free culture medium, i.e. any medium for industrial-scale production and an\y medium, rich or minimal, to which lysine has been added.

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

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44 4 00 0l 04 44 .4 *O 44 41 4 44 4 a 0 0 e ft -3- A dapD" strain (with one of the 9 genes of the bio-synthetic pathway of lysine, the gene D, corresponding to tetrahydropicolinate-N-succinyl transferase, deleted) is constructed and the dapD gene on a commonly used expression plasmid (carrying the gene to be expressed under the control of the promoter is introduced. Under selection conditions, it can be shown that the plasmid is stable for at least 150 generations. The cloning of another gene (dapA), coding for an enzyme for the biosynthesis of lysine, on a plasmid, has already been carried out, with a view to increasing the production of lysine (Dauce-Le Reverend et al. 1982); however, these authors have not been able to control the stability of their plasmid.

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

(1984); however, the object of this work is only to study the regulation of this gene.

For this reason, the present invention relates to a method for stabilizing a plasmid vector contained in a bacterium, wherein the bacterium comprises a dap" chromosomal mutation and wherein the plasmid vector carries a dap gene.

Accordingly the invention provides in one broad aspect a bacterium, the genome of which comprises a dap gene inactivated by mutation and which is transformed by a plasmid comprising a functional dap gene.

In a further aspect the invention provides a process for preparing a hozIgenous culture of bacterium comprising culturing said bacterium in a dap- free medium in the presence of a substantial amount of lysine.

Among dap- chromosomal mutations, dapD- will preferably be mwspe#8170 9181 employed; however, other genes coding for an enzyme for DAP biosynthesis could also be employed, for example dapA, on condition that a vector plasmid carrying the corresponding gene is used; in the case of a dapD" strain, the gene to be inserted into the plasmid will be dapD.

The dap stabilization system does not interfere w.th the expression of the protein, it may therefore be used with any expression vector.

In order to avoid possible homologous recombinations between the dapD1 plasinid and the mutated dapD gene of the chromosome, it is preferable to provide for a substantial deletion of the dapD gene from the 0 4 10 chromosome.

For this reason, the present invention relates more particularly to a method for the stabilization of a plasmid vector contained in a bacterium as described above, wherein *wp#87 91 1 *0i 400## mwspO810 918 1i' 4 the dap chromosomal mutation is a deletion of at least a portion of the dapD gene and wherein the plasmid vector carries an intact dapD gene.

It is clear that in order to avoid any recombination, a total deletion of the dapD gene will be the most satisfactory solution.

However, a selection system, however: strong it may be, cannot overcome the inherent instability of a plasmid.

Because of this, in order to increase the stability of the expression vectors in question by genetic means it is possible to introduce into said vectors a sequence which maintains them in the monomeric state, especially the "car" sequence.

The "cer" (Summers and Sherratt, 1984) is an element which does not code for any protein and the presence of which promotes the maintenance of a plasmid in the monomeric state. If the plasmid forms multimers, the number of units O which can be distributed to daughter cells drops and 0 plasmid-free cells are obtained more easily. Therefore, M2, cer stabilizes the plasmid indirectly by ma ,ntaining it in the monomeric state.

Fo. this reason, the present invention also relates

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0 to plasmid vectors which additionally comprise the gene coding for a protein of industrial value and to the elements which ensure its expression in the host bacterium and, in particular, to the gene coding for a hirudin or 'one of the natural or synthetic variants thereof, for example, C2,30, interferon-gamma or alpha-antitrypsin, and to strains transformed by these different vectors and *SB8 to the methods for the preparation of industrial proteins by culturing said transformed strais in a complete medium and recovering the protein after culture.

in the following text, the expression system comprises i the leftward promoter, PL, of phage lambda, however, it could be another promoter which is active in the bacterium in question. This explains the reason for not giving a complete description of the expression elements of the heterologous protein, this type of plasmid now being widely known. IL will be advisable simply to insert dapD or

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0*44 94 9 49 9 94Q 9 99 9 9989 9 94* 9 44 44 9 4 44 4 49 *9 9 9), 1* 4* 0 9 *044 4 St other gene into a non-essential site of the plasmid vector.

The plasmids in queStion may be employed to transform any E. coLi strain which has been rendered dapD-.

By virtue of the method according to the invention, bacterial strains in which the expression plasmids are stable in a complete medium without the need, for example, for a selection pressure by an antibiotic or for a specifi; amino acid-free medium, are obtained. Additionally, the method according to the invention exerts a counter selection Ggainst bacteria which have Lost the plasmid.

Therefore, the invention also relates to bacterial strains, in particular dap E. coli, transformed by a plasmid vector carrying a dap gene and to elements which ensure the expression of a protein of industrial value, carried by this plasmid.

The invention also relates to a method for the preparation of a protein of industrial value from a bacterium, wherein a bacterium transformed by a piasmid according to the invention is cultured in a complete medium, s-id plas- 20 mid additionally comprising the gene of said protein and control signals for the expression of this protein in the host bacterium.

The object of the examples below is to demonstrate the stability oq plasmids carrying the dapD gene in the 25 dap- bacteria, in comparison with plasmids carrying the Ampr genp.

Additionally, th se plasmids carrying the dapD ge'nu have been used to express the genes coding for hirudin and interferon-gamma.

30 These two genes are placed under the control of the phage lambda promoter PL and the E. coli host strains contain the heat-sensitive repressor CI857; this system makes it possible to induce the expression of the gene controlled by PL by increasing the temperature.

The E. coli strain TGE 900 which contains the repressor C1857 was modified to become dapDo by giving the strain TGE 7615 or TGE7214 and strain 5969 was modified to becne TGE7303.

The foLlowing plasmids were then prepared according to the diagrams attached hereto:

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4 F ig u re F ig u re F ig ur e F i gur e F igure F i gure F ig ur e F ig ur e 1 prG764 2 pTG720 2 p rG 77 1 3 -pTG775 3 -pTG776 4 -pl'G766 4 -pTG767 5 -PTG7671 -6- Amp r 0 0 dapD 0 Foreign protein 0 H ir ud in H i r udc i n H i ru d in H i ru d in 0 0 I FN-gamma F igu re 6 F igu re 7 F igu re 8 Restriction map of the HindIII-BamHI fra~iment of p D 66 Diagram of PstI inserts of pDB6 in Ml3mp8 and position of EcoRI sites introducea therein.

Restriction map for the KpnI-BgLII fragment of pTG47.

11 D 0 -210 0 0 o 35 Figure 9: Demonstration of the deLetion of the dlap gene in chromosomaL DNAs of different strains, by "Southern bLot2" au toradiography.

Figure 9A: probe =kan R gene carried by the EcoRt fragment of pTG47 bands 6 to 9 DNA cut with Pstl to 14 DNA cut with BamHI and H in dI I bands 6 and 10 =strains dC4540 7 and 11 T GE 7 213 8 and 12 TGE72 14 9 and 13 TGE901 14 pDO6 moLecuLar weight markers Figure 98: probe side of the dapo gene carrieO by the EcoRi fragment of M13TG620 bands 4 to 7 DNA tut with PstI 8 to 11 DNA cut with BamHI and Hiidlll bands 4 and 8 strains GC4540 and 9 !GE7213 6 arid 10 Ta.E7214 0 a -7- 7 and 11 TG E Figure 9C: go 254 Ii a 430 a 0 -00 0 Figure 90: bands 13 moLecuLar weight markers 1 and 12 p066 cut with PstI or BamHI and HindI 2 M,13TG620 cut with Pstl 3 M13TG597 cut with Pstt probe fLanking regions of the dapD gene carried by the KpnI-6g111 fragment of pTG47,, ChrornosomaL DNAs cut with PstI.

bands 4 =strains GC4540 T GE 7 213 6 T G E 7214 7 T G E9O01 bands 8 =molecular wei ght narkeos 1 BamHt-HindII I f rogment of pD66 cut with P s t I 2 M13TG620 cut with PstI 3 M13TG597 cut with Pstl prQbe fLanking regions of dapD gene carried by the KpnI-Bgl 11 fragment of pTG47.

