FR2685919A1 - Protein with cytokine-type activity, recombinant DNA encoding this protein, transformed animal cells - Google Patents

Abstract

The subject of the present invention is a protein with cytokine-type activity, or a precursor of this protein which comprises the following amino acid sequence (a1): or a sequence having a high degree of homology with the sequence (a1).

Description

 The subject of the present invention is a novel cytokine-type protein, the genetic engineering tools for producing it, namely a recombinant DNA, an expression vector carrying this recombinant DNA, the prokaryotic microorganisms and the eukaryotic cells containing this DNA. recombinant and a drug, useful in particular as an immunomodulator, containing as active ingredient this protein.

It is well known that the immune system includes cellular elements and soluble substances secreted by them, called cytokines. These are proteins that provide communication between an emitter cell and a target cell belonging either to the immune system or to another biological system of the body. Cytokines generally have a pleiotropic biological activity: they can have multiple actions on the target cell: proliferation, differentiation, cytolysis, activation, chemotaxis, etc. Several of these molecules have already found applications in therapeutics: for example , interleukin-2 or interferon-a used for the treatment of certain tumors by immunotherapy and myelopoietic factors such as GCSF (Granulocyte Colony Stimulating
Factor) or GMCSF (Granulocyte Monocyte Colony Stimulating Factor) which stimulate the growth and differentiation of blood cells and thus allow the enrichment of depleted blood in them, following, for example, chemotherapy.

In particular, we have the following knowledge about cytokines:
1) These correspond to secreted proteins. The secretion of a protein, and therefore in particular of a cytokine, is most often carried out by a mechanism which comprises the cleavage of a hydrophobic amino-terminal region of the translated protein, called pre peptide sequence or signal peptide, during the excretion of the mature protein in the medium (Von Heijne G. 1986, Nucl.

Ac. Res., 14, 4683-4690). Most peptide sequences of known cytokines do indeed comprise a signal peptide.

2) The cytokines generally correspond to inducible proteins, or other cytokines (K. Matsushima et al., 1988, J. Exp Med., 167, 1883-1893 - Shimizu H. et al, 1990, Mol. Cell Biol., 10, 561-568), or by chemical activators of cell proliferation and differentiation such as lipopolysaccharides, calcium ionophores, for example calimicin, cyclic dibutyryl AMP, dimethylsulfoxide, acid retinoic, concanavalin A, phytohemaglutinin (PHA-P) and phorbol esters, for example phorbol myristate-2-acetate-3 (PMA) (Muegge K. and
Durun SK 1990 Cytokine 2, 1-8 - CB Thompson et al., 1989, Proc. Natl.

Acad. Sci., 86, 1333-1337), or by antibodies against surface molecules, such as surface antigens CD2, CD3, CD28 and CD40 (Thomson
CB et al, reference above and Rousset F. et al, 1991, J. Exp. Med., 173, 703-710).

 3) As the induction of cytokines is transient, the messenger RNAs of cytokines are unstable. They have, for the most part, cytokines rich in regions A and U, notably the AUUUA sequence, a consensus instability sequence demonstrated by D. Caput et al., 1986, Proc. Ntl. Acad. Sci.

USA, 83, 1670-1674; G. Shaw et al, 1986, Cell, 46, 659-667 and K.

Peppel et al., 1991, J. Exp. Med., 173, 349-355.

 4) The vast majority of known cytokines are synthesized by the cells of the immune system, including monocytes and helper T cells of peripheral blood (Ullman K.S. et al., 1989, Ann.

Rev. Imm., 8, 421-452 and the work "Macrophage derived cell regulatory factors" by C. Sorg, published in 1989 by Karger-BâIe-Suisse). Their synthesis is induced by the inducing agents mentioned in 2).

It is also known that it is possible to construct from peripheral blood mononuclear cells consisting mainly of lymphocytes and monocytes, cultured and stimulated using different inducing agents such as phytohemaglutinin, phorbol myristate-2 acetate-3 and the anti-CD2 monoclonal antibody, a complementary DNA library subtracted ubiquitous sequences of complementary DNA in animal cells, and thus enriched in complementary DNA encoding cytokines (HC Chang et al, Eur J Imm., 1989, 19, 1045-1051, PF Zipfel et al, 1989,
Mol. Cell. Biol 9: 1041-1048).

On the other hand (CB Thompson et al., 1989, Proc., Ntl Acad Sci., 86, 1333-1337 and Lindsten T. et al., 1989, Science, 244, 339), it is known that the production of cytokines ( called lymphokines) by T lymphocytes, is larger, so corresponds to larger amounts of messenger RNA, with stimulation using the binary combination: phorbol myristate-2 acetate-3 and phytohemaglutinine or phorbol myristate-2 acetate-3 and the anti-CD28 antibody, only with phorbol myristate-2 acetate-3 only and with the binary combination phorbol myristate-2-acetate-3 and phytohemaglutine than with the tertiary combination: phorbol myristate-2 acetate- 3, phytohemaglutin and cyclosporin A, cyclosporin A apparently having an inhibitory effect on the transcription of messenger RNAs of cytokines (Thompson et al, cited above and Mattila et al., 1990,
EMBO J., 9, 4425-4433).

 This differential expression of the cytokine messenger RNAs under different stimulation conditions can be used for a screening, called differential screening, of a complementary DNA library containing sequences coding for cytokines in order to select these latter sequences (see, for example, Dworkin et al., 1980, Dev Biol., 76, 449-464, Cochran BM et al, 1983, Cell, 33, 939-947 and Zipfel PF et al, 1989, Mol Cell Biol., 9, 1041-1048. It is indeed possible to construct from two messenger RNA corresponding to two states of stimulation of the cells, here called stimulation state 1 and stimulation state 2, the stimulation state 2 being supposed to be richer in messenger RNA coding for a cytokine that the state 1, two radioactive probes called respectively probe 1 and probe 2, which are transcripts obtained using the reverse transcriptase of these two messenger RNAs. These two probes are hybridized with bacterial colonies containing DNA sequences complementary to the library. The bacterial colonies containing a complementary DNA whose messenger RNA changes in abundance between the two activation states will give differential signals with both probes, the hybridization being more important for the retained clones (which correspond to inducible proteins). ) for probe 2 only for probe 1.

 It is also known that COS cells, monkey kidney cells expressing SV40 virus T antigen (Gluzman, Y. Cell, 23, 1981, 175-182), which allow the replication of vectors containing the origin of replication of SV40 DNA, are prime hosts for the study of gene expression in animal cells and secretion of expressed proteins. The secretion of a protein in these cells indicates that it is secreted by the cell that produces it naturally (H.C. Chang et al., Eur.

Imm., 1989, 19, 1045-1051 and W.Y. Weiser et al., 1989, Proc. Ntl. Acad. Sci.

USA, 86, 7522-7526)
The subject of the present invention is a cytokine-type activity protein, or a precursor thereof, which comprises the following amino acid sequence (a1):
Gly Pro Pro Pro Pro Ser Thr Ala Leu Arg Glu Leu Glu Glu Leu
1 5 10 15
Val Asn Thr Island Gn Asn Gln Lys Ala Pro Leuk Cys Asn Gly Ser Met
20 25 30
Val Trp Ser Asn Island Leu Thr Ala Asp Met Tyr Cys Ala Ala Leu Glu
35 40 45
Ser Leu Island Asn Val Ser Gly Cys Ser Ala Island Glu Lys Thr Gln Arg
50 55 60
Met Leu Ser Gly Phe Cys Pro His Lys Ser Val Ala Gly Gln Phe Ser
65 70 75 80
Ser Leu His Arg Val Asp Thr Glu Island Gla Val Ala Gln Phe Val Lys 85 90 95
Asp Leu Leu Leu Leu Leu Lys Leu Phe Arg Glu Gly Arg Phe Asn
100 105 110 or a sequence having a high degree of homology with the sequence (a1)
A high degree of homology here means homology of identity (ratio of identical amino acids to total number of amino acids) of at least 80%, and preferably at least 90%, of the sequences. amino acids, when aligned according to maximum homology, according to the optimal sequence alignment method of Needleman and Wunsch, 1970,
J. Mol. Biol., 48, 443-453. This algorithmic method, which considers all the possible alignments and creates an alignment in which as many identical amino acids as possible are paired and the number of holes in the aligned sequences is minimal, is used in particular in the software.
UWGCG from the University of Wisconsin: Devereux et al, 1984, Nucl. Ac. Res., 12, 8711-8722- GAP option.

 The peptide sequence already known closest to that of the sequence (a1) of 112 amino acids is that of 132 amino acids deduced from the DNA complementary to the mouse P600 protein, described by KD Brown et al., 1989, J. Imm., 142, 679-687 (protein expressed by mouse TH2 lymphocytes whose function has not been elucidated). A comparison of these peptide sequences using the method of Needleman and Wunsch, 1970, J.

Mol. Biol., 48, 443-453 shows that 63 amino acids are identical on 112, an identity homology of about 56%.

The protein of the present invention is a protein secreted in particular by T lymphocytes and inducible in the latter by phorbol myristate-2-acetate-3, this induction being - as expected for a cytokine (see text above) - amplified by phytohemaglutinin or monoclonal antibody CD28 with an inhibitory effect of this increase in the case of stimulation by phytohemaglutamine in the additional presence of cyclosporin A. It has a cytokine-type activity: it has activities vis-à-vis at least two key cells of the immune system: B cells and peripheral blood monocytes. It inhibits the synthesis of interleukin-1b and interleukin-6 by activated peripheral blood monocytes. to LPS and it increases the expression of antigen
These two properties are also those of Interleukin-4 (Paul W., 1991, Blood, 77, 1959 and Waal
Malefyt et al., 1991, J. Exp. Med., 174, 1909-1220).

 The protein of the invention is a new interleukin, lymphokine type. It is of interest as a principle of a drug useful for the immunomodulatory treatment of tumors and certain infectious or inflammatory conditions.

The protein of the invention may comprise, immediately upstream of the sequence (a1) one or more amino acids, in particular the sequence
Ser-Pro.

 This protein is preferably in a form which has an apparent molecular weight, determined by SDS polyacrylamide gel electrophoresis of 9.0 +2 or 16.0 + 2 kDa. It is advantageously Nglycosylated, in particular when it has an apparent molecular mass of 16.0 ± 2kDa.

