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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
  • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
• • C—CHEMISTRY; METALLURGY
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  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA

Definitions

• the present invention relates to the field of molecular biology. More particularly, it relates to a new DNA sequence exhibiting transcriptional promoter activity, expression vectors containing this sequence, and its use for the production of recombinant proteins, and for example heterologous proteins. The invention also relates to the recombinant cells containing this DNA sequence.
• This situation can cause various drawbacks, and in particular limit the activity of the promoter due to the absence of certain elements of the transcriptional machinery (for example of trans-activators), present a certain toxicity for the host cell due to an absence of regulation, or affect the stability of the vector.
• the Applicant has now identified, clone and sequence xa region of the genome of Kluyveromyces lactis exhibiting transcriptional promoter activity (see SEQ ID No. 1). More specifically, this region corresponds to the promoter of the gene for a protein involved in the transport of sugars. This region, or derivatives or fragments thereof, can be used very efficiently for the production of recombinant proteins in yeasts of the genus Kluyveromyces. It is understood that this sequence can also be used in other host organisms.
• an advantage of the promoter activity obtained lies in its regulable nature. Therefore, according to the conditions of use (medium, strain), it is possible to control the activity of the promoter, and therefore to trigger or repress the expression of a recombinant gene.
• An object of the present invention therefore resides in a DNA sequence comprising all or part of the sequence SEQ ID No. 1 or of its complementary strand, or of a derivative thereof, and having a transcriptional promoter activity.
• the term “derivative” means any sequence obtained from the sequence SEQ ID No. 1 by modification (s) of genetic and / or chemical nature, retaining promoter activity.
• modification of a genetic and / or chemical nature is meant any mutation, deletion, substitution, addition, and / or modification of one or more nucleotides. Such modifications can be carried out for different purposes, and in particular that of preparing portable promoters, or that of preparing promoters suitable for expression in a particular type of vector or host, that of reducing the size, of increasing transciption promoter activity, generate inducible promoters, improve the level of regulation, or to change the nature of regulation.
• Such modifications can be made, for example, by in vitro mutagenesis, by the introduction of additional control elements or synthetic sequences, or by deletions or substitutions of the original control elements.
• a derivative as defined above When a derivative as defined above is produced, its transcriptional promoter activity can be demonstrated in several ways, and in particular by placing under the control of the sequence studied, a reporter gene whose expression is detectable. Any other technique known to those skilled in the art can obviously be used for this purpose.
• the sequence SEQ ID No. 1 was obtained from a fusion library between fragments of the genome of Klactis 2359/152 and the lacZ gene of Ec li according to the protocol described in the examples.
• the invention also relates to a DNA fragment comprising all or part of the 3 kb BglII-BamHI fragment described in FIG. 3 and having a transcriptional promoter activity.
• Another subject of the invention relates to a recombinant DNA comprising a DNA sequence as defined above.
• This recombinant DNA can contain, for example, the promoter sequence SEQ ID No. 1 or a derivative thereof, into which a restriction site is inserted, facilitating the use of this sequence as a "portable" promoter.
• this recombinant DNA also contains one or more structural genes. In particular, they may be genes coding for proteins of pharmaceutical or agrifood interest.
• enzymes such as in particular superoxide dismutase, catalase, amylases, lipases, amidases, chymosin, etc.
• blood derivatives such as serum albumin, alpha- or beta-globin, factor VIII, factor IX, von Willebrand factor, fibronectin, alpha-1 antitrypsin, etc.
• insulin and its variants lymphokines (such as interleukins, interferons, colony stimulating factors [G-CSF, GM-CSF, M-CSF ...], TNF, TRF, etc.), growth factors (such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, etc.), apolipoproteins, antigenic polypeptides for production of vaccines (hepatitis, cytomegalovirus, Eppstein-Barr, herpes, etc.), or even fusions of polypeptides such as in particular fusions comprising an active
• the recombinant DNA also contains signals allowing the secretion of the expression product of said structural gene (s).