ChromosomaL DNA s cut with PstI.

bands 4 strains RH5345 5 RL58 6 T GE 7 615 12 and 3 (as in 9C) Diagrm manlecular wieight ni kerfG, D ag am ndrestriction map arp 9 D iag r am and restr ict ion map f or pTG792.

D ia g ram and rest r ic tion. map focr pTG7922.

Diag r am and restriction map 1(o r pTG769.

Dia gr a o and restriction map f or pTG74O6.

SODS p oLy a cry La m id e ge L e Le ct ro p hor e s is, v is ua Li zed with Coomassie bLue, of proteins synthesized by TGE7213/prG7407.

bands 1 arid 8 moLecuLah weight markers bands Z to 5 =cul ture at 30 0 C for 4 hours (2 and 3 or 7 hours (4 and bands 6 to 12: cul ture initiated at 42 0 C for 4 hours 11 and 12 or 7 hours F i gur e F i govre F ig ur e F i g u r F ig ur e F ig ur e

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9 and C insoluble fractions; 20 pl S soluble fraction; 40 pl Figure 16: Diagram and restriction map for pTG7675.

Figure 17: Diagram and restriction map for pTG7914.

METHODS

Unless otherwise mentioned, the differef,! enzymes are employed according to known techniques.

Transformation Competent cells were prepared according to the method of Hanahan (1983). Their competence ranges up to 104 to 105 transformants/pg DN for the RL58 strain (Bollen et aL. 1979) and 105 to 106 transformants/4g DN for the TGE 7615, TGE7214 or TGE 7303 strain.

In generaL, 1 to 10 GI of a soLution, at the appropriate dilution, of a DNA preparation containing the plasmid which is desired to be introduced into the strain, is added to 0.2 ml of a stock of competent cells. The cells are allowed to react with the DNA for 15 minutes at O°C and they are then incubated at 37°C for 90 seconds. They are returned to 0 C for 5 minutes and then 0.8 ml of 1.

medium is added thereto. For the strain RL58 or for the o strain TGE 7615, this medium must contain DAP at a rate of S2 9 8 gamma/ml. This is necessary for the growth of dapDcells, even for those which contain the plasmid carrying 4 oO1 the dapD gene. The cells are generally incubated at with vigorous stirring (however, they may be placed at 37 0 C if the plasmid does not contain the promoter PL p0 The incubation period is 60 minutes, but it can be extended to 90 minutes for growth at 30 0 C for the strain TGE 7615.

Subsequently, defined volumes (0.1 ml) are spread on solid LB medium. In the case of plasmids contaninng the gene for ampicillin resistance, the clones are selected by adding 100 gamma/ml of ampicillin; the, clones containing the dapD gene are selected from a culture which is dapDO on the whole, on the LB medium without'other additions. The dishes are incubated for 24 hours at 30 0 C. The colonies are then transferred with toothpicks into 3 ml of LB medium 4 9 and, after growth at 30'C overnight, the plasmid is isolated and its structure confirmed on agarose gel, after digestion with appropriate restriction en-ymes.

Plasmids containing the dapD gene may be transformed in any E. coli which carries the dapD mutation (for example RL58, TGE75150, TGE7214 or TGE7303); if they additionally contain the bacteriophage lambda promoter PL, they must necessarily be transformed in dapD E. coli strains which express the bacteriophage lambda repressor c1857 either on a plasmid or in the chromosome, or cl and Roc mutation (for example RecA441).

EXAMPLE 1 Introduction of a mutated dapDO gene in E. coli strain TGE900 The E. coli host strain TGET900 was rendered dapby conjugation with a known cdapD mutant, RbLS.

The strain TGO1900 is a derivative of strain N4830 described by Gottesman at al. (1980), the characteristics of which are as follows: Su F his ilyv bio (A cl857 A Dam A I) Smr It is commonly used as the host for expression vectors in which the foreign gene is placed under the control of the lambda promoter Itboas it enables the expression to be induced at will by increasing tha temperature (above 37QC), which inactivatos the heat-sonistive repressor (lambda c1857) carried by the bacterium.

The dap' RML58 (met B dapD) met Q279 Ur P 4

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0 *04 strain has been described by Oollen et al, (1979), Startrig with this strain, a trimethoprim-resistant 4 a Itospontaneous mutant Was selected (after spreading the strain on a dish containing 2 gamma/ml of trimethopriim and confirmin5 the resistance of a colony chosen from a medium containing 4 gamnma/ml trimethoprim). In fact, the gene coding for this easistance is sufficiently close to the dapDP mutation so that if the recipient Otraino after conjugation, becomes, resistant to trimethoprim, it also becomes dapDt.

The reaistant strain has been called 7t755; the useful characteristics there',, are: Her, dapD ii

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-9A r TpAfter conjugation Of the strains TG900 Srr dap+)f and TGE755, stopped after 15 minutes, by spreading 4 44 44 4 44 4. 4 44 44 44 4 444 4 44 a $4.4 44 '4 44$ 44 44 4 444 4 44 44 4 44 4444 4 4 4444 4 444444 4 4 *4 4 4 44 4 44 10 gn a minimal medium containing DAP, streptomnycin' and trime'choprim, Smr T mpr colonie-s are selected; the clap nature of the strains seLect'&?d and the presence of auxotrophic mutations in the parent strain (his iLv) in the rriniinal medium containing DAP, are verified on on LB medium.

one strain (his ilv dap )was chosen: TGE7615.

EXAMPLE 2 construction of a vector plasmid carrying *the E. coLi dapD gene (Figure 1) a) Con-truction of a muLtiple-use cloning pLasmid comprising a poLyLinker with 12 restriction sites The construction was initiated starting with a pML2 pLasmid (Lusky and Botchan, 1981), derived from pBR322 by deleting the nucleotidles 1089 to 2491. This pLasmid retamned the origin of replication of pBR322 s,7td the betaactaMase gene (ampiciLLin resistance), 0 4 The PstI enzyme recognition sequence was removed by inserting an Ahal II-AhaiII fragment of pUC8 which carries o Pstl 0 ckttation induced with ethanesuLfonate (Vieira ~~2O and Mess ing, 1982), between the two AhalIl sites of~ pML 2 which cdeLetes 19 base pairs from pML 2 The resulting *~spLasmid, pTGI,90, carries the Pstt 0 mutation of pUC8.

A BgLlLlinker (51-dCAGATCVG-30; Collaborative Re- 0. search) was inserted into the2 only NruI site, by the "Linker tailing" technique described by Lathe et aL., 1984. The resulting construction is pTG191.

0 The PTG191 was opened with EcoRl and 6g111 (whic.h 0 deLetes the tetracyclirie-resistance gene) aind reLigated w it h the EcoRl-BgLII segment of phage M13TG131 (Kieriy et 1983) 'which comprises a polyLinker contairiin,, 12~ restriction enzyr- recognition sequences.

The resulting pLasmid is pTG192.

b) Cloning of a dapD gene in the p1TG192 pLasmid The dapD chromosomaL gene of E. coLi was inserted into the pLasmid pACYCI84 to give pD8.6 (Bendiak and Friesen, 1981). This dapD gene was recovered from p066 in the form of a 1.3-kb ALul fragment and inserted into the EcoP~t site of PTG192 (the EcoRI ends having been repaired beforehand by treating with the Kienow fragment of DNA poLymerase 1).

S 11- From the resulting plasmids, pTG764, in which a single EcoRI silte is reconstituted, is chosen.

Thus, the pTG764 carries the dapD gene and the ampicillinresistance gene of pTG192 (originally of pUC8).

EXAMPLE 3 Expression plasmid of hirudin contaIning dapD and Ampr genes (figure 2) The pTG720 plasmid described in French Patent No. 84/04,755 essentially comprises the hirudin gene under the control of the lambda promoter Pb.

The pTG720 plasmid digested with glII and BgLI, gives a 2.74-kb fragment which carries the hirudin gene and a portion of the amp gene. This fragment, treated with phosphatase, is religated to the BglII- UglI fragment of the pTG764 plasmid which carries the dapD gene and another fragment of the amp gene.

The resultant plasmid, pTG771, carries the reconstituted while amp gene, the dapD gene and the gene coding ~for hirudin.