 It is interesting that this protein has a degree of purity, determined by polyacrylamide gel electrophoresis in the presence of SDS and silver detection, greater than 70%, preferably greater than 90%.

 The subject of the invention is also a recombinant DNA characterized in that it codes for the preceding protein, which can then be obtained from the cell lysate or, advantageously, for a precursor of the preceding protein. This precursor preferably comprises a signal sequence.

 This signal sequence, chosen according to the host cell, has the function of allowing the export of the recombinant protein outside the cytoplasm, which allows the recombinant protein to take a conformation close to that of the natural protein and considerably facilitates his purification. This signal sequence can be cleaved, either in several steps by a signal-peptidase that releases the mature protein, or in several steps when this signal sequence comprises in addition to the sequence eliminated by the signal-peptidase, called signal peptide or pre-sequence, a sequence eliminated later during one or more proteolytic events, called pro sequence.

The coding sequence for the mature protein is, for example, the following sequence (Nal):
GGCCCTGTGC CTCCCTCTAC AGCCCTCAGG GAGCTCATTG
AGGAGCTGGT CAACATCACC CAGAACCAGA AGGCTCCGCT
CTGCAATGGC AGCATGGTAT GGAGCATCAA CCTGACAGCT
GACATGTACT GTGCAGCCCT GGAATCCCTG ATCAACGTGT
CAGGCTGCAG TGCCATCGAG AAGACCCAGA GGATGCTGAG
CGGATTCTGC CCGCACAAGG TCTCAGCTGG GCAGTTITCC
AGCLTGCATG TCCGAGACAC CAAAATCGAG GTGGCCCAGT TTGTAAAGGA CCTGCTCTTA CATTTAAAGA AACTITITCG
CGAGGGACGG TTCAAC
For expression in prokaryotic microorganisms, such as for example Escherichia coli, this signal sequence may be either a sequence derived from the sequence encoding a natural precursor of a protein exported by a prokaryotic microorganism (e.g. signal peptide
OmPA [Ghrayeb et al., 1984, EMBO Journal 3, 2437-2442] or the signal peptide of alkaline phosphatase [Michaelis et al., J. Bact. 1983, 154, 366-374]), a non-endogenous sequence originating from a sequence coding for a eukaryotic precursor (for example the signal peptide of one of the natural precursors of human growth hormone), or a sequence coding for a synthetic signal peptide (for example that described in French Patent Application No. 2,636,643, of sequence ::
Met Al Ser Ser Gly Lys Ser Leu Leu Leu Leu Leu Leu Leu Leu Leu
1 5 10 15
Cys Leu Ser Pro Asn Ala Gly Ala
20 25
For expression in eukaryotic cells such as ascomycetes, for example the yeast Saccharomyces cerevisiae or the filamentous fungus Cryphonectria parasitica, this signal sequence is preferably a sequence derived from a sequence coding for a natural precursor of a protein secreted by these cells, for example for yeast the precursor of invertase (patent application EP 0123289) or the precursor of the prepro sequence of the alpha pheromone (patent application DK 2484/84), or for Cryphonectria parasitica, the precursor of the prepro sequence of endothiapepsin, sequence ::
Met Ser Ser Pro Leuk Lys Asn Ala Leu Val Thr Ala Met Leu Ala Gly
1 5 10 15
Gly Ala Leu Ser Pro Ser Lys Gln His Val Gly Pro Val Asn
20 25 30
Ala Ser Pro Glu Val Gly Pro Gly Lys Tyr Ser Phe Lys Gln Val Arg
35 40 45
Asn Pro Asn Tyr Lys Phe Asn Gly Pro Leu Ser Val Lys Lys Thr Tyr
50 55 60
Leu Lys Tyr Gly Val Pro Pro Ala Trp Leu Glu Asp Ala Val Gln
65 70 75 80
Asn Ser Ser Ser Gly Leu Ala Glu Arg
85
For expression in animal cells, the signal sequence used is a signal sequence of an animal cell protein known to be exported, for example a sequence coding for the signal peptide of one of the natural precursors of the hormone. of human growth, already known to allow the secretion of interleukin-2 (see French patent application 2,619,711), one of the four signal sequences (b1), (b2), (b3) and ( b4): (b1)
Met His Pro Leu Leu Asn Leu Leu Leu Al Leu Leu Leu Met Ala
1 5 10 15
Leu Leu Leu Thr Thr Val Ala Leu Thr Cys Lcu Gly Gly Phe Ala
20 25 30 (b2)
Met His Pro Leu Leu Asn Pro Leu Leu Leu Ala Lcu Gly Lcu Met Ala
1 5 10 15
Leu Leu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly Phe Ala
20 25 30
Ser Pro (b3)
Met Ala Leu Leu Leu Thr Thr Val Ala Island Leu Thr Cys Lcu Gly Gly
1 5 10 15
Phe Ala (b4)
Met Ala Leu Leu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly
1 5 10 15
Phe Ala Ser Pro
Advantageously coded respectively by the following sequences (Nbl), (Nb2), (Nb3) and (Nb4) :: (Nbl)
ATGCATCCGC TCCTCAATCC TCTCCTGTTG GCACTGGGCC
TCATGGCGCT TPrGTTGACC ACGGTCATTG CTCTCACTTG CCTTGGCGGC TTGCC (Nb2)
ATGCATCCGC TCCTCAATCC TCTCCTGTTG GCACTGGGCC
TCATGGCGCT TTTGTTGACC ACG GTCATFG CTCTCAC? TG
CCTTGGCGGC lll GCCTCCC CA (Nb3)
ATGGCGC1ZT TGTTGACCAC GGTCATTGCT CTCACTTGCC
TTGGCGGCTT TGCC (Nb4) ATGGCGCTTT TGTTGACCACG GTCATTG CT CTCACITGCC
TTGGCGGCIT TGCCTCCCCA
The invention also relates to an expression vector which carries, with the means necessary for its expression, the recombinant DNA defined above.

 For expression in prokaryotic microorganisms, in particular in Escherichia coli, the recombinant DNA must be inserted into an expression vector comprising in particular an effective promoter, followed by a ribosome binding site upstream of the gene to be expressed. as well as an effective transcriptional stop sequence downstream of the gene to be expressed. This plasmid must also include an origin of replication and a selection marker. All these sequences must be chosen according to the host cell.

 For expression in eukaryotic cells, the expression vector according to the invention carries the recombinant DNA defined above with the means necessary for its expression, its replication in eukaryotic cells and the selection of transformed cells. Preferably, this vector carries a selection marker, chosen for example to complement a mutation of the eukaryotic receptor cells, which allows the selection of cells which have integrated the recombinant DNA into a high copy number either in their genome or in a multicopy vector.

 For expression in eukaryotic cells such as yeast, for example Saccharomyces cerevisiae, it is appropriate to insert the recombinant DNA between, on the one hand, sequences recognized as an effective promoter, on the other hand, a transcription terminator. The promoter-coding sequence-terminator assembly, called expression cassette, is cloned either in a monocopy plasmid vector or mimeograph for the yeast, or integrated in multicopy in the genome of the yeast.

 For expression in eukaryotic cells such as those of filamentous fungi, of the ascomycetes group, for example those of the genera Aspergillus, Neurospora, Podospora, Trichoderma or Cryphonectria, the expression vector according to the invention carries the recombinant DNA defined above. with the means necessary for its expression, and optionally a selection marker and / or telomeric sequences. it is indeed possible to select the transformants having integrated a DNA of interest using a selection marker located on the same vector as the DNA of interest, or on another vector, these two vectors being The recombinant DNA of the invention can be either integrated into the genome of the filamentous fungi, or preserved in extrachromosomal form by means of sequences allowing the replication and partition of this DNA.

For expression in animal cells, especially in CHO Chinese hamster ovary cells, the recombinant DNA is preferably inserted into a plasmid (eg derived from pBR322) having either a single expression unit in which is inserted the recombinant DNA of the invention and optionally a selection marker, in front of an effective promoter, two expression units. The first expression unit comprises the recombinant DNA above, preceded by an effective promoter (for example the SV40 early promoter). The sequence around the initiating ATG is preferably selected according to the consensus sequence described by Kozak (M. Kozak (1978) Cell., 15, 1109-1123). An intronic sequence, for example the intron of mouse α-globin, can be inserted upstream of the recombinant DNA as well as a sequence comprising a polyadenylation site, for example a polyadenylation sequence of the mouse.
SV40, downstream of the recombinant gene. The second expression unit comprises a selection marker, for example a DNA sequence coding for dihydrofolate reductase (abbreviated enzyme DHFR). The plasmid is transfected into animal cells, for example CHO dhfr cells (unable to express DHFR). A line is selected for its resistance to methotrexate: it has integrated into its genome a high copy number of the recombinant DNA and expresses it at a sufficient level.

 The invention also relates to the prokaryotic microorganisms transformed by the expression vector defined above, in particular those of the Escherichia coli species as well as to the eukaryotic cells which contain, with the means necessary for its expression, the defined recombinant DNA. previously. This may have been introduced by transformation with the expression vector previously or by the recombinant DNA of the invention itself, which can sometimes be integrated directly into the genome into a locus that allows its expression.

 Useful eukaryotic cells are animal cells.

The recombinant DNA may, for example, have been introduced into these cells by transfection with the above expression vector, by infection with a virus or retro-virus carrying it, or by microinjection. Preferred animal cells are CHO cells, particularly cells
CHO dhfr- from which it is possible to obtain highly productive lines in the protein of the invention. COS cells are also an advantageous host for obtaining this protein.

 Other interesting eukaryotic cells are yeast cells, in particular Saccharomyces cerevisiae.

 The invention also relates to a process for the preparation of the previously defined protein which comprises a step of culturing the above animal cells, or this yeast, followed by the isolation and purification of the recombinant protein.

 The invention also relates to the recombinant protein that can be obtained by a process that comprises a step of culturing these animal cells, or this yeast, followed by the isolation and purification of the recombinant protein.