• signals may correspond to the natural secretion signals of the protein in question, but they may be of a different origin.
• secretion signals derived from yeast genes can be used, such as those of the killer toxin genes (Stark and Boyd, EMBO J. 5 (1986) 199i) or the alpha pheromone (Kurjan and Herskowitz, Cell 2 ⁇ (1982) 933; Brake et al., Yeast 4 (1988) S436).
• the recombinant DNA is part of an expression plasmid, which can be autonomously replicating or integrative.
• autonomously replicating vectors can be obtained using autonomous replicating sequences in the chosen host.
• these may be origins of replication derived from plasmids (pKDl, 2 ⁇ , etc.), or else chromosomal sequences (ARS).
• Integrative vectors can be obtained in particular by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector.
• Another subject of the invention relates to recombinant cells containing a DNA sequence as defined above.
• the cells are chosen from yeasts, and even more preferably from yeasts of the genus Kluyveromyces. It is understood, however, that the invention covers all the recombinant cells in which the promoter regions of the invention are active, whether they are eukaryotic or prokaryotic cells.
• the eukaryotic cells mention may be made of plant, animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces, Pichia, Schwanniomyces, or Hansenula.
• animal cells mention may be made of COS, CHO, C127 cells, etc.
• fungi capable of being used in the present invention, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
• bacteria such as Escherichia coli, or those belonging to the genera Corynebacterium, Bacillus or Streptomyces can be used.
• promoter of the transcription of the sequences of the invention in these different hosts can be verified for example by introducing into the host cell considered a recombinant DNA comprising, under the control of the promoter sequence studied, a reporter gene, the expression can be highlighted in the host considered.
• the recombinant cells of the invention can be obtained by any method which makes it possible to introduce foreign DNA into a cell. It may especially be transformation, electroporation, conjugation, fusion of protoplasts, or any other technique known to those skilled in the art. With regard to transformation, various protocols have been described in the prior art. In particular, it can be carried out by treating the whole cells in the presence of lithium acetate and of polyethylene glycol according to the technique described by Ito et al. (J. Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and dimethyl sulfoxide according to the technique of Durrens et al. (Curr. Genêt. Lj £ (1990) 7).
• sequences of the invention can be used for the expression of genes coding for proteins of pharmaceutical or agrifood interest.
• proteins of pharmaceutical or agrifood interest By way of example, mention may be made of the proteins listed above.
• the present invention also makes it possible to carry out a method for producing recombinant proteins, according to which a recombinant cell as defined above is cultivated and the protein produced is recovered.
• a recombinant cell as defined above is cultivated and the protein produced is recovered.
• a particularly advantageous aspect of the invention lies in the possibility of regulating the activity of the promoters.
• the Applicant has indeed shown that the promoter of the sequence SEQ ID No. 1 was repressed by glucose and active in the presence of lactose.
• the promoter of the sequence SEQ ID No. 1 is 50 to 100 times more active in the presence of lactose than in the presence of glucose.
• This result is interesting because it makes it possible to carry out a process for producing recombinant proteins in which the phases of cell growth and expression of the desired recombinant gene are separated.
• glucose medium the recombinant cells divide up to a stationary phase. At this time, the concentration of glucose in the culture medium has dropped sharply, which allows the expression of the gene of interest to be depressed.
• the method of the invention is applicable to the production of human serum albumin, or one of its molecular variants.
• molecular variant of albumin is understood to mean the natural variants resulting from the polymorphism of albumin, truncated forms, or any hybrid protein based on albumin.
• SEQ ID No. 1 Nucleotide sequence of the 1 kb fragment corresponding to the promoter of a sugar transporter gene from K. lactis.
• Figure 1 Preparation of the Mini Mu MudIIZKl transposon.
• Figure 2 Restriction map of the Mini Mu MudIIZKl transposon.
• Figure 3 Restriction map of clone 2C5.