The colonies of TGE7615 transformed by this plasmid 0 may be selected either on an LB medium (selection for the dap nature) or on an LB ampicillin medium.

*1 0*0After induction, the transformed strain produces hirudin.

EXAMPLE 4 Expression plasmid of hirudin containing the dapD gene and no longer containing the Ampr gene (Figure 3) SThe starting plasmid is pTG771.

0 4A digestion is carried out with Ahalil in order 3 to remove the small fragment comprising the 3' end rj of the gene coding for Amp and the plasmid is closed again by ligation in the presence of an EcoRI "linker":

CCGAATTCGG

The pYG775 plasmid is obtained.

An EcoRI digestion followed by ligation enables the gene coding for Anfpr t. be removed completely.

The pTG776 plasmid is obtained.

The TGE7615 bacteria transformed by this plasmid may be selected on an LB medium, without addition (selection for the dap+ character).

LU~IL~J~£'L\£ri lii u1~1UiNU 1 1 4 A6i"I CONTAINED IN A BACTERIAL STRAIN AND STRAIN OBTAINED The following statement is a full da,%;cr~ption of this, invention, including the best met~iod of performing it known to 1-- 'Noto: The description is to be typed in double spacing, pica typeface, in an area not exceeding 250 mm in de~pth and 160 mmlIn widt on tough white paper of cood quality and it is to be inserted inside this form.

1 17 10/73-L CJ.'riomNCommowa hGovernmlent PrncrCanbeff 12 Aftor 'duction, tLhe transformed strain produces hirudin.

j EXAMPLE Construction of p lasmid!- carrying the da pD gene and not "I containing the Amp gene (x?,'gure 4) The starting plasmid is the plasmid pTG192.

fl After digesting with Sinal and AhaII and then treating with phosphatase, the 0.95-kb vector fragment is isolated and ligated with the 1.3-kb AluI fragment r of pD86 which carries the dapuD gene. The amp gene has therefore been completely cV-leted., ThG TGE8615 strain is transformed w~ith this new plasmid and selection is carried out on an LB medium in order to obtain clones which carry the dapD gene.

Plasinids which carry the dap gene in the two orientations are obtained; the orientation of the insert may be determined by digesting with PstI.

0 0 aFor two candidates chosen: pTG766 re! -ses 1.34-kb and 0.9-kb fragments, and pTG767 releases 1.85-kb and 0.4-kb fragments.

*EXAMPLE 6 Expression plasmid of interferon-gamnma, carrying the dapD gend. (Figiure The pTG40 (PED) (Il P-A-Oi46462) plasid carries, on a 1 .2-kb UlindIll fragment, the interferon-gamma gerie under the .ontrol of the promoter P L' After digesting with IlindTI1, this fragment is recovered 4,'*,and inserted into the HindlIT site of pTG767 which has previously been treated with phosphatase. After4 t #436ligation, TGE7615 is transformed1 and selection f2or 44 6 *4 4 the presence of the dQapD gone is carried out on anI LB medium. Two plasmids aarrying the insert in oppositew 41orientations are chosen: pPG7671 (which, hia; 1 h e P pomao the replication origin and in the same~ oritentation, and pTG7672.

The TGE7615 bacteria transformed by these plasmids may be selectod on an LB mediuim,, without addition (selection for the dap +charactor).

After inclucttion, the transformdid strain produces 44 -13intergeron-gamma.

The followinq examples are intended to demonstrate the characteristics Qf the strains transformed according to the present invention.

All the plasmids carrying the dapD gene are in the TGE7615 strain and the others are in the TGE900 strain.

The stability of the different plasmids was tested in suitable media: "selective medium" twill refer to the LB medium in the case of the plasmids according to the invention and to the LB ampicillin medium in the case of plasmids and pTG40; and "non-wselective medium" will refer to the LB medium DA: in the case of the plasmids according to the invention and to the LB medium in the case of other plasmids.

a A study of stability over 110 genereations is carried 0o*0, out by several successive cycles of dilution and Sq multiplication of the bacteria. At the end of this multiplication phase, the stability of the plasmid 4, is determined as follows: the number of viable cells in the culture is determi,Ned by suitable dilution and counting the cells in a non-selective agar medium; and after a growth, a significant sample of these colonies Is transferred into selective and non,-selective agar media, and the percentage of colonies which have lost the plasmid, and therefore the capacity to grow in a "3 0 selective medium, is determined.

0* 4 The restits obtained with the plasmids carrying s o the interferon gene are given in Table I: pi 14 TABLE I Determination of pLasmid Loss by bacteria after 110 generations 04 0 9 0k 64 *9 Strain TGE 900 PLasMid pTG40 SeLection Amp LB medium p-/c/generation* 2xl 1O 3 LB medium T G E76 15 pTG766 dapD 5x 10 T G E76 15 prG 76? 5x 10 T GE 76 pTG7671 dapD 3 x 10 DAP 2 x10- 3 5 x10 5 x10 3 x105 *c number of viable ceLLs p- ceLLs which have lost the pLasmid p-/c/generation =(number of p-/number of c tested) x number of generations SimiLar tests carried out with pLasm ids carrying the hirudin gene and with the clon ing vector pTG766, af ter the muLtipLication of the bacteria over 170 generations in an LB medium, givee the foLlowing results: TABLE 2 Determination of pLasmid Loss by bac te r ia after 170 generations in an LB medium Strain TGE900 TGE7615 TGE7615 TGE7615 PLasmid pTG720 pTG771 pTG776 pTG766 96 985 100 100 p-/c/generation 2x.0 1.50 <1lxlO 4 <1.lx10 4 The results presented in TabLes 1 and 2 show that: 1) the stability of dap vectors is increased by a factor of 10 reLative to the plasmid carrying the amp r gene; 2) the difference between the selective and natnseLective media is nflnimaL; 3) the ctoning vectors pTG766 and pTG3767 have a Losg of Less than 5 x 10- p-/c/generation; 4) the stability of pTG4O is significantly Less than that of the clap vectors; however, it is worth pointing out that the phenomenon becomes particularly marked after 50 generations; at the early stages of culture, the '1 Loss is only 2.5 x 10 p-/c/generation; and the loss of ampr.control plasmids pTG40 and pTG720, carrying the interferon and the hirudin genes respectively, is of the same order of magnitude; this plasmid loss with time is reduced when they carry the dapD gene (pTG766, 767, 7671 and 776).

However, it should be pointed out that the stability of the plasmid carrying the dapD gene and the gene for ampicillin resistance (pTG77'i is lower and remains comparable to that of pTG-720. This result is explained by an analysis of the plasmid content in the agarose gel; the latter shows that pTG771 forms tetramers, which phenomenon has repercussions on the partition of plasmids and which increases their loss (Summers and Sherratt, 1984). It should be added that over a smaller number of generations this multimerization phenomenon does not take place, and that over a tota.L of 70 generations, -4 the loss of pTG771 remains less than 2.8 x 10 p-/c/ t generation.

t"26 Therefore, the stability of the plasmids which c o, contain the dapD gene is increased relative to the plasmids carrying the ampr gene, and their loss becomes minimal.

EXAMPLE 7 Stability of plasmids at 37 or 420C 26 The effect of temperature on the stability of plasmids cannot be studied in the case of those which contain a gene coding for a foreign protein placed under the control of the promoter PL without inducing the expression of this foreign protein. Nevertheless, it should be pointed out that, under the operating conditions, the induction is followed by an increase in 0.D. from 0.3 to 3.6, which coriresponds to 3.6 generations. Therefore, in fact, the bacteria form the largest number of generations at 300C, before induction, with a loss which has already been determined in the preceding exampe.

When the expression of a foreign protein is i.nduced (for exampLe hirudir for pTG720 and interferongamma for pTG40), a mortality of the E. coli culture, at a rate of 90% to 95%, is observed after a certain period I- II-;--I 16 of time (2 to 4 hours). These are cells containing the pLasmid (because the p- host cell is capable of growing at 37 to 42 0 Inevitably, this mortality increases the proportion of p- cells relative to the living cells by a factor of 10 to 20. When the total number of living cells is significantly decreased, and the p- cells form a significant fraction of. the population, the multiplication of the p- cells is determined, which decreases significantly the ratio (see for example Table 3, results obtained after 5 h 30 min and 7 hours).