 The invention therefore also relates to the medicinal product, useful in particular oncology and in the immunomodulatory treatment of certain infectious conditions and certain inflammatory conditions which contains as active ingredient the protein defined above in a pharmaceutically acceptable excipient. These can be used alone or in combination with other active agents: for example one or more other cytokines.

 The invention will be better understood from the following discussion, divided into sections, which includes experimental results and a discussion thereof. Some of these sections relate to experiments carried out for the purpose of carrying out the invention, others of the embodiments of the invention given of course purely by way of illustration.

 Many of the techniques described in these sections, well known to those skilled in the art, are described in detail in the book by Sambrook, Fritsch and Maniatis: "Molecular cloning: a Laboratory manual" published in 1989 by Cold Spring Harbor Press in New York (2nd edition).

 The following description will be better understood with reference to FIGS. 1a, 1b, 1c, 2, 3, 4 and 5.

 Figure 1aX shows an assembly map of the plasmid pSE1, cloning plasmid in E. coli and expression in animal cells, the sites having disappeared by ligation being noted in parentheses. The symbols used in this figure will be specified when describing this plasmid (section 2).

Figure lb shows the sequence of the synthetic fragment
HindIII- HindIII, used for the assembly of plasmid pSE1.

 Figure ic represents the sequence of the synthetic fragment HindIII- "BamHI-binding site".

FIG. 2 represents the nucleotide sequence of the NC30 cDNA and the deduced amino acid sequence, with the two metabolizable M1s being underlined, the probable cleavage sites of the signal peptide being indicated by vertical arrows and the four possible sites of
N-glycosylation being underlined in dotted lines.

 FIG. 3 and FIG. 4 respectively represent the alignment according to the maximum homology according to the method of Needleman and Wunsch, 1970, J. Mol. BioI., 48, 443-453 of the deduced amino acid sequence of the NC30 cDNA (upper line) and the deduced sequence of the mouse P600 protein cDNA (lower line) and the alignment according to this method of the coding part of the NC30 cDNA (upper line) and the coding part of the cDNA of the P600 protein (lower line).

 Figure 5 shows the sequence of fragment B, the silent mutation site relative to NC30 cDNA being indicated by an asterisk.

SECTION 1: Culture and stimulation using PMA and PHA-P cells
mononuclear peripheral blood. RNA preparation
messenger used to make the complementary DNA library 1) Culture and stimulation of peripheral blood mononuclear cells:
From peripheral blood bags (taken from three healthy volunteers in a blood transfusion center), having previously undergone cytapheresis and a Ficoll gradient (Pharmacia Boyum A., 1968, Scand.

Clin. Lab. Invest., 21, p. 77-89) a fraction of cells enriched in peripheral blood mononuclear cells (PBMNC) peripheral blood mononuclear cells of approximate composition: 70% lymphocytes, 25% monocytes and 5% granulocytes (cell count at using the Coulter-Model S-Plus IV cell counter)
The cells are collected in a 250 ml flask and then centrifuged at 300 g for 10 min at 37in. The supernatant is removed and the cell pellet is rinsed with 50 ml of glucose medium, mineral salts, amino acids and vitamins, called RPMI medium (RPMI 1640 Gibco BRL medium), and then centrifuged again under the same conditions.

 The cell pellet is then taken up with 500 ml of RPMI medium supplemented with 10% fetal calf serum (Gibco BRL-ref 013-06290H), supplemented with 10 units of penicillin and 10 g of streptomycin (penicillin solution / Gibco Streptomycin No. 043-05140D) per ml of medium as well as L-glutamine (Gibco BRL-043-05030D) up to 2mM final.

 Part of the cell suspension is distributed for separation of the adherent cells and the non-adherent cells, at a rate of about 100 ml per dish, in four large square culture dishes (245 x 245 x 20 mm-Nunc - ref 166508) and incubated for 1 hour at 37 ° C. It is known that most monocyte cells adhere to the culture dish while most lymphocyte cells remain in suspension.

 The non-adherent cells are aspirated by pipette and grown in 175 cm 2 surface Falcon culture flasks in the presence of RPMI medium supplemented as described above, supplemented with 10 ng / ml of phorbol myristate. 2 acetate-3 (PMA) (Sigma-P8139) and 5 μg / ml phytohemaglutinin (PHA-P) (Sigma-ref L8754) at 37 ° C in the presence of 5% CO 2 for 24 h .

On the adherent cells are added 100 ml of RPMI medium supplemented as described above supplemented with 10 ng / ml of PMA and 5 μg / ml of
PHA-P. The cells are incubated at 37 ° C. in the presence of 5% (v / v) of CO2 for 5 hours.

The rest of the cell suspension, hereinafter referred to as the total cells, is divided into 4 large square culture dishes and incubated in the presence of RPMI medium supplemented as described above, supplemented with 10 ng / ml of
PMA and 5 g / ml PHA-P at 37C in the presence of 5% (v / v) CO 2 for 5 h for the first two cans and 24 h for the other two.

 About 2 hours before the end of the incubation, these different cells are added to the culture medium with 1 μg / ml of cycloheximide (Sigma C6255) (translation inhibitor which increases the stability of the RNAs of cytokines: see Lindsten et al. 1989, Science 244, 339, 344) and the incubation is continued for 2 hours at 37C.

2) Preparation of the messenger RNA:
a) Extraction of messenger RNA
The cells are recovered in the following manner: the adherent cells are washed twice with PBS buffer (phosphate buffered saline 04104040-Gibco BRL) then scraped with a rubber scraper and centrifuged. This gives a cell pellet, called a A pellet.

- For non-adherent cells, after shaking the bottle containing the cell suspension, the cell suspension is removed and centrifuged. A cell pellet, called the NA cell pellet, is thus obtained.

for the total cells, the adhering fraction is washed twice with buffer
PBS and scraped as before and then centrifuged. The non-adhered fraction is centrifuged. The two cell pellets obtained will be joined later. Their meeting is called cell pellet T (5 h) for the total cells incubated for 5 h and T (24 h) for the total cells incubated for 24 h.

Cell pellets A, NA, T (5 h) and T (24 h) are frozen and
kept at -80iC.

Each frozen cell pellet is suspended in the lysis buffer of the following composition: 5M guanidine-thiocyanate; Tris (hydroxy
50mM methyl-aminomethane pH 7.5 EDTA 10mM. The suspension is sonicated using an Ultra Turax n 231 256 sonicator (Janke and Kunkel) at maximum power for 4 cycles of 20 s. S-mercaptoethanol is added to 0.2M and a 30 s sonication cycle is repeated. Lithium chloride is added up to 3M. The suspension is cooled to 40C and allowed to stand at this temperature for 48 hours. The RNA is then isolated by centrifugation for 60 min. The RNA pellet is washed once with a solution of 3M lithium chloride, recentrifuged and then taken up in a buffer of the following composition: 1% SDS, 5 mM EDTA and Tris HCl 10mM pH 7.5, supplemented with
1 mg / ml Proteinase K (Boehringer Mannheim, GmbH). After incubation at 400C for 1h, the RNA solution is extracted with a phenol / chloroform mixture. The ART contained in the aqueous phase is precipitated at -20 ° C. using a final 0.3M ammonium acetate solution and 2.5 volumes of ethanol. Centrifuge at 15,000 xg for 30 min and hold pellet.

b) Purification of the poly A + fraction of the RNA
The pellet is taken up in 1 ml of 10 mM Tris-HCl buffer pH 7.5 EDTA 1 mM, called TE buffer and suspended by vortexing. The oligo dT-cellulose type 3 (marketed by Collaborative Research
Inc., Biomedicals Product Division) is prepared according to the manufacturer's recommendations. The RNA is deposited on the oligo dT-cellulose, stirred gently to suspend the beads, and then heated for 1 min at 650C.

 The suspension is adjusted to 0.5 M NaCl and then stirred gently for 10 min. The suspension is then centrifuged for 1 min at 1000 g, the supernatant is removed, the pellet is washed twice with 1 ml of TE buffer containing 0.5 M NaCl. Supernatants are eliminated. The elution of the polyadenylated fraction of the RNAs (consisting of the messenger RNAs) is obtained by suspending the beads in 1 ml of TE buffer and then heating this suspension at 600 ° C. for 1 min, followed by stirring for 10 min on a tilting plate. . It is then centrifuged for 1 min at 1000 g, which makes it possible to recover the supernatant containing free messenger RNAs in solution.

All the above operations (from elution) are repeated twice. The supernatants thus obtained are pooled, the residual beads are removed by centrifugation and the supernatant is precipitated with 3 volumes of ethanol and a final 0.3M NaCl solution.

Thus, from the cell pellets A, NA, T (5 h) and T (24 h), four samples of poly A + RNA, hereinafter referred to as RNA-poly A ± A, are obtained.
RNA-poly A ± NA; RNA-polyA ± T (5 h) and RNA-poly A ± T (24 h).

SECTION2: Preparation of an enriched complementary DNA library
specific sequences of blood mononuclear cells
peripheral 1) Construction of the pSEI cloning vector
The strategy used uses fragments obtained from publicly available pre-existing plasmids and fragments prepared by synthesis according to the techniques now commonly used. The cloning techniques employed are those described by Sambrook et al, in "Molecular Cloning, a Laboratory Manual" (Cold Spring Harbor Laboratory, 1989). The synthesis of the oligonucleotides is carried out using a Biosearch 8700 DNA synthesizer.