• Figure 4 Restriction map of the 3 kb BglII-BamHI fragment carrying the sequence SEQ ID No. 1.
• the pBR322 and pUC type plasmids are of commercial origin (Bethesda
• the DNA fragments are separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA.
• phage T4 ligase (Boehringer) according to supplier's recommendations.
• the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of the DNA Polymerase I of £. coli (Boehringer) according to the supplier's specifications. Destruction of the prominent 3 ′ ends can be carried out in the presence of phage T4 DNA Polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends can be carried out by gentle treatment with nuclease SI.
• K. lactis The transformations of K. lactis are carried out by any technique known to a person skilled in the art, an example of which is given in the text. Unless otherwise indicated, the bacterial strains used are Ecoli DH1
• the yeast strains used belong to budding yeasts and more particularly to yeasts of the genus Kluyveromyces.
• the strains K. lactis 2359/152 and K. lactis SD6 were particularly used.
• the yeast strains transformed by the plasmids are cultured in Erlenmeyer flasks or in pilot fermenters of 21 (SETRIC, France) at 28 ° C in rich medium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.
• YPD 1% yeast extract, 2% Bactopeptone, 2% glucose
• YPL 1% yeast extract, 2% Bactopeptone, 2% lactose
• sequence SEQ ID No. 1 was isolated from a fusion library between fragments of the genome of K lactis 2359/152 and the lacZ gene of Exoli. This example describes in (A) the preparation of the fusion library, and in (B) the selection and characterization of a clone of this library carrying the promoter sequence SEQ ID no.
• the Mini Mu MudIIZKl was built from the Mini Mu Mud ⁇ ZZl described by Daignan-Fornier and Bolotin-Fukuhara (Gene £ 2 (1988) 45). It was obtained by replacing the origin of replication of the MudIIZZI mini transposon with an origin of functional replication in Kluyveromyces: the origin of replication of the plasmid pKDl (EP231,435).
• A.1.1 Construction of a cassette carrying the origin of replication of the plasmid pKD1 (fragment SU).
• the SU fragment (carrying the origin of replication of the plasmid pKD1) was put into the form of a NotI cassette.
• a derivative of the plasmid pUCl ⁇ was constructed in which the external sites of the cloning multisite (Hindi ⁇ and EcoRI sites) were changed into NotI sites. This was done by digestion with the corresponding enzyme, action of the Klenow enzyme and ligation with a synthetic oligonucleotide corresponding to a NotI site [oligo d (AGCGGCCGCT); Biolabs].
• the plasmid obtained is designated ⁇ GM67.
• the SU fragment of 960 bp obtained by digestion with the enzyme Sau3A of the plasmid KEp6 was then inserted at the BamHI compatible site of the plasmid pGM67.
• the plasmid thus obtained, designated pGM68 contains, in the form of a NotI cassette, the SU fragment.
• the plasmid pGM15 carrying the mini Mu MudIIZZl was deleted from the 2 ⁇ regions by digestion using the enzyme Sali.
• the unique SalI site thus obtained was then transformed into a NotI site by ligation of a synthetic oligonucleotide corresponding to a NotI site after the action of the Klenow enzyme.
• the resulting plasmid is called pGM59.
• the plasmid obtained designated pGM83, carries a mini Mu, called MudIIZKl, which is adapted to the yeast Kluyveromyces lactis, as well as a functional copy of the LEU2 gene from S. cerevisiae capable of complementing a leu2 mutation in Klactis
• MudIIZKl is shown in Figure 2.
• the strain JM109 :: (Mute) was transformed by the plasmid pGM83 containing the mini mu MudIIZKl in the presence of calcium chloride. After transformation, the transposition was induced by thermal shock according to the technique described by Castilho et al. (J. Bacteriol. 15S (1984) 488). The phage lysate obtained after induction is then used to superinfect the JM109 strain: :( Mucts).