It is rovious that the parameter p-/cell/generation has lost its significance under such conditions because the p+ cells no longer multiply. The following parameter is proposed: p-/mL/optical density unit, which measures only the p-cells at each culture period. Additionally, it enables two different cultures to be compared for their plasmid loss. This parameter is calculated according I to the following formula: p-/ml/OD x viable cells/ml/OD.

t ir ?1 ?0 Finally, if this parameter is expressed on the 8 t basis of the total number of cells (which is 4 x 10 under t r the present conditions), it is possible to obtain the percentage of (Fp cells in the culture and to determine the degree of contamination of the culture with p- cells ,25 at each period. Fp is calculated as follows: t 4* 0 Fp~ 4 x 108 c/ml/OD x 100(p-/ml/OD).

Fp- is an objective parameter which makes it pos- I sible to decide whether a culture intended for the production of a molecule is sufficiently pure to make it worthwhile to develop it further and which makes it posisible to compare different plasmids with one another under the conditions of induction.

In the inductions described in the following exampLes, the number of viable cells and the percentage of p+ will be determined and then p/ml/OD and Fp will be calculated. These data will be presented in the form of tables.

jI 17 EXAMPLE 8 induction of hirudin The results for hirudlin induction will be presented, first with a vector containing the ampiciLLin resistance gene and then with the vector which also, contain,the dapD gene.

1) Stab i Lity of pLasmids a) Induction of hirudlin expression in a vector which contains the ampiciLLin resistance gene (TGE900/PTG72O) in an LB medium at 37 0

C

Typical results for TGE900/pTG72O are given in ')bLe 3.

TABLE 3 StabiLity parameters for the pLasmids during hirudlin expression at 42 0 C in TGE900/pTG72O Time in c/ m L/O0D P+ m L0 D F p- hours 0 h 8.3 x 1 Q100 1 .7 x 10 6 Q .4 1 h 30 2.4 x 10 8 100 4 .8 x 10 6 1.2 2 t 3 h 1. x 10 8 96 8.4 x 106 2. 1 4 h 30 4.7 x 107 82 8.5 x 10 6 2. 1 7 6~ 0005 h 30 1 .8 x10 48 9.4 x 106 2.35 7 h 2.8 x 10 Z-11 2.2 x 10 5 It is s e en that after 3 hours, t he number of vi abLe cells per 0D uni t decreases concurrenttly with the pro- 4. portion of P+ n eLL s After 5 h 30 min, Fp- of p- ceLLs #4re~ative to the total number of cells) is of the order of 2.35%. After 7 hours, this number wi L have doubLed and it is probable that this is due to the growth of tfle p- cells only.

b) induction of hirudlin express ion in a vector which contains the dapo gene (TGE7615/pTG771) in an LB medium at 37 0

C

The results given in Table 4 are comparable to those obtained for pTG72O.

18 TABLE 4 Stability parameters for the plasmids during hirudin expression at 42 0 C in TGE7615/pTG771 Time in c/ml/OD p+ p-/ml/OD Fp_ D%) hours 0 h 3.4 x 106 99.8 7.7 x 105 0.2 1 h 30 2.1 x 107 99.2 1..7 x 105 0.04 3 h 9.7 x 10 6 100 <1.0 x 10 5 <0.025 4 h 9.3 x 106 100 <8.0 x 10 4 <0.005 5 h 7.5 x 106 98 1.5 x 105 0.04 It is seen from Table 4 that despite a mortality comparable to that recorded for TGE900/pTG771, the loss of plasmid is low (98% p+ after 5 hours), which is also the case for the absolute quantity of p- cells ml/OD). The quantity of Fp- after 5 hours is 0.04%, which is 50 times less than that in the case of pTG720 at the same period. This demonstrates the greater stability of pTG771 compared with pTG720. It should be pointed out that in an induction experiment pTG771 does not form t ti '.20 tetramers and ijts loss is supposed to be less than that determined over 170 generations at 30°C (see Table 2).

'o ,The results show a greater stability of the plasmid pTG771 compared with pTG720.

2) Plasmid content Plasmids pTG720 and pTG771 were isolated at difr ferent times during the induction. In general, in the initial stages of induction, during the exponential phase, a low plasmid content per cell is observed, which increases substantially with time. Hirudin is produced during this period.

It is interesting to note that this increase in plasmid content occurs in a population in which more than of cells are dead.

3) Activity induced The hirudin activities produced from p.TG720 and pTG771 are not significantly different. The values (in' antithrombin units, ATU) are 2720 ATU/l/OD for pTG720 and 2380 ATU/l/OD for pTG771, after 5 hours of induction.

In conclusion, it may be stated that pTG771 shows bands 4 and 8 and 9 6 and 10 strains GC4540 TGE7213 TGE7214 .1 19 an increased stability relative to pTG720, while maintaining the same production .capacity and the same properties with respect to increasing its copy number as pTG720, at the end -f the exponential phase.

EXAMPLE 9 Induction of interferon-gamma The data for interferon-gamma induction are presented in the same way as those of hirudin.

1) Stability of plasmids a) Induction of interferon-gamma expression in a vecta containing ampicillin resistance gene (TGE900/pTG40) in an LB medium at 42°C 0i..

0o0 a o o 0 0, 0 0 0 V 01 The results given in Table 5 show that the loss of plasmids is comparable to that for pTG720, and that Fp- rises to 6% after 5 hours. However, unlike pTG720, the loss of viability is quicker and a minimum value for the number of viable cells is already reached after 3 hours (as compared with 5 hours observed in the case of pTG720); additionally, the p- cells present in the culture remain viable and are already dividing significanly after 3 hours, and contribute 6% to this Fp- after hours.

TABLE Stability parameters for the plasmids during interferon expression at 42°C in TGE900/pTG40 o o 0 a 0 Time in hours 0 h 1 h 30.

3 h h c/ml/OD p+ (No. of coLp-/m D tuiies tested) 1.8 x 106 6.6 x 106 2.5 x 107 4.3 x 1.3 x 10 8 100 (202/202) 10 (2/20) 2.7 (10/366) 1.7 (11/645) 0.9 (4/465) <106 6.0 x 106 2.4 x 107 7.3 x 107 1.3 x 108 1 6 18 32 6 h 8 h I-

B

i;~j 4I 4 4o 4 '4 446 b) Induction of interferon expression in a vector containing the dapD gene (TGE7615/ pTG7671) in LB at 42 0

C

The data for a TGE7615/pTG7671 indu :tion at 420C in LB appear in Table 6. It is observed that maximum mortality is not reached until 5 hours have elapsed and that at this period, although is comparable to that in the case of pTG40 the number of p- cells (p-/ml/OD) is one fifth that obtained after 3 hours in the case of pTG40. In fact, after hours, Fp- is 0.3% whereas Fp-for pTG40 is already 6% after this period. Therefore, there is a difference of a factor of 20, which is a reflection of the increased stability of pTG7671, which observation confirms the stability data at 30 0 C (see Table 1).

TABLE 6 Stability parameters for the plasmids during interferon expression at 420C in TGE7615/pTG7671 Time in c/ml/OD p+ p-/ml/OD hours (No. of colonies tested) 0 h 1.7 x 10 8 100 <3.8 x 10 6 1 (50/50) 1 h 30 1.35 x 10 8 100 <2.7 x 106 (50/50) 3 h 4.2 x 10 7 100 <8.4 x 105 0.i (50/50) h 1,.3 x 10 6 12 1.1 x 106 0.3 (6/50) 6 h 2.2 x 10 6 5 2.1 x 106 (5/100) Under conditions of TGE7615/pTG7671 induction at 42 0 C, the stability of the plasmid is practically identical in the selective and the non-selective media, the stability of pTG7671 being very high even in the absence of selection. Plasmid loss is therefore reduced to a level which is very satisfactory and compatible with production on an industrial scale.

i -21- 2) Plasmid content Analysis of plasmid content on agarose gel shows that the strains transformed by the plasmids pTG40 and pTG7671 contain equal quantities of material and that this plasmid content per cell increases with time during the induction pTG40 is present in the dimer fran (as shown on agarose gel).