The following description will be better understood with reference to FIG.
This plasmid was constructed by successive ligations of the following elements: a) - a PvuII-PvuII fragment - symbolized by +++++++ in FIG. 1a - of 2525 bp, obtained by complete digestion of the plasmid pTZ18R (Pharmacia) at 1 using the restriction enzyme PvuII. This fragment contains the origin of replication of phage fl (denoted ORI F1 in FIG. 1a), a gene (denoted AmpR in FIG. 1a) bearing the ampicillin resistance and the origin of replication (denoted ORI pBR322 on the FIG. 1a) allowing the replication of this plasmid in E. coli. The first free site PvuII disappears by ligation with the free site
EcoRV (which also disappears) of the fragment described in g).

sur la figure 1a - de 1060 pb de l'ADN d'adénovirus type 5 entre les positions 11299 (site de restriction PvuII) et 10239 (site de restriction HpaI) (Dekker et Van Ormondt,
Gene, 27, 1984, 115-120) contenant l'information pour les ARN VA-I et
VA-II. Le site franc HpaI disparaît par ligation avec le site franc PvuII (qui disparaît également) du fragment décrit en c). Le site ApaI en position 11 218 a été enlevé par clivage à l'aide de l'enzyme ApaI, traitement à l'aide de l'exonucléase : ADN polymérase du phageT4 et religation.b) - a fragment PvuII-HpaI - symbolized by

in Figure 1a - 1060 bp of adenovirus type 5 DNA between positions 11299 (PvuII restriction site) and 10239 (HpaI restriction site) (Dekker and Van Ormondt,
Gene, 27, 1984, 115-120) containing information for VA-I RNAs and
VA-II. The free website HpaI disappears by ligation with the free website PvuII (which also disappears) of the fragment described in c). The ApaI site at position 11 218 was removed by cleavage using the ApaI enzyme, treatment with exonuclease: phageT4 DNA polymerase and religation.

c) - a fragment PvuII-HindIII - symbolized by 11111111 in Figure 1a - 344 bp, derived from the SV40 virus DNA obtained by complete digestion with the restriction enzymes PvuII and HindIII. This fragment contains the origin of replication and the SV40 early virus DNA promoter (ref B.J. Byrne et al., Proc, Ntl Acad Sci, USA (1983), 80, 721-725).

 The HindIII site disappears by ligation with the HindIII binding site of the fragment described in d).

d) - a synthetic fragment "HindIII binding site" - HindIII - symbolized by
in FIG. 1a - of 419 bp, the sequence of which, given in FIG. 1b, contains a sequence close to the 5 'untranslated sequence of the HTLV1 virus (R. Weiss et al., Molecular Biology of Tumor Viruses - part 2-2e ed 1985 - Cold Spring Harbor Laboratory - p 1057) and the distal intron of the mouse α-globin gene (Y. Nishioka et al, 1979, Cell, 18, 875-882).

e) - a synthetic fragment HindIII- "BamHI binding site" - symbolized by
XXXXXXX in FIG. 1a containing the phage T7 RNA polymerase promoter and a polylinker containing in particular the cloning sites.
ApaI and BamHI, the sequence of which is shown in Figure 1c.

sur la figure 1a -, petit fragment obtenu par digestion complète à l'aide des enzymes
BclI et BamHI du virus SV40 qui contient le site de polyadénylation tardif de ce dernier. (M. Fitzgerald et al. Cell, 24, 1981, 251-260). Les sites BamHI et
BclI disparaissent par ligations avec respectivement le "site liant à BamHI" du fragment décrit en e) et le site BamHI (qui disparaît également du fragment décrit en g).f) - a BamHI-BclI fragment of 240 bp - represented by

in Figure 1a -, small fragment obtained by complete digestion with the help of enzymes
BclI and BamHI of SV40 virus which contains the site of late polyadenylation of the latter. (Fitzgerald M. et al., Cell, 24, 1981, 251-260). The BamHI sites and
BclI disappear in ligation with respectively the "BamHI binding site" of the fragment described in e) and the BamHI site (which also disappears from the fragment described in g).

g) - BamHI-EcoRV fragment - symbolized by 000000 in Figure 1a of 190 bp, small fragment from plasmid pBR322 after complete digestion with the enzymes EcoRV and BamHI.

The plasmid pSE1 therefore contains the elements necessary for its use as a cloning vector in E. coli (origin of replication in
E. coli and ampicillin resistance gene, from plasmid pTZ18R) as well as expression vector in animal cells (promoter, intron, polyadenylation site, origin of replication of SV40 virus), and for its copy in single strand for sequencing purpose (replication origin of phage fl).

2) Constitution of a complementary DNA library enriched with specific sequences of peripheral blood mononuclear cells:
The cloning technique used is that described by Caput et al., (Primer-adapter technique): Caput et al, Proc Natl.

Acad. Sci. U.S.A., 1986, 83, 1670-1674.

 It consists on the one hand in digesting the pSE1 vector with ApaI, adding a polydC tail on the protruding 3 'end, and then digesting the plasmids thus obtained with BamHI endonuclease. The fragment corresponding to the vector is purified on a column of Sepharose CL4B (Pharmacia). It therefore comprises a polydC tail at one end, the other end being cohesive, of the BamHI type.

On the other hand, the polyA + RNAs obtained at the end of section 1 are subjected to reverse transcription from a primer whose sequence is as follows.
5 '<GATCCGGGCC LII llllllll TT <3'
Thus, the cDNAs have at their 5 'end the complementary GATCC sequence of the BamHI cohesive end.

 The RNA-DNA hybrids obtained by the action of the reverse transcriptase are subjected to an alkaline hydrolysis which makes it possible to get rid of the RNA.

The single-stranded cDNAs are then subjected to a terminal transferase treatment, so as to add polydG at 3 'and purified by 2 cycles on a CMB sepharose column.

 These cDNAs are hybridized with polyA + RNA from cells of the COS3 line (monkey kidney cell line expressing the SV40 virus T antigen: see Y. Gluzman, 1981, Cell, 23, 175-182. ), prepared as described in Section 1 (2).

 Unhybridized cDNAs are isolated (DNA enriched fraction complementary to messenger RNAs specific for peripheral blood mononuclear cells).

 These cDNAs are inserted in single-stranded form into the vector pSE1. A second oligonucleotide (adapter) complementary to the primer is required to generate a BamHI site at the 5 'end of the cDNAs. After hybridization of the vector, the cDNA and the adapter, the recombinant molecules are circularized by the action of T4 phage ligase. The single-stranded regions are then repaired using T4 phage DNA polymerase. The plasmid pool thus obtained serves to transform E. coli strain MC 1061 (Casabadan and S.

Cohen, J. Bact. (1980), 143, 971-980) by electroporation.

Protocol for the preparation of the complementary DNA bank
a) Preparation of the complementary DNA
From 5 g poly-A + RNAs of peripheral blood mononuclear cells obtained at the end of section 1 of the following composition
RNA-poly A + A: 0.5 erg, RNA-poly A + NA: 2, ceg, RNA-poly A + T (5 h): 2, ug and RNA-poly A + T (24 h): 0.5, erg 32P-dCTP-labeled single-stranded cDNA (the resulting DNA obtained has a specific activity of 3000 dpm / ng) was prepared with the following synthetic primer (including a BamHI site): ## STR2 ## TTT <3 'in a volume of 100 μl of composition buffer: 50mM Tris HCl pH8.3, 5mM MgCl2, 10mM DTT, containing 5mM each of the trisphosphate oxynucleotides, 100 Ci of dCTP a32P and 100 U of RNasin (Promega). After incubation for 30 min at 4 ° C. with 100 units of the enzyme reverse transcriptase (Genofit-El 022), 4 ml of 0.5 M EDTA are added. The mixture is first extracted with phenol (saturated with TE buffer) and then with second time with chloroform. 10 g of calf liver transfer RNA, 1/10 volume of a 10M ammonium acetate solution and 2.5 volumes of ethanol are added to precipitate complementary RADON. The pellet is centrifuged and dissolved in 30 μl of TE buffer and the small molecules such as salts, phenol and chloroform are removed by exclusion chromatography on a P10 polyacrylamide column (Biogel P10-200-400mesh, Cat. Biorad).

b) Alkaline hydrolysis of the RNA template
4.6 l of a 2N NaOH solution are added, the mixture is incubated for 30 min at 68 ° C., then 4.6 l of 2N acetic acid are added and the resulting solution is passed through a P10 polyacrylamide column.

c) Homopolymeric addition of dG
The 3 'complement DNA was extended with a dG "tail" with 66 units of the terminal enzyme transferase (Pharmacia 27073001). Incubate in 60 l of buffer composition: Tris HCl 30mM pH 7.6; 1 mM cobalt chloride, 140 mM cacodylic acid, 0.1 mM DTT, 1 mM dGTP, for 30 min at 37 ° C., then 4 l of 0.5 M EDTA is added.

d) Purification on CMB Sepharose Column
In order to eliminate the synthetic primer, the complementary DNA is purified on two successive columns of 1 ml of CMB sepharose (Pharmacia), equilibrated with a 30mM NaOH / 2mM EDTA solution.

 The first three radioactive fractions (about 80, 1 each) are pooled and precipitated with 1/10 volume of an ammonium acetate solution and 2.5 volumes of ethanol. The amount of complementary DNA is 1 μg.

e) Hybridization The pellet of complementary DNA is suspended in 25 μl of buffer TE, 15 μg of RNA-polyA + extracted from cells of the line are added.
COS, then 1 / 10th volume of a 3M NaCl solution, 5 volumes of ethanol and allowed to precipitate at -20 ° C.

 It is centrifuged, washed with 70% ethanol, dried and the residue is dissolved in 5 μl of buffer of the following composition: 0.1M Tris-HCl pH 7.5; 0.3M NaCl, 1mM EDTA, the resulting solution is placed in a capillary tube which is sealed and incubated at 650C for 40 h.

 The contents of the capillary are diluted in 100 μl of TE buffer to which is added 300 μl of 50 mM sodium phosphate buffer pH 6.8. The resulting solution is passed through a hydroxylapatite column (Biorad Ref 130.0520) at 60 ° C., equilibrated with this phosphate buffer. The single strand (the unhybridized complementary DNA) and the double strand (messenger RNA of hybridized COS to the complementary DNA) are separated by a 0.1M to 0.2M phosphate buffer gradient across the column. The fractions corresponding to the single-stranded complementary DNA (25% by weight of the eluted cDNA, which corresponds to about 4-fold enrichment in specific sequences of peripheral blood mononuclear cells) are added. , ug of transfer RNA, the total volume is precipitated with 1 / 10th volume of a solution of 10M ammonium acetate and 2.5 volumes of ethanol. After centrifugation, the pellet is dissolved in 200 μl of TE, the residual phosphate is removed on polyacrylamide P10, precipitated again with 1/10 volume of a 10M ammonium acetate solution and 2.5 × ethanol.