• the JM 109 "strain (Mutes) being recA, the linear DNA packaged by the phage cannot be closed to give a replicative plasmid.
• the integrants [strain JM109 :: (Mutes)" (MudIIZKl)] are therefore selected as chloramphenicol clones resistant (Cm ⁇ ), sensitive ampicillin (AmpS).
• the plasmid DNA of each pool produced in DH1 is extracted (Maniatis).
• the fusion library is produced by extensive transposition of the Mini Mu MudIIZKl onto the plasmids forming the genomic DNA library of Klactis.
• the mini-muductions were made according to the protocol described by Castilho et al. (J.
• LBAC LBAC (LB medium (Gibco BRL) supplemented with 50 mg / 1 of ampicillin and 30 mg 1 of chloramphenicol), the marker Amp ⁇ being provided by the plasmid, and the marker Cm ⁇ by the mini-mu.
• transpositions are made in series, and between 10,000 and 20,000 transductants are recovered per pool.
• the DNA of the transductants is then extracted from a 100 ml preparation, purified by precipitation with polyethylene glycol (Maniatis et al, 1989) and resuspended in 100 ⁇ l of water. This
• the fusion DNA prepared above was used to transform, by electroporation, a receptor strain of Klactis.
• This receptor strain designated SD6
• This last mutation prevents the strain from growing on a medium containing lactose as the only carbon source, but it can be complemented by overexpression of the lacZ ⁇ .coli gene coding for ⁇ -galactosidase (Chen et al., J. Basic Microbiol £ 2 (1988) 211). Therefore, the expression of a protein fused to ⁇ -galactosidase must allow the growth of the strain SD6 on lactose after transformation. This positive screen was used to rapidly select clones carrying strong promoters.
• the strain SD6 (Chen et al., Mol. Gen. Genêt. 222 (1992) 97) was obtained by crossing the strain Klactis CXJ1-7A (a, lac4-8, ura3A, adel-1, Kl, K2, pKDl) (Chen and Fukuhara, Gene £ 2 (1988) 181) with the strain AWJ-137 (leu2, trpl, homothallic) (Kâmper et al, Curr. Genêt. 12 (1991) 109), and selection of spores having the ADE + genotype, uraA, leu2, lac4-8.
• the strain CXJ1-7A As the spores obtained were not capable of regenerating after transformation by protoplasts, a back crossing was made with the strain CXJ1-7A. After mass sporulation, the spores of the selected genotype were tested by transformation with lithium chloride with the plasmid KEp6 according to a technique derived from that described by Ito et al. (J. Bacteriol. 153 (1983) 163) (the LiCl concentration is 20 mM, ie 10 times less than that used by Ito for S. cerevisiae). The strain CXJ1-7A served as a transformation control.
• the strain SD6 selected on these criteria, transforms correctly: 1 to 3. 10 ⁇ transformants per ⁇ g of DNA; and the transformants have satisfactory stability: 30 to 40% of the colonies retain the [Ura + ] phenotype after 6 generations in a non-selective medium.
• strain SD6 was transformed by electroporation according to Becker and
• clone 2C5 was studied by restriction (see FIG. 3) and by analysis of the sequence of the junction between the Klactis protein and the ⁇ - galactosidase.
• sequence of the junction starting from the lacZ end of the mini-m ⁇ (double-stranded sequence) was determined by sequencing, using the following oligonucleotide located at -59 nucleotides from the junction:
• the different Kluyveromyces strains used were transformed by treating the whole cells in the presence of lithium acetate and polyethylene glycol, according to the technique described by Ito et al. (J. Bacteriol. 153 (1983)
• a portable promoter is prepared by PCR, by insertion on the 3 kb BglII-BamHI fragment of a restriction site in position +1 relative to the ATG codon.
• This site makes it possible to generate a fragment comprising the promoter region and to introduce any gene which it is desired to express downstream of the promoter thus obtained.
• NAME RHONE-POULENC RORER S.A.

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