3) Selection of p+ cells containing the dapD gene I, order to demonstrate the selective capacity of the dapD system taking into account the growth of pcells, which will no longer be generated as soon as the major part of cells have lost their viability, an induction is carried out, during which the culture is diluted by a factor of 5, three times, at 2-hourly intervals, Safter which this culture is allowed to reach a stationary phase. The results for TGE7615/pTG7671 induction at 42 0 C are presented in Table 7 for growth in a selective medium and in Table 8 for growth in a non-selective LB S+ DAP medium.

TABLE 7 Stability parameter, for the plasmids during interferon S expression at 42°C in TGE7615/pTG7671 in a selective SLB' medium Time in c/mL/Ob p+ p-/l/OD Fp- hours (No. of colonies tested) 0 h 3.8 x 1 00 6 x 105 0.2 (50/50) 7 6 2 h 8.3 x 10 97 1.4 x 10 0.4 (74/76) 4 h 1 6 h 10 6 9 h 3.7 x 10 6 100 <3.7 x 104 0.01| (60/60) i f: 22 TABLE 8 S tab i Lity pa rame ters f or the pi asmids dur ing i nterf eronexrres si an a t 42 0 C i n TGE76 15/pTG767 1 in an LB +r DAP med ium me in c/mi/GD p+ p-/mi/GD Fpurs (No. of coL- T i ho c%) 0 h 2 h 5 .5 x 1G 7 9.9 x 107 106 1 .7 x10 onies tested) 10G (50/50) 99 (74/75) 5 <1,2 Y, 106 1 .3 x 1G 6 1 .6 x10 G.3 0.3 4 4 h 6 h 9 h (5/10OG) lt 15 it is observed in Table 8 that, after 9 hours of induction, in the selective LB medium a(L the viable cells contain the pLasmid (the presence of the pLasmid is confirmed by mini preparation from 30 colonies out of a totaL of 60 positive cm~onies in an LB medium and iden- Q Of ication of the pLasmid by' agarose gel eLectrophoresis).

In contrast, in the non-se~ective med ium, the viability is 5 times as great, but the culture conta ins onLy 5% of the cels. containing the pLasmid. Th is greater viability i n LB DAP medium than in LB medium shows the growth of p- cells in the medium to which DAP has been added and a f o rt i o r the mortality of the p- cells in the se~ective m ed i um. Therefore, the LB medium enables an effective *,counter-selection of p- cells to be carried out.

4) tnterferon production pTG7671 reta ins the same interfeon-gamma production capacity as the original plasmid pTG4G.

EXAMPLE 10: DeLetion of the dapD gene from the chromosome of the bacterium Sub-cLoninq of the dapO gene on 2 Pstf( fragments 3$ PLasmid p066 and Ml3mP8 are cut with Pstl and L igated. The Ml3s containing the 5' end and the 31 end of the gene are screened with oLigonucLeot ides specIf ic to these regions, TG596 (GCGCTTAATAAC AGTTG) and TO$98 -23 (TGTGCATACTTTAGTC) respectiveLy. The candidates S896 arnd S898 are chosen, and the insertion of the desired fragments confirmed by sequencing. (See diagram in Figure 6) Introductidi of an EcoRI site into the 5' and 3' ends of the dapD genie The EcoR.l sites are introduced into S696 and S698 by point mutation: into S698 with the o,igonucLeotide TG597:

GTACGCAGGAATTCCTTAATGCCG

whi cm pa irrs wit'h the 3' region of the end of the gene, but before an assumed transcription terminator; and into S896 with the o~igonucteotide TG620: AGAGGCC CGAATTC CAAACG it 0:04which pairs with the 5' region upstream of the @9 assumed promoter for the dapD gene.

The transf ormants are anaL ysed w ith the same probes as those used for introducing the EcoRI sites. The pres- 4 t 20 ence of this EcoRl site is confirmed by a DNA minipreparation and then by sequencing the M13 candidates chosen 4 (see drawing in Figure construction of a de~etion vector The kanamycin resistance gene of pLasmid pUC-4K (soLd by Pharmacia) is recovered in, the form of anl EcoRI fragment The M13TG597 and 620 are cut with EcoRI and Psti, wh ich, reLeases the 3' end of the dapD gene (without the assumed terminator) and the 5' end of the gene (with a, 04 its assumed promoter) respectiv e Ly 9 *4 00* 30 The cloninj vector pTG192Z (described in ExampLe 2a) is cut, with PstI.

ALIL these fragments are L g a t ed. After the transformation of 5K eeLLs 4nd spreading on an LB medium ampiciLLin 0.1*g/mL. kanamycin 0.02 the coLonies are screened with oLigonucteotide TG596.

One construction was seLected: pTG47. Its structure is anaLysed by a DNA rinilpreparation and the orientation of the kanamycin resistance gene is determined by digesting the pLasmid with HindItI, which reLeases 2

I

I

L

24 bands of sizes K3kb and~ 4.0 kb, The orientation of 1.kanamycin resistance gene in pTr,,47 is the same as tha of the dapU gene in pDB6 (in the other orientation, is of sizes 4.9 kb and 4.4 kb wou~d have been obta~ined).

The, diagram for pTG47 is shown in Figure 8.

IDeL-ptjon of the dapD gene from the chroi~some Deletion of the dlapD gene of the chro(- some from arn intermediate straih.

R~H5345 cels, the competence of which amounts to 25 x 10' transformants/og of DNA of pTG47, arekl transformed by the pLasmid pTG47 previously cut with Kpnl and g Ilt (s.ee Fiqure 8).

This digestion reLease-' a fragment which contains the flanking regions of the 6apD gene, the gene itself ~1 15 being replaced by the kanamycin resistance gene.

After spreading on an LB medium DAP kananiycin 0Q-01 Axg/mL, 9 candidates are selected: TGE721 to TGE729, 440: the Hap-, kanR phenotype of which is confirmed.

The absence of the dapD gene is confirmed after za 0' transformation~of these strains by pTG764 1 by their capacity to multiply in the LS medium.

A DeLetion of the dapD gene from the strains TGiS9O1 and N5969 The jjapD deletion of the strain TGE721 is then transduced into strains TGE901 and N5969 with the phage 6transducer p1 vir/TGE721, and the dap- recombinants selec- 46 ted by their resistance to 0.01 4Ag/mL of kat amyc in.

44 The candidates chosen are TGE7213, 7214 and TGE- 7303 respectively. For one candidate, TGE7214, the iLe, VaL, his requirements of the parent strain, TGE9O1, in addi- 30tion to the dlap- kanR character, were confirmqjd on suitable 4 m ed i a EXAMPLE 1'1: Confirmation of the deletion of the dap gene in different strains by chromosome blotting.

The various strains beLow were analysed: the parent strains TGE901 and ,RH5345, dapD the deleted strailins TGE7213 and 7214, the parent strain RLS8 which was used to introduc'e the dapDo mutation into TGE9OI, and the dapDo mutant, TGEI615, derived therefrom, and 25 a GC4540 strain which is resistant to kanamycin by the integration of Tn5, whereas in TG..,strains, the kanamycin resistance gene is derived from Tn903 (the 2 genes should not give cross hybridization owing to lack of homology. Beck et al. 1982).

The choice of restrict-' enzymes is based on the sequence of pDBR; a cL, h: BamHI and HindIII releases a 9-kb chromosomal fragment containing the dapD gene and its flanking regions, in the case of wild strains; in the case of deleted strains, the. resistance gent must be released by digestion with BamHI and cut into 2 frr. ments by digestion with HindIII; and PstI releases 2 fragments, each containing a portion of the dapD gene (2.8 kb from the region and 3.4 kb from the 3' region) for the wild type strains; in the case of the deleted strains, digestion with PstI releases the kanmycin resistance gene and the 2 flanking regions (a S;.p 2.4-kb fragment on the 5' side and a 2.8-kb fragment on the 3'side) (see Figures 6 and 8).