 The pellet is dissolved in 30 μl of a 30 mM NaOH solution; EDTA 2mM. Complementary DNA was loaded onto a 1 ml CAB (Pharmacia) sepharose column, equilibrated with 30mM NaOH solution; EDTA 2mM, to eliminate the rest of synthetic primer. The first 3 radioactive fractions of about 80 μl each are grouped together. The cDNA contained in these fractions is precipitated with 1 / 10th volume of a 10M ammonium solution and 2.5 volumes of ethanol. The amount of complementary DNA thus recovered is 20 ng.

f) Pairing of the cloning vector PSE1 and the complementary DNA in the presence of the adapter
The pellet is centrifuged and dissolved in 33 μl of TE buffer, 5 μl (125 ng) of cloning vector pSE1, 1 μl (120 ng) of the following sequence adapter (including an ApaI site) are added.
To a solution of 200 mM NaCl, the mixture is incubated for 5 minutes at 650 ° C. and then the reaction mixture is allowed to cool to ambient temperature.

g) Ligation
The cloning vector and single-stranded cDNA were pooled in a volume of 100 μl with 32.5 units of T4 phage DNA ligase enzyme (Pharmacia ref: 270,8002) overnight at 15C in a buffer of composition: Tris HCl 50mM pH 7.5, 10mM MgCl 2, 1mM ATP.

h) Synthesis of the second strand of the cDNA
The proteins are removed by phenol extraction followed by extraction with chloroform, then 1 / 10th volume of a 10 mM ammonium solution and then 2.5 volumes of ethanol are added. The pellet is centrifuged and dissolved in the Tris acetate pH 7.9 33 mM buffer, 62.5 mM potassium acetate, 1 mM magnesium acetate and 1 mM dithiothreitol (DTI), the second strand of complementary DNA is synthesized in a volume of 30, with 30 units of phage T4 DNA polymerase enzyme (Pharmacia, ref 27-0718) and a mixture of 1mM of the four deoxynucleotide triphosphates of dATP, dCTP, dGTP and dTTP, as well as two units of the phage gene 32 protein
T4 (Pharmacia - ref 27-0213), for 1 hour at 37 ° C. Phenol is extracted and traces of phenol are removed by a PlO polyacrylamide column (Biogel P10200-400 mesh- Ref 15011050 - Biorad).

i) Electroporation transformation
E. coli MC1061 (Clontech) cells are transformed with the recombinant DNA obtained previously by electroporation using the apparatus.
Biorad Gene Pulser (Biorad) used at 2.5 kV under the conditions prescribed by the manufacturer, then the bacteria were grown for 1 hour in medium called LB medium (Sambrook, op cited) composition: bactrotryptone 10 g / l; yeast extract 5 gn; NaCl 10 g / l, then for 6 h 30 in LB medium supplemented with 100 g / ml of ampicillin.

 The number of independent clones is determined by plating a 1 / 1000th dilution of the transformation after the first hour of incubation on a box of LB medium supplemented with 1.5% agar (w / v) and 100 μg. ml of ampicillin, subsequently called LB agar medium. The number of independent clones is 500,000.

SECTION 3: Screening of the complementary DNA bank subtracted and
selection of clone NC30 1) Realization of replicas of bacterial colonies of the cDNA library on nylon filter:
About 40,000 recombinant bacteria were distributed from the cDNA library onto (245 x 245) mm Petri dishes containing LB agar medium (approximately 2000 colonies / dish).

 From each of these dishes, colonies are transferred to a nylon membrane (Hybond N-Amersham) by depositing the membrane on the surface of the can and setting markers by piercing the membrane with the aid of a needle. The membrane is then removed and deposited on the surface of a new Petri dish containing LB agar medium. It is left for a few hours at 37 ° C. to obtain regrowth of the colonies. From this first membrane four replicas are made on new membranes (previously deposited on LB agar medium to moisten them) by successive contacts with the first membrane. The membrane replicas obtained are finally deposited on plates of LB agar medium and incubated overnight at 30C.

 Membrane replicas are plated with the colonies upward on a 3MM Whatman sheet saturated with a solution of composition: 0.5M NaOH; 1.5M NaCl, for 5 min, which allows to lyse the bacteria and fix the DNA. The membrane replicas are then placed on a second sheet of Whatman 3MM, saturated this time with a neutralizing solution of composition: 1.5M NaCl; Tris-HCl pH 8.0, 5M, for 5 min. The membrane replicas are then immersed in a solution of 2 x SSC (composition of the SSC solution: 0.15M NaCl, 0.015M sodium citrate) and the bacterial debris are partially removed by gently rubbing with cotton wool. cleaning.

Membrane replicas are then treated with proteinase K (Boehringer Mannheim GmbH) at 100 μg / ml in a solution of composition:
10mM Tris-HCl pH8; EDTA ™; 50 mM NaCl; 0.1% SDS at 20 ml per membrane. Incubate for 30 min at 37 ° C with stirring. The diaphragm replicas are again immersed in a solution of 2 x
SSC to permanently eliminate all traces of bacterial debris. They are finally dried on filter paper for a few minutes and then for 30 min under vacuum at + 80in. Thus, for each box, four membrane replicas, hereinafter called replica 1, replica 2, replica 3 and replica 4, are obtained.

2) Preparation of the RNA used for the production of the cDNA probes:
a) Culture and stimulation of peripheral blood mononuclear cells using PMA, (PMA and anti-CD28), (PMA, PHA-P and cyclosporin A) or (PMA and PHA-P):
Non-adherent cells are prepared as previously described (section 1.1). They are cultured for 5 h in Falcon flasks with a surface area of 175 cm 2 in the presence of RPMI medium supplemented with 10% fetal calf serum (Gibco BRL-Ref 013-06290H), supplemented with 10 units of penicillin and 10 μg. streptomycin (Penicillin solution / Gibco streptomycin-Ref 043-05140D) per ml of medium as well as L-glutamine (Gibco BRL-Ref 043-05030D) up to 2 mM final.The cells are stimulated for 5 minutes. h under one of the following stimulation conditions 1) 2) 3) and 4): 1) in the presence of 10 ng / ml phorbol myristate-2-acetate-3 (PMA) (Sigma, P8139) 2) in presence of 10 ng / ml PMA and 1 μg / ml of the anti-CD28 monoclonal antibody (Reagents and Systems SA ref ACM0280NC050) 3) in the presence of 10 ng / ml PMA, 5 μg / ml of phytohemagglutinin (PHA-P) (Sigma, ref. L8754) and 1 μg / ml cyclosporine A (Sandoz) 4) in the presence of 10 ng / ml PMA and 5 μg / ml PHA-P
It is indeed known that the addition of the anti-CD28 antibody increases the amount of the messenger RNAs of several cytokines, in particular IL-2, IFNy, TNFo and GMCSF by an increase in the stability of their RNAs. messengers (Lindsten T. et al., (1989), Science, 244, 339) in lymphocytes
T, and that the immunosuppressant cyclosporin A inhibits the increase of cytokine messenger RNAs induced by activation with PMA and PHA-P in T cells (Thompson CB et al, 1989, Proc Natl Acad Sci USA, 86, 1333-1337).

 The cells are collected under the stimulation conditions above and the cell pellets, called cell pellet 1, cell pellet 2, cell pellet 3 and cell pellet 4, are retained.

b) Preparation of poly A + RNA
From the cell pellets above, the RNA is extracted and the polyA + fraction is purified as described in section 1-2) a) and b). There are thus obtained four RNA-polyA + fractions, referred to as polyA + 1 fraction, polyA + 2 fraction, polyA + 3 fraction and polyA + 4 fraction respectively.

3) Preparation of radiolabelled cDNA probes:
Radiolabeled cDNA probes, designated probe 1, probe 2, probe 3 and probe 4 respectively, are synthesized from the four fractions.
RNA-polyA above, prepared as described below.

 1 μg of polyA + RNA is hybridized with 200 μg of 12-18 oligo (Pharmacia) in 2 to 34 μl of 50 mM Tris-HCl pH 7.5 buffer and 1 mM EDTA by incubation for 2 min at 65. * C and cooling to room temperature. The synthesis of the radiolabeled cDNA is carried out in a reaction volume of 10 4 in a composition buffer: 50 mM Tris-HCl pH 8.3; 5mM MgCl2; 10 mM dithiothreitol, containing 50 μM each of the 3 dATP, dGT and dlTP deoxynucleotides (Pharmacia), 10 μM dCTP and 150 μCi dCTP a32P (3000 Ci / mmol-Amersham) and 40 units RNase (RNAse-inhibitor). Genofit). The reaction is carried out at 46 ° C. for 30 minutes in the presence of 10 to 20 units of reverse transcriptase (Genofit). This synthesis is followed by the alkaline hydrolysis of the RNA by a solution of 0.3M NaOH in a final volume of 20 l for 30 min at 65 C. It is neutralized by addition of 3M acetic acid. The volume is adjusted to 504 with TE medium. Extraction is carried out with the same volume of phenol followed by a second extraction with the same volume of a chloroform / isoamyl alcohol mixture (in the respective proportions 24/1). Unincorporated a32P dCTP was removed during synthesis of the cDNA strand by exclusion chromatography on a P10 polyacrylamide column (Biogel-200-400 mesh-Biorad).

 The amount of cDNA is 60 to 100 ng with a specific activity of 1 x 109dpm / μg.

4) Hybridization of replicas of bacterial colonies with cDNA probes:
Membrane replicas are prehybridized for 2 hours at 42C in a composition buffer: 50% formamide; 6 x SSC; 5 x Denhardt solution; 0.1% SDS and 100 μg / ml of sonicated salmon sperm DNA, added after denaturation for 10 min at 100 ° C. The membrane replicates are hybridized for two days: replica 1 with probe 1 and the replica 2 with probe 2, replicate 3 with probe 3, replicate 4 with probe 4, these probes being used at a concentration of 4 ng / ml in the buffer above. The 5 x Denhardt solution (see Sambrook, cited above) has the composition: Ficoli (type 400-Pharmacia) 1 g / l, polyvinylpyrrolidone 1 g / l and bovine serum albumin (BSA) 1 g / l.

 Prehybridization and hybridization are carried out in tubes in a hybridization oven (Hybaid) with respectively 25 ml and 10 ml of buffer per membrane.