Probes were chosen in order to reveal the dap gene or its flanking regions or the kanr gene: in order to probe the kanamycin resistance gene, an EcoRI fragment of pTG47 which contains only this resistance gene (Figure 8) is isolated; in order to probe the dapo gene, a 2.4-kb EcoRI I t fragment of M13TG620 which contains only the region of the dapD gene and which must be 10 completely deleted in TGE721, 7213 and 7214 (Figure 7) is employed; and in order to probe the chromosomic gap D gene and its flanking regions, a KpnI-BglXI fragment of pTG47 which contains the kanamyoin resistance gene and the 2 regions (the 5' and the 3' sides) of the chromosome which flanks the dapD gene (Figure 8) is employed.

Therefore, revelation of the following is expected: in the wild strains, a 2.8-kb band corresponding 44'! 26 tG the 5' region of the dapD gene and a 3.4-kb band corresponding -to the 3' region of this gene (Figure and in the deleted strains, the kanamycin resistance gene, the 5' region flanking the dapD gene which still exists (2.4 kb) and the 3' region flanking the dapD gene which stiLL exists (2,8 kb) (Figure 8).

Demonstration of the incorporation of the kanamycin resistance gene The chromosomal DNAs of GC4540, TGE7213, TGE7214 and TGE901, cut with PstI or BamHI and HindIII were compared and probed with the EcoRI fragment of pTG47.

Psti relldses the 1.3-kb band; a BamHI and HindIII restriction gives 2 fragments of 0.7 kb and 0.6 kb for the deleted strains only, No band is revealed in TGE901 or in GC4540 (Figure 9A).

SThese results prove that the kanamycin resistance gene is incorporated into the chromosome of the deleted -20 Sstrains and that this gene originates from pUC-4K.

Demonstration of the deletion of the dapD gene from the chromosont The chromosomal DNAs of GC4540, TGE7213, 7214 and "TGE901 are compared with, as controls, M13TG620, M13TG597, the BamHI-HindIII fragment of pDB6 and the same fragment cut with Pstl (Figure 6).

tt The chromosomal DN/s are cut with Pstl or BamHI and HindIII.

The M13TG620 cut with EcoRI is isolated from the 30 'band specifically containing the 5' side of the dapD gene in order to use it as probe (Figure 98).

After digestion with Pstt, it is seen that in the wild strains, a 2.8-kb band corresponding to the 5' region of the dapD gene and also an unexpected 1.7-kb band are revealed.

After digestion with BamHI and HindIII, an apprbximately 12-kb band whi. is larger than the band (9 kb) originating from pD86 is i; .aaled. Furthermore, an additional band is also present in the wild strains (Figure 98).

u~ 27 For the deleted strains, no significant homology is apparent in any digestion fragment.

These observations show that: a probe which covers the 5' portion of the dapD gene does not reveal any band in the chromosome of strains TGE7213 ai.d 7214, which demonstrates the deletion of the 5' side of this gene; and in the wild strains, as a second band specific for the dapD gene is revealed in addition to the expected band it seems that this gene is duplicated in these strains.

As a duplication of the dapD gene was unexpected, we have confirmed this duplication in some wild strains and confirmed the deletion of the dapD gene from the 3' side in addition to the 5' side.

The same chromosomal and control DNAs are emoloyed 4 a after digestion with PstI (Figure 8).

4, The probe i pTG47 cut with KpnI and BgLII. In S."o0 addition to the bands predicted above, this probe must reveal at least a 1.7-kb band. In fact, pTG47 contains 300 bp and 100 bp homologous to the dapD gene, from the side and the 3' side respectively, which must be revealed in the deleted strains.

5 Figure 9C shows that: 2.8-kb and 2.4-kb bands are revealed in the deleted strains and 3.4-kb and 2.8-kb bands in the wild strains; additionally, a 1.3-kb band corresponding to the kanamycin resistance gene is ret '0 vealed. This proves the deletion in strains TGE7213 and 7214; and two less intense bands, due to the duplicated dapD gene, are also revealed in the wild strains only.

As it is known that the 1.7-kb band is revealed with the 5' portion, the 2.1-kb band must originate from the 3' side. This proves that these strains indeed contain a duplication which has disappeared in the deleted strains.

}i

H

28 The chromosomal DNA cut with PstI of strains RH5345 and RL58, and the dapD mutant obtained by conjugation of RL58 with TGE901 were compared. These DNAs were probed with the KpnI, BgLII fragment isolated from pTG47 which contains the kanamycin resistance gene (which should not reveaL anything) and the regions flanking the dapD gene. Figure 90 shows that in RH5345, only the 3.4 and 2.8-kb bands are revealed and not the bands due to the duplication of the gene, which are present in GC4540 or TGE901. Additionally, only a 7-kb band is observed for RL58 and TGE7615, which indicates loss of a PstI site; this prove, that these 2 mutants are identical and are affected at least in the PstI site of the dapD gene.

In conclusion, these experiments show that: strains TGE7213 and 7214 are deleted for the dapD D gene and they contain the kanamycin resistance gene; the dapD mutation of RL58 and TGE7615 is present at least at the PstI site of the dapD gene; and some strains of E. coli have a duplication of the 0 4 dapD gene and this duplication is not present in the deleted strains.

As the 2 dapD genes of the recipient strain are 25" successfuLLy deleted by transduction, it may be concluded that the duplicated gene must be close (at less than 2' on the chromosomal map of E. coli) to the first dapD gene.

EXAMPLE 12: Cloning of the cer gene.

The cer gene is recovered from the Col El plasmid, the I sequence for which has been published by Chan et al. I r (1985); in the form of a 1.85-kb HaeII fragment. The HaeII fragment is then cut with HpaII, treated with Klenow and a 0.4-kb band is recovered. The M13mp130 is cut with EcoRV and treated with phosphatase.

The 0.4-kb fragment of Col El is Ligated to M13mpl30 and it is introduced into the strain J 103. The presence of the Col El cer fragment was confirmed by sequencing the' 0.4-kb band released by cutting with Smal and HindIIt.

i| The cer g ne inserted into the polylinker of M13m)131 is then isolated after digestion with Smal and i -29 HindIII and ligated to the pTG720 vector (carrying the hirudin gene, Figure 2) cut with BgLII and treated with KLenow.

The resuLting pLasmid is pTG720cer.

EXAMPLE 13: Construction of cloning vectors containing the cer gene and the dapD gene.

Inversion of the orientation of the M13mp131 polylinker in a vector coding for ampicillin resistance The pTG192 (Figure 1) is cut with EcoRI and BgLII in order to reLease the M13mpl31 polyLinker and is shortened by HaeIII digestion. A plasmid carrying the ampicillin resistance gene, for example pTG730 (expression vector of hirudin, described in French Patent 86/16,723) is employed; this pLasmid is cut with BgLII and EcoRI and ligated to the EcoRI-BgLII fragment of pTG192. In this way, the expression block comprising the PL and the hirudin struc- *o tural gene of pTG730 are Lost, being replaced with the *0 M13mp131 polylinker. This new plasmid is called pTG790 (Figure I" Introduction of the cer fragment into the cloning vector 2',20 pTG790 is cut with SstI and KpnI and treated with S phosphatase. The fragment resulting from this digestion is ligated to pTG720cer, cut with SstI and KpnI (which releases the cer fragment) and shortened by BglII digestion. The resulting vector, pTG792, contains the cer fragment (Figure 11).

Introduction of the dapD gene into a vector containing the cer fragment pTG792 is cut with EcoRI, treated with Klenow and phosphatase. The resulting fragment is Ligated with the 1.3-kb 0O ALul fragment resulting from pDB6 which contains the dapD gene (Figure 2 plasmids, pTG7922 and pTG7923, result therefrom, and differ only by the orientation of the dapD gene located between 2 EcoRI sites. For pTG7922, the Promoters for the 3 genes, replication origin, ampicillin resistance and dapD are oriented in the same way (Figure 12).

Deletion of the ampicillin resistance gene The following constructions have a multiple aim:; to delete the ampicillin resistance gene from vectors containing the dapo gene; to obtain a dapD cloning vector conitairing the cer gene; to obtain a dapD cloning vector which contains the M13mpl31 poLylinker (minus the EcoRV site, used for the cloning of cer); and to obtain a dapD vector with a single EcoRI site.

A PstI fragment is recovered from pTG7922 and pTG7923 containing, respectively, the 3' and 5' portions of the dapD gene as well as the cer gene in order to introduce it into a dap vector containing an analogous fragment but without any EcoRI or Aval site, or cer. The analogous vectors are respectively pTG767 and pTG766 described above.

The pTG7922 and pTG7923 are cut with PstI, shora 0tened by BglII digestion and ligated respectivey to pTG767 and pTG766 cut with Pstt and treated with phosphatase.

The cloning vectors which result therefrom are pTG769 and pTG768 respectively (pTG769 is shown in Figure 13).

EXAMPLE 14: Application of the dap model to the construction of expression vectors for catechol 2,3-oxygenase

(C

2 o Vector without BamHI site upstream of C2,30 Tho structural gene for C 2 3 0 is recovered from a 25 pTG444, to be introduced into the dap vector pTG7671 (described above).

pTG444 is identical to pTG445 described by tukowski et at. (1984) except for a non-regenerated XmaIt isite. The pTG444 is cut .with 8amHI and HindIl and liga- S30 ted to pTG769 cut with BamHI and Hindill and treated with phosphatase. The resulting plasmid, called pTG7401, does not contain the structural gene for C2, 3 0.

pTG7671 contains 2 BglII sites: a site upstream of PL forming part of the polylinker and a site downtream of PL Located in the ribosome binding site, upstream of the structural gene for interferon-gaema.

The pTG7671 is cut with BglI, and shortened by KpnI digestion. The resulting mixture is 'Ligated to pTG7401, cut at its polylinker, with BglIt and aamHI and treated with 31 phosphatase. The BgLII site is reconstituted by this Ligation, but the BamHI site Ligated to the BgLII site is Lost. Two orientations of PL with respect to the structuraL gene for C 2 3 0 are possible. In order to distinguish the construction in which C2,30 is under the controL of PL, cutting is carried out with BamHI and BgLII (in fact, for the orientation desired, a BamHI site is present near the BgllI site and in practice, the digestion will give only a 4.3-kb band; in the other orientation, the dige3tion releasL, a 3.9-kb band and a 0.4-kb band.

The plasmid chosen, pTG7407, having C2, 3 0 under the control of PL, has a structure cLose to pTG7406 described Later (compare with Figure 14), however, it has Lost its BamHI site upstream of C 2 3 0.

Vector with a BamHI site upstream of C 2 3 0 a pTG769 is cut with BgHII and BamHI, treated with i 0 phosphatase and Ligated with a BamHI-BgLII fragment of Sany expression plas id (pTG907) which contains PL and the Scomplete N gene of X. A construction, pTG7400, results S1f2QO therefrom, which may be identified by a BamHI and BgLII cut which releases 2 bands, of 2.6 kb and 1.3 kb. This construction contains the PL and the complete N gene.

i The PTG7400 is then cut with Hpat and a BamHI Linker, CCGGATCCGG (sold by BRL), which has been phosphory ated and hybridized, is inserted therein. This 1 egives pTG7402 which has Lost its HpaI site but which contains 2 BamHI sites.

The pTG7402 is cut with BamHI and religated to S wgive pTG7404. By this procedure, a BamHI site is removed 30 and the N gene is truncated.

Introduction of the C 2 3 0 gene into a dap-cer vector The pTG7402 is cut with BamHI and HindIII, treated with phosphatase, and the BamHI-HindIII fragment of pTG444 is introduced therein to give pTG7406 (see Figure 14).

It differs from pTG'407 in that the C 2 30 gene may be removed by a BamHI-HindIrl cut so as to recover the fragment introduced fromn pTG444.

32 Expression of C2, 3 0 in dap E. coLi strains transformed by the plasmid pTG7407 The expression of the C 2 3 0 gene in the bacteria TGE7213/pTG7407 was determined at 30°C after 4 h and 7 h of culture and during induction at 42 0 C after 4 h and 7 h. For each determination, a sample is harvested from the culture and centrifuged; the pellet is washed and taken up with phosphate buffer (as described by Zukowski et al., 1983) and then treated with ultrasound 3 times for 20 seconds each.

0066 O c.

o fe Z0o C,

VU

0O 0600 oC o Oso After centrifuging for 10 min at 10,000 g, the pellet is considered as the insoluble fraction and the supernatant as the soluble fraction The proteins present in each fraction are ana- 15 lysed by electrophoresis on SDS poly&crylamide gel. The bands are visualized by staining with Coomessie blue.

The results are presented in Figure 15. The intensity of the band for MW 35,000 is noted, especiealy after 7 h of induction at 42 C. The "scanning" of the gel gives 20 approximately .64% and 75% of C2, 3 0 in fractions S and P respectively.

In the richer sample 7 h at 42°C), the specific activity of C2, 3 0 is determined (according to the method described by Zukowski et al., 1983), by adding catechol 25 as substrate. A specific activity of 28 to 35 U/mg is *0

I

r~l 6 ye 60 OU measured.

6 0 The specific activity of a pure enzyme preparation being 280 U/mg, the extracts analyzed contain approximately 12% of active C 2 3 0.

EXAMPLE 15: Introduction of the cer fragment into an expression vector of interferon-gamma.

The pTG7671 described above is cut at its polylinker with SstI (identical to Sacd) and Kpnl and then treated with phosphatase and ligated to fragments pTG720cer cut with SstI and Kpnl. After transfor ation in TGE7615, one candidate, pTG7675, which releases 2 fragments of 400 bp and 3.4 kb after digestion with SstI and Kpnl (Figure 16) is chosen.

IV

111 0 to 0 4 4 *0 *aa~ 4 14 33 EXAMPLE 16: Demonstration of the stability of t-e plasmids during the induction of interferongamma expression.

a) Induction of interferon-gamma expression in .B medium at 42°C for a vector carrying the ampicillin resistance gene: TGE901/pTG40.

The results are given in Table 9. The total number of celLs/ml/OD unit is determined in order to ensure that the loss of viability is a true phenomencii and not, 3 for example, a change in cell volume. These data are also given in Table 9. Fp was defined in examnle 7 and relates the number of p cells to the total number of cells present at a given time.

It will be noted that Fp- after 7 h 30 min of induction reaches a value of approximately Therefore, it is less than in the previous experiment (see Example which can be attributed only to a difference in the structure of the pLasmid pTG40 in these 2 experiments; in fact, although the quantity of plasmid is the same at different times of induction, in the first experiment 'h'e plasmid was in the dimeric form, whereas in the experiment described here it is mainly in the monomeric form (as shown by analysis on gel). The condition of the plasmid does not affect interferon-gamma production, but illustrates conclusively the loss of stability if the monomeric form of a p!asmid is not maintained.

b) Induction of interferon-gamma expression in a strain mutated for the dap gene and transformed by a vector containing the dapD gene and the cer gene, TGE7615/ 0 pTQ7675 The results are given in Table 10, The total number of cells/OD unit/ml are determined; there is no marked difference with TGE901/pTG40. A real loss of viability is thus confirmed: after 7 h 30 min of induction, only 0.01% of the culture remains viable. This should be compared with TGE901/pTG40 in which case the value obtained is This manifests itself in Fp', which is i decreased by a factor of 1000 in the case of TGE7615/pTG- 7675 compared with TGE901/pTG40. Nevertheless, some p 4 3( a a Level which is very satisfactory and compatible with production on an industrial scale.

34 cells still remain, which appear the end of induction.