Then the membrane replicas are successively washed several times for 15 min at 200C in the 2 x SSC composition buffer; 0.1% SDS, then twice for 15 min in a 0.1 x SSC solution, 0.1%
SDS at 55il, dried on Whatman 3MM paper and autoradiographed on films
Kodak XAR5 5) Hybridization with a mixture of oligonucleotides corresponding to most known cytokines:
To identify clones that contain complementary DNAs
Messenger RNAs of known cytokines, another series of membrane replicas, prepared as described above, is hybridized with a mixture called C-mixture of 28 oligonucleotides each comprising 20 nucleotides, corresponding to the complementary DNAs of the following cytokines ::
Interleukin-1a (Fumtani Y. et al, 1985, Nucl Ac Res., 13, 5869-5882), Interleukin-1 (Auron P. et al, 1984, Proc Natl Acad Sci USA, 81, 7907-7911), Interleukin-2 (Degrave W. et al, 1983, EMBO J., 2, 3249-3253), Interleukin-3 (Yang YC et al, 1986, 47, 3-10), 4 (Yokoto T. et al., 1986, Proc.Natl.Academ Sci., 83, 5894-5898), Interleukin-5 (Hirano T. et al, 1986, Nature, 324, 73-75), Interleukin-6 (May L. et al, 1986, Proc Natl Acad Sci USA, 83, 8957-8961), Interleukin-7 (Namen A.

et al., 1988, Nature, 333, 571-573), Interleukin-8 (Matsushima K. et al, 1988,
J. Exp. Med., 167, 1883-1893), Interleukin-9 (Yang YC et al., 1989, Blood, 74, 1880-1884), TNFα (Pennica D. et al, 1984, Nature, 312, 724-729), TNFss (Gray P. et al, 1984, Nature, 312, 721-724), G-CSF (Nagata S et al, 1986, 319, 415-418), M-CSF (Kawasaki E. et al, 1985, Science, 230, 291-296),
GM-CSF (Wong G. et al., 1985, Science, 228, 810-815), LIF (Grough N. et al, 1988, Proc Natl Acad Sci.USA, 85, 2623-2627), Interferon a (Goeddel D. et al, 1981, Nature, 290, 20-26), Interferon t31 (Taniguchi T. et al, 1980, Gene, 10, 11-15), Interferon y (Gray P. et al, 1982, Nature , 295, 503-508),
TGFα (Derynck R. et al, 1984, Cell, 38, 287-297), TGFB1 (Derynck R. et al, 1985, Nature, 316, 701-705), bFGF (Prats, H. et al, 1989, Proc. Acadian National Sci.

USA, 86, 1836-1840), Erythropoietin (Jacobs K et al, 1985, Nature, 313, 806-810), BCGF (Sharma S et al, 1987, Science, 235, 1489-1492), MIF (Weiser W. et al., 1989, Proc Natl Acad Sci USA, 86, 7522-7526), MCP-1 (Yoshimura T. et al., FEBS Lett., 244, 487-493), Oncostatin-M (Malik N et al., 1989, Mol Cell Biol., 9, 2847-2853) and EDF (Murata M. et al, 1988, Proc.

Natl. Acad. Sci. USA, 85, 2434-2438).

These oligonucleotides, made using the DNA synthesizer
Biosearch 8700, are coupled with horseradish peroxidase EC 1.11.17 (Boehringer Mannheim-Ref 814-407) according to the following protocol:
oligonucleotides are reacted on the synthesis column with carbonyl diimidazole (Aldrich-11, 553-3) and diamino-1,6-hexane (Aldrich-H1.169-6) according to the method of Wachter et al. 1986, Nucl. Ac. Res., 14, 7985-7994.

 after deprotection of the bases and cleavage of the support by ammoniac treatment, the oligonucleotides are purified on an ion exchange resin (Quiagen-Diagen-500051) with a change of the ammonium counter-ion to lithium ion.

 the 5'-amino-oligonucleotides are coupled to horseradish peroxidase (Boehringer Mannheim-814407) according to the method of M. Urdea et al., Nuci. Ac.

Res., 1988, 16, 4937-4956.

 The oligonucleotide mixture hybridizes with about 10% of the clones of the library.

 Clones giving a stronger autoradiographic signal with probe 2 than with probe 1, stronger with probe 4 than with probe 1 and stronger with probe 4 than with probe 3 and not hybridizing with the mixture C were partially sequenced as described in section 4 below. One of these clones, hereafter called clone NC30, was retained. This clone contains a plasmid called pSE1-NC30.

SECTION 4: Expression of messenger RNA from clone NC-30 in cells
mononuclear peripheral blood
The non-adherent cells (consisting mainly of lymphocytes) are prepared as described in section 1-1) and stimulated as described in section 3-2) -a) with, in addition, unstimulated control cells (stimulation conditions 0). )). The messenger RNAs of these cells under stimulation conditions are prepared as described in Example 1-2) -a) and analyzed by electrophoresis on 1% agarose gel in the presence of formaldehyde (Sambrook, op cited), followed by transfer to a nylon membrane (Hybond N ± Amersham) and hybridization according to the protocol described hereinafter.

 This membrane is hybridized with a radiolabeled probe with dCTPa32P made from NC30 cDNA (Amersham) by partial cleavage of the latter using DNAse I, followed by polymerization using the enzyme. DNA polymerase I (so-called "nick-translation" technique or hyphenation), as described by Sambrook et al, op cit.

Hybridization was at 42 ° C for 16 h in aqueous medium containing 50% formamide, 1M NaCl, 5 x Denhardt solution and 0.1% SDS. The membranes are washed several times at room temperature with a 2 × SSC solution containing 0.1% SDS, and then washed twice at 500 ° C. for 15 minutes with a 0.1 × SSC solution containing 0.1% SDS. The solution of 5 x
Denhardt has the following composition: Ficoll (type 400 - Pharmacia) 1 g / l, polyvinylpyrrolidone 1 g / l and BSA 1 g / l. 1 x SSC solution contains 0.15M of
NaCl and 0.015M sodium citrate.

 For unstimulated cells and cells stimulated under conditions 1), 2), 3) and 4), an autoradiographic band corresponding to an RNA of about 1.4 kb is observed. The expression of this RNA is increased in the presence of PMA (intensity band of at least 5 times higher for the stimulation condition 1) than for the stimulation condition 0)), this increase being amplified with the additional presence of PHA-P or anti-CD28 (intensity bands about 5 times stronger for stimulation conditions 2) and 4) than for stimulation condition 1)) and not changed with the additional presence of PHA -P and cyclosporine (bands of similar intensity for stimulation conditions 1) and 3)).

 In another experiment carried out with a population of purified T lymphocytes (more than 95%), the expression of messenger RNA NC30 after costimulation with PMA and anti-CD28 is also found.

SECTIONS: Sequencing and sequence analysis of clone cDNA
NC30: 1) Sequencing of the cDNA of clone NC30:
a) preparation of the single-stranded DNA
Clone NC30 contains the vector pSE1, which carries a cDNA between Apal and BamHI sites, hereinafter called NC3O cDNA.

The vector pSE1, which contains the origin of replication of the phage fl, makes it possible to produce single-stranded DNA by culturing the clone NC30 in the presence of bacteriophage M13K07 (Pharmacia-ref: 27-1524) as follows:
The clone NC30 is cultivated in a 15 ml tube, with stirring at 37.degree. C. in 2 ml of 2 × YT medium of composition: bactotryptone 16 g / l, yeast extract 10 g / l NaCl 5 g / l (described in Sambrook et al, op cited), supplemented with 0.001% thiamine and 100, zeg / ml ampicillin to an optical density at 660 nm of about 0.60.

 100 μl of this culture are infected with bacteriophage M13K07 (Pharmacia ref: 27-1524) at a multiplicity of infection of the order of 10 in a 15 ml tube. The crop is shaken at 37in.

After 1 hour, 2 ml of medium are added. The culture is then incubated for about 16 hours at 37 ° C. with stirring.

1.5 ml of the culture is centrifuged in a microtube at 15,000 g for 2 min.

1 ml of supernatant is transferred to a microtube and 250 ml of a solution of polyethylene glycol of molecular weight 6000, 20% containing 2.5M NaCl are added. The mixture is incubated for 5 min at 40C to facilitate the precipitation of the phage and then centrifuged for 5 min at 15 000 g. The supernatant is removed and the pellet is resuspended in 500 μl of 10 mM Tris-HCl pH8 buffer, 1 mM EDTA.

The suspension is extracted once with phenol saturated with 100 mM Tris-HCl pH8 and then twice with chloroform.

The preparation is then precipitated by adding 1/10 volume of a solution of 3M sodium acetate pH 4.8 and 2.5 volumes of ethanol. Precipitation is carried out at -20 ° C. for a minimum of 20 minutes. The DNA is centrifuged for 10 min at 15,000 g, the pellet is washed with 70% ethanol solution and then resuspended in buffer of composition: 10 mM Tris-HCl pH8, 1 mM EDTA.

b) sequencing
Sequencing reactions are performed using the sequencing kit
United States Biochemical (ref: 70770), which uses the method of Sanger et al,
Proc. Ntl. Acad. Sci. USA, 1977, 14, 5463-5467. The primers used are oligonucleotides of 18 nucleotides, complementary to either the pSE1 vector in the region immediately 5 'of the NC30 cDNA, or to the sequence of the NC30 cDNA.

2) Analysis of the sequence of the NC30 cDNA:
The description below will be better understood with reference to FIGS. 2, 3 and 4.

Sequence Analysis of the NC30 cDNA (1) The NC30 cDNA has 1282 nucleotides and ends with a polyA (2) sequence. This number of nucleotides is in agreement with the size of the corresponding messenger RNA (about 1.4 kb) (see section 4).

(3) At position 1264-1269 the AATAAA sequence, which corresponds to the consensus sequence described by M. Birstiel et al., 1985, Cell, 41, 349 is a polyadenylation signal. In positions 855-861, 872-878, 1133-1139 and 1152-1158 there are 7 nucleotide sequences: TATITAT, TATlTAA, AATlTAT and TATlTAA, containing the ATITA sequence corresponding to the AUUUA consensus pattern of instability described by G. Shaw. et al., 1986, Cell, 46, 659-667. The DNA complementary to most known cytokines has a sequence corresponding to this consensus pattern of instability.

(4) The DNA sequence has an open reading phase for translation of a protein from ATG at position 15-17 to TGA at position 453-455 which corresponds to a stop codon translation. In this reading phase there are two ATG codons at positions 15-17 and 57-59, capable of initiating the translation, corresponding respectively to translated proteins of 146 and 132 amino acids. Of these, the nucleotide environment of ATG at positions 57-59 is closest to the consensus sequence described by Kozak M., 1978, Cell, 15, 1109-1123, for the initiation of translation into eukaryotic cells (5) A signal peptide search software, hereinafter referred to as PS software, was developed by the Applicant according to the method and the information described by Von Heijne, 1986, Nucl. Ac. Res. 14, 483-490. This software provides in this reading phase a hydrophobic region resembling a signal peptide and four likely sites of protein cleavage at positions 74-75 (between Thr and Thr), 86-87 (between Ala and Leu), 110-111 (between Ala and Ser) and 116-117 (between
Pro and Gly). The expected signal peptide is between one of two Met likely to initiate translation and one of these four cleavage sites. The predicted mature protein (translated protein cleaved from its signal peptide) thus comprises a sequence of 126, 122, 114 or 112 amino acids.

 The same predictions are obtained using UWGCG Software from the University of Wisconsin: Devereux et al., 1984, Nucl. Ac. Res., 12, 8711-8721-Option: Signal peptide search according to G. method.

von Heijne (reference above)
Comparison with other known sequences
The peptide sequence already known that is closest to that of the 112 amino acid sequence of the mature protein is that of the 132 amino acid protein deduced from the cDNA of the mouse P 600 protein described by
KD Brown et al., 1989, J. Imm., 142, 679-687. This protein is expressed in a subclass of mouse T lymphocytes: Th2 cells, activated using concanavalin A.

A comparison of these peptide sequences using the method of
Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443-453, implemented in UWGCG software from the University of Wisconsin: Devereux et al, 1984, Nucl.

Ac. Res. 12, 8711-8721, GAP option, shows that 63 amino acids are identical out of 112, an identity homology of about 56%. This algorithmic method considers all possible alignments and creates an alignment, shown in Figure 3, in which as many identical amino acids are matched and the number of holes in the aligned sequences is minimal.

A comparison of the nucleotide sequences by this method shows an identity homology of about 70% between the coding sequence of the NC30 cDNA and the cDNA of the mouse P600 protein (see FIG.
SECTION 6: Analysis of Secretion in COS Cells of Protein
encoded by NC30 cDNA
COS cells are monkey kidney cells expressing antigen
T of SV40 virus (Gluzman Y., Cell, 23, 1981, 175-182). These cells, which allow the replication of vectors containing the origin of replication of the SV40 virus DNA (case of the vector pSE1) are prime hosts for the study of gene expression in animal cells.

1) Transfection of COS Cells and Transient Expression of the Protein Encoded by Nu30 cDNA:
5 x 10 5 COS cells are seeded in a 6 cm diameter petri dish (Corning) in 5 ml of modified Dubelcco Eagle medium, hereinafter referred to as DMEM (Gibco's Dulbecco's Modified Eagle medium ref # 411-01965). ), which contains 0.6 g of glutamine, 3.7 gui NaHCO 3 and is supplemented with fetal calf serum (Gibco) at 5%. After about 16 h of culture at 37 ° C in an atmosphere containing 5% carbon dioxide, the culture medium is removed by aspiration and the cells are washed with 3 ml of PBS buffer (Gibco Phosphate Buffered Saline). the following mixture is then added: 1000 μl of (DMEM + 10% fetal calf serum (Gibco)), 110 μl of diethylaminoethyl-dextran with a mean molecular weight of 500,000 (Pharmacia), at a concentration of 2 mg / ml, 1.1 μl of 100 mM chloroquine (Sigma) and 6 μg of plasmid DNA of clone NC30 prepared according to the alkaline lysis technique followed by purification of the plasmid DNA on a cesium chloride gradient (Sambrook et al. op cited).

After 5 h of incubation at 370C in an atmosphere containing 5% carbon dioxide, the mixture is removed from the cells. 2 ml of buffer is then added
PBS containing 10% dimethyl sulfoxide (Spectroscopy grade, Merck).

After 1 min of incubation at room temperature, the mixture is removed and the cells are washed twice with PBS and are incubated in medium.
DMEM containing 2% fetal calf serum. The incubation is continued for 40 h at 37 ° C. under an atmosphere containing 5% of carbon dioxide.

 On the other hand, control COS cells were prepared by carrying out the operations described above with the plasmid pSE1 DNA.

2) Protein labeling:
The set of operations described below is carried out with the COS cells transfected with the plasmid DNA of clone NC30 and the cells
COS witnesses.

The culture medium is removed by aspiration and the cells are washed twice with 3 ml of PBS buffer. 5 ml of MEM medium (Minimal
Eagle's Medium) without methionine (Gibco - ref.041-01900H), supplemented with 3 g / ml glucose and 4 mM glutamine. Incubate for 2 hours at 37 ° C. The culture medium is removed and 2 ml of the same medium supplemented with 200 μl of 35 S methionine (ref SJ1015 Amersham) is added. Incubate for 6 h at 37C. The culture medium is removed, centrifuged for 5 min to remove cell debris and suspended cells, and the supernatant is retained.

3) Analysis of radiolabelled proteins of COS cells transfected by polyacrylamide gel electrophoresis:
1 ml of the supernatant of the transfected COS cells are precipitated and 9 ml of acetone at -20 ° C. Centrifuge and recover the pellets of proteins. They are taken up in a composition buffer: 0.125M Tris-HCl pH 6.8, 4% SDS, 20% glycerol and heated at 100in for 10 min. An aliquot of the suspension obtained, corresponding to a radioactivity of 200,000 cpm, is analyzed by electrophoresis on a 15% polyacrylamide gel in the presence of
SDS, according to the technique described by UK Laemmli, Anal. Biochem., 1977, 78, 459. The gel is dried under vacuum. The radiolabelled proteins are revealed by autoradiography.

The autoradiogram shows the presence of two supernumerary bands for the cells transfected with the plasmid DNA of the clone
NC30 compared to the control cells: a high intensity net band corresponding to an apparent molecular mass of 9.0 ± 2 kDa and a low intensity diffuse band corresponding to an apparent molecular mass of 16.0 ± 2 kDa. Clone NC30 therefore codes for a secreted protein, hereinafter called NC30 protein. The apparent molecular weight for the form of the protein of the invention corresponding to the 9 + 2 kDa band is less than the calculated molecular mass for the mature 112 amino acid protein of 12,366 Da. This difference is probably caused by an unexpected electrophoretic mobility of this form of the protein of the invention.

The band of 16.0 + 2 kDa apparent molecular weight may correspond to an N-glycosylated form of the protein of the invention. This has indeed four possible N-glycosylation sites highlighted in dots in Figure 2 and corresponding to the consensus sequence described by
Donner et al., 1987, J. Cel. Biol., 105, 2665.

4) Demonstration of the probable N-glycosylation of the 16 + 2 kDa apparent molecular weight protein.

 Protein labeling is performed as in 2) above but in the presence of 10 μg / ml tunicanycin (Sigma-ref T7765), protein N-glycosylation inhibitor.

 The analysis of the proteins on polyacrylamide gel is carried out as in 3).

 The autoradiogram shows the presence of a single 9 + 2 kDa supernumerary band for the cells transfected with the plasmid DNA of clone NC30, relative to the control cells. These results show that the form of the recombinant protein observed in 3), which corresponds to a molecular mass of 16 + 2 kDa, is N-glycosylated.

SECTION 7: Production of NC30 Protein in COS Cells
4.3 × 10 7 COS cells are seeded in a cylindrical culture flask, usually called "roller", surface 850 cm 2 in 150 ml of DMEM medium, which contains 0.6 g / l glutamine, 3.7 g / l NaHCO 3 and is supplemented with fetal calf serum (Gibco) at 5%, and then buffered with carbon dioxide.

 After about 16 hours of culture at 370C on a chassis (rotation speed of about 0.2 rpm), the culture medium is removed by suction and the cells are washed with PBS buffer (Phosphate Buffered Saline). The following mixture is then added: 35 ml of DMEM medium + 10% fetal calf serum (Sigma), 4 ml of diethylaminoethyl-dextran (Pharmacia, average molecular weight 500,000) at a concentration of 2 mg / ml, 40, 100 mM chloroquine (Sigma) and 128 μg plasmid DNA of clone NC30, prepared according to the alkaline lysis technique, followed by purification of the plasmid DNA on a cesium chloride gradient (Sambrook et al, op. city).

After 5 h of incubation at 370C in an atmosphere containing 5% carbon dioxide, the mixture is removed from the cells. 35 ml of 4% PBS buffer containing 7% dimethylsulfoxide (Spectroscopy grade,
Merck). After 1 min and 30 s of rotation at room temperature, the mixture is removed and the cells are washed twice with PBS. 150 ml of DMEM medium (Sigma) containing 1% fetal calf serum (FCS) is added by roller and the cells are incubated at 370 ° C. in the presence of 5% CO 2 (rotation at 0.2 rpm). One day after the transfection, the medium is removed by aspiration, the cells are rinsed twice with PBS. 150 ml of serum-free DMEM (Sigma) medium are then added and the rollers are put back at 37 ° C. in the presence of 5 ml. % CO2 (rotating at 0.2 rpm) for 5 days.

 Harvesting takes place on the 6th day after transfection.

The culture medium is centrifuged at 7000 rpm for 10 minutes; the supernatant is filtered through a 0.2 μm filter of Nalgene.

 By analysis of the proteins of the supernatant by SDS polyacrylamide gel electrophoresis, the same band profile is observed as in section 8.