The plasmid content has the, same tures as before, i.e.

increase in the copy number at end of growth, but the multimeric forms, which are present in variable numbers with the plasmids without cer, are almost absent in this case. The interferon-gamma production is slightly greater than that obtained with c) Induction of interferon-gamma expression in a host cell deleted for the dapD gene and transformed by a vector containing the dapD gene and cer, TGE7213/ pTG7675 The results are given in Table 11. The conclusions are identical to those drawn from the comparison between TGE901/pTG40 and TGE7615/pTG7675: the mortality reaches a factor of 3.5 x 10 and the value for total number of cells/ml/OD Lnit does not vary significanty 0 during the induction. In contrast, even after 7 h 30 min of induction, p cells do not appear (after 24 h, the entire culture became p in the case of TGE901/pTG40 and remained 100% p+ in the case of TGE7213/pTG7675). The plasmid content is comparable to that of TGE7615/pTG7675 in the absence of multimers. Interferon gamma production is slightly greater than that obtained with TGE7615/pTG7675.

iil 1 tI t ;I~s-p

I-

a a l a a C *C 0 0 a 1 a" 0o TabLe 9 Induction of TGE901IpTG40 at 42 0 in LB medium 0.1) 600 nm mi min mTin min min min min 0-370 1.29 1-67 2-13 2-42 2-89 3-08 3-9 x 108 3-8 108 3.0 1Gx 7 2-7 x 10 6 2.9 x 10 6 7-0 x 10 6 9-5 x 16 total number of ceLlslmtIO- D- Fp- 6.5 x 108 7-2 x 108 6.7 x 108 6.5 x 108 1.,4% 0.03% 0-11% 0.23% 0-70% 1-73% NOTE: Fp- is the percentage of pat a given timecetis over the total number of cells present

X;

5 0 5 05 0 00 S 0 0 00 Table NGE7615/pTG7675 at 420 C in LB medium 600 nm dm1 10. 0. total number of cetLs/mLIO.D- 00I min 00 min 00 m i 00 mi P.

00 min m~in, sia 0-310 1 .07 1-.64 2.31 2-67 3-26 3-44 3-5 x1 3-3 x 108a 1.5 x 1 1-44 x 10 7 7-1 x 0 ,6-1 x 710 6-4 x1 4.7x 10 8 5-1 x 10 8 ,6-1 x10~ 801 0.0018% I- -ZZUA64 TabLe 11 induction, of TGE7214/pTG7675 at 42 0 C in LB medium Ofl.. 60C nm 0 h 00 min 1 bi 00, min 2 h 00 min 3 h 00 min 4 h 00 min 6 h, 15 min 7 h 30 min 0-400 1 .25 1-71 2-45 3,32 4-.25 cImLI0,D..

3- 4 108 3. x 10 3-8 x107 30x 10 6-.6 x 10 4 7-3 x 1 1.2 x 10 5 1.1% total number of ce'LLs~mL/0-D- Fp- 5-8 x 0 12-.8 x 5-8 xc 10 8 6 x 10- 7.

-38 JXAMPT_ 17: Application of the dap model to the construction of an expression vector i for alpha-i antitrypsin.

The PstI fragment containing~ the alpha-i antitrypsii expression block, i.e. the phage lamba promiotcr PL the truncated N gens, a riboz.me binding site and the structural get -4 for alpha-i antitrypsin (Arg 356 orignating from pTG29O1 (truncated derivative of 1?TG983, described in French Patent 85/07,393) is introduced into pTG792 (described abov-e), cut with PstI and treated with phosphatase.

The resulting expression vector, pTG7SI'3, is then cut with BglII and Sstl and the expression bio~ck containing the alpha 1 antitrypsin and the cer gene introduced into pTG767, cut with BglII and SsLI and tr'-ated with phosphata- The re-Oiltinti plasmici, (17 9 A contains the dapD gene, the oor jene arid~ alpha-~1 antitrypsln expression block (rarg 35 ka isir 17).

2Q Deposit ion. of ruLrouertqtd j rL -L riirn of tho invnt Ion The followin g strains e d e tositedl at the Collection Nationale de Cultuires does Mcroorqanismes (National Collection of Mioroh'Ial Cultures) (25 Rue du Dr. Roux, Paris): 'TGE7615/p'."Q7671 uaiilr thc number 1-586 )on TG87615/pTG771 under the number 1-585 )25.07.86 TGE7214. dapD gene-deleted coli. strain, under the number 1-652; TGE,7303, dapD gen_-deloted coli strain, under the number 1-6534 (the 2 strains are transformed by the plasmid pTG768 which carries the dapO and cor gene); and TQ1E7214/pTG7407, dapl'D strain tronsformad by the expression plasmid for C 2 3 0, Qnder the numbor 1-655 on 10.03.87.

I- i-ihw~ 9914 9I 4* 09 9 9 94 4$ *t 4 4r

I

$6i 4a I 1 $54' I~r 39

REFERENCES

1. Jones Primrose Robinson Ellwood D.C. (1980) Mol. Gen. Genet, 180, 579-584.

2. Nilsson Skogman G. (1985) European patent applicaticn n' 84850313.2.

3. Skogman Nilsson J. (1984) Gene 31, 117-122.

4. Miwa K. Nakamori Sano Mvimose H. (1984) Agric. BioJ.. Chem. 48, 2233-2237.

Miwa Nakamori Sano K. Mc~mose -1984) Cene 31, 275-277.

6. Hersihberger Rozteck P.R. (1984) United States Patent no 4 436 815.

7. Work E. (1950) Pature 165, 74-75.

8. Davis Dulbecco Eisen rinsbezg Wood W.B. (2nd Ed. 1973) Microbiology Ed.

Harper International p. 72.

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15f 227-231.

Richaud Rihaud Martin Haziza Patte, J.C. (1984) J. Biol. Chem. 259, 14824-14828.

11. danahan D. (1983) J. Mol. Biol. 166, 557-580.

12. Bolion bathe Herzog Denicourt D., 25 Lecocg J-11, uomarev. Iavalle R. (1979) J.

Mol. Biol. 132, 219-233.

13. Gottesman Adhya Das A. (1980) J. Mol.

Biol. 140, 57-75.

14. Lusky Botchan M,(1981) Nature 293, 79-6i.

15. VAeira Messing 0. (1982) Gene 22, 259-268.

16. Lathe, Kieny Skory, Lecocj J-P.

(1984) DNA 3, 173-182.

V. Kieny Lathe eCCc J-P. (!983) Gene 2-E, 91-99.

18. Bendiak Friosen J.D. (1981) Mo]. Gen. Genet.

181, 356-362.

19. Summers Shrratt D.J. (1984) Cell 36, 1097- 1103C: 17 404 40 Beck, Ludwig, Auerswald, Reiss, B. Schaller, M. Gene 19, 327-336 (1982).

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181 356-362 (1981).

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4ii( 2 IA 4 4 1ii

Claims (13)

1. A bacterium, the genome of which comprises a dap gene inactivated by mutation and which is transformed by a plasmid cormprising a functional dap gene.

2. A bacterium according to claim 1, the genome of which comprises a dapD gene inactivated by mutation and which is transformed by a plasmid comprising a functional dapD gene.

S.3. A bacterium according to claim 2, the genome ;f which comprises a 1 0 dapD gene which is inactivated by deletion of at least part of the gene.

4. A bacterium according to any one of claims 1 to 3, in which the 9 plasmid comprising a functional dap gene also comprises a sequence which maintains said plasmid in a monomeric state.

IS. A bacterium according to claim 4, in which said sequence is a "cer" sequence.

6. A bacterium according to any one of claims 1 to 5, which is an E. coli bacterium.

7. A bacterium according to any one of claims 1 to 6 in which the plasmid comprising a functional dap gene also comprises a gene which encodes a protein of industrial interest and the elements ensuring its expression.

8. A bacterium according to claim 7 in which the plasmid comprising a functionml dap gene also comprises a gene which encodes a protein of industrial interest selected from hirudin, gamma-interferon, catechol 2, 3 oxygenase and alpha-antitrypsin.

9. A process for preparing a homogenous culture of bacterium according mwspe#8170 917 31 T 42 to any one of claims 1 to 8 which comprises culturiig said bacterium in a DAP-free medium in the presence of a substantial amount of lysine.

A process for preparing a protein of industrial interest which comprises culturing in a DAP-free medium containing a substantial amount of lysine, a bacterium according to claim 7 or 8 and recovering said protein from the culture.

11. A process according to claim 8, which comprises culturing in a rich medium, a bacterium according to claim 7 or 8, and recovering said protein from the culture. 10

12. A bacterium according to any one of claims 1 to 8 substantially as 00 hereinbefore described.

13. A process according to any one of claims 9 to 11 substantially as 0 Shereinbefore described. DATED this July 31, 1991 CARTER SMITH BEADLE Fellows Institute of Patent Attorneys of Australia Patent Attorneys for the Applicant: TRANSGENE SA. mwspe#8170 91 7 31