SECTION 8: Purification of NC-30 protein produced in cells
COS, and determination of its amino-terminal sequence 1) Purification of NC30 protein
The majority form of the recombinant protein of apparent molecular mass 9 + 2 kDa was purified from 500 ml of the supernatant obtained in section 7 by the following steps: - ion exchange chromatography on a column S fast flow (Pharmacia) (15 x 100 mm), previously equilibrated with a solution of 50 mM sodium acetate pH 4.0, eluting with a 1M NaCl solution in a 50 mM sodium acetate buffer pH 4.0 with a flow rate of 2 ml / min.

the eluate is concentrated on a centriprep membrane (Amicon) to a volume of approximately 1 ml and then subjected to HPLC chromatography (reverse phase on C4 column (Bownlee) (100 x 2.1 mm), previously equilibrated with a solution containing 30% acetonitrile and 0.1% TFA, with a linear gradient of 30 to 70% acetonitrile in a solution containing 0.1% TFA The fractions containing the recombinant protein (determined by analysis polyacrylamide gel electrophoresis in the presence of SDS) are collected.

2) Determination of the aminoterminal sequence of the NC30 protein of apparent molecular mass 9 + 2 kDa:
The protein obtained at the end of 1) was subjected to a 16% polyacrylamide gel electrophoretic analysis in the presence of SDS. Proteins are transferred for 1 hour at an intensity of 0.8 mA / cm 2 in a buffer of 25 mM Tris-borate pH9.0 and 10% methanol on an Immobilon membrane (Millipore), then revealed in Coomassie blue.

The 9 + 2 kDa apparent molecular weight band was cut and deposited on an Applied Biosystems model 470A sequencer coupled to an Applied Biosystems Model 120 A phenylthiohidantoic analyzer.
The aminoterminal sequence obtained is the following sequence (sorting):
Gly Pro Pro Pro Pro Ser Thr Ala Leu Arg Glu Leu Glu Glu Leu
Which corresponds to the fourth cleavage site provided by the PS software (see section 2).

SECTION 9: Construction of an NC30 cDNA expression vector in the
yeast, plasmid pEMR673 and transformation of a strain of
yeast using this plasmid:
I) Construction of the plasmid pEMR673
Plasmid pEMR583 (described in patent application EP-435776) was subjected to complete digestion with HindIII and BamHI enzymes. The large fragment (hereinafter referred to as fragment A) comprising the origin of replication and the STB locus of 2 ', the LEU2d gene, the ampicillin resistance gene, the origin of pBR322, the terminator of the PGK gene , the URA3 gene, the artificial promoter and the beginning of the prepro region of alpha pheromone has been purified.

The HindIII-BamHI fragment (hereinafter referred to as fragment B) comprising the end of the prepro region of the alpha pheromone and the cDNA encoding the mature protein, flanked by the 3 'BamHI restriction site, was obtained by amplification by the PCR technique from the plasmid pSE1-NC30. The sequence of this fragment is specified in FIG. 5. The fragments A and B were ligated to obtain the plasmid pEMR673. A) Description of the Polymerase Chain Reaction (PCR) Technique
The technique of the polymerase chain reaction (PCR) is a method well known to those skilled in the art which makes it possible to simultaneously copy the two strands of a previously denatured DNA sequence by using two oligonucleotides as primers (cf. including HA Erlich's book "PCR Technology: Principles and Applications for DNA Amplification" published in 1989 by Macmillan Publishers Ltd, United Kingdom and MA Innis et al., "PCR Protocols" published in 1990 by Academic Press
Inc. San Diego, California 92101, USA). The principle of this technique is summarized below.

The PCR technique is based on the repetition of three steps, allowing, after 10 to 30 cycles, to obtain hundreds of thousands of copies of the original matrix, using a Thermus aquaticus DNA polymerase, usually called Taq polymerase. The three steps are:
- Denaturation of the matrix:
The double-stranded DNA is denatured to single-stranded DNA by incubation at high temperature (92C to 96C) for approximately 2 minutes.

- Hybridization of the primers:
These primers are a pair of synthetic oligonucleotides that hybridize with the ends of the region to be amplified. Both primers hybridize with opposite strands. The primers are added in excess, so that formation of the primer-template complex is favored.

- Primer extension:
The step in which the Taq polymerase extends the primer-template complex from 5 'to 3' is carried out at 72μl.

 In the PCR technique, the product of interest appears in the third cycle and is then amplified significantly. During the course of the cycles, the amplification product quickly becomes the matrix with which the primers hybridize.

b) Description of the primers used
Two synthetic oligonucleotides were prepared.

The first oligonucleotide, called primer 1, whose sequence is as follows: CAGTGAATTC A AGC TG GAT AAA AGA TCC CCA GGC CCT GTG CCT CC
Ser Leu Asp Lys Arg Pro Pro Pro Gly Pro
Region 1 Region 2 has two distinct regions: Region 1, which contains the end of the prepro pheromone region has altered from the natural sequence described by Kurjan et al, 1982, 30, 933-943 by a silent mutation that allows to introduce a HindIII cleavage site just before the coding part of region 1 (eleventh nucleotide of region 1), and region 2, which is a region intended to hybridize with the coding region corresponding to the beginning of the mature protein of 114 amino acids (see section 5), non-coding strand of cDNA
NC30.

The second oligonucleotide, called primer 2, whose sequence is as follows:
CGACGGATCC CAAATAATGA TGCITTCGAA G
Region 1 Region 2 also consists of two distinct regions: Region 1 which carries on the fourth nucleotide a BamHI site, and Region 2, which carries a nucleotide sequence corresponding to the 3 'untranslated region of the NC30 cDNA.

This region is intended to hybridize with the coding strand of NC30 cDNA.

c) Obtaining the amplified HindIII-BamHI fragment representing the end of the prepro region of the pheromone a and the cDNA coding for the mature protein
NC30
The plasmid pSE1 - NC30 carrying the cDNA coding for the NC30 protein is used as template.

 100 μg of pSE1-NC30, 100 ng of primer 1, 100 ng of primer 2.5, and 10 μl of reaction mixture were added to a tube (final concentration: 67 mM Tris-HCl pH 8.8 16.6 mM (NH 4) 2 SO 4, 1 mM S mercaptoethanol, 6.7 mM EDTA, 0.15% Triton x 100, 2 mM MgCl 2, 0.2 mM dNTP, 200 ng gelatin). The volume of the mixture is then raised to 50 I by adding water.

0.5 μl, or 2.5 units of Taq Polymerase (Boehringer) is added.
Manheim ref 1146 - 173). The mixture is then covered with paraffin in order to avoid evaporation of the aqueous solution.

The amplification is carried out during 15 reaction cycles, the stages of which are as follows:
- 1 min at 940C -> denaturation
- 1 min at 55 ° C -> hybridization
- 1 min at 720C -> polymerization
After 15 cycles, the enzymatic reaction is stopped by the addition of 20 mM EDTA.

The amplified DNA fragment, which has the expected size of approximately 380 bp, is then isolated and purified on a 1% agarose gel, subjected for dialysis to chromatography on a P10 polyacrylamide gel column (Pharmacia), and then completely and simultaneously hydrolyzed by the HindIII and BamHI enzymes according to the usual techniques well known to those skilled in the art (Sambrook, 1983) to form the HindIII and
Bam. After hydrolysis, the fragment is purified on a P10 column.

 The sequence of the fragment B obtained is shown in FIG. 5. It comprises in its part coding for the NC3O protein a silent mutation with respect to the NC30 cDNA, indicated by an asterisk in FIG.

 Fragments A and B were ligated to obtain plasmid pEMR673.

2) Transformation of the yeast strain EMY761 with the plasmid pEMR673 and expression of the NC30 protein by the transformed strain
The EMY761 strain (Mat alpha, leu2, ura3, his3) described in the patent
EP-0408461, which can be obtained by plasmid dissolving of the strain deposited at the CNCM on December 27, 1989 under No. I-1021, contains mutations (leu2 and ura3), which can be complemented by the defective selection marker LEU2d and the Selection marker URA3, present in plasmid pEMR673.It was transformed with plasmid pEMR673 with selection for leucine prototrophy according to a variant of the transformation technique described by Beggs et al. (Beggs et al., 1978, Nature, 275, 104109) which consists in subjecting the yeasts to a protoplastisation treatment in the presence of an osmotic stabilizer, sorbitol in 1M concentration.

The precise transformation protocol is specified below:
a) 200 ml of the liquid YPG medium (see Table 1 below) are inoculated with approximately 5 × 10 6 cells of a stationary phase culture and the culture thus inoculated is placed overnight with stirring at 30 ° C.

 b) When the culture reaches about 107 cells per ml, the cells are centrifuged at 4000 rpm for 5 min and the pellet is washed with a 1 M sorbitol solution.

 c) The cells are suspended in 5 ml of 1 M sorbitol solution containing 25 mM EDTA and 50 mM diothiothreitol and incubated for 10 min at 30in.

d) The cells are washed once with 10 ml of sorbitol solution 1
M and suspended in 20 ml of sorbitol solution. Zymolase100T (a preparation marketed by Seykagaku Kogyo Co. Ltd., obtained by partial purification on an affinity column of the Arthobacter luteus culture supernatant and containing B-1,3-glucanase-laminaripentahydrolase) is added. at a final concentration of 20 μg / ml and the suspension is incubated at room temperature for about 15 minutes.

 e) The cells are resuspended in 20 ml of a medium containing sorbitol called YPGsorbitol medium (see Table I below) and incubated for 20 min at 300 ° C. with gentle stirring.

 f) Centrifuge for 3 min at 2500 rpm.

 g) The cells are resuspended in 9 ml of transformation buffer of composition: 1 M sorbitol, 10 mM Tris-HCl pH 7.5 and 10 mM CaCl 2).

 h) 0.1 ml of cells and 5 .mu.l of DNA solution (about 5 cl) are added and the resulting suspension is allowed to stand for 10-15 min at room temperature.

i) 1 ml of the solution is added: polyethylene glycol PEG 4000 20%,
10 mM Tris-HCl pH 7.5 and PoC, 10 mM.

 j) 0.1 ml of the suspension obtained in i) is poured into a tube containing regeneration solid medium without leucine (see Table I below), previously melted and kept liquid at approximately 45 ° C. The suspension is poured onto a petri dish containing a solidified layer of 15 ml of solid regeneration medium without leucine.

 Transforms begin to appear after three days. A transformation was thus retained, called strain EMY761 pEMR673.

Table I
Composition and preparation of the main media used in the protocol
yeast strain EMY761 - liquid YPG medium 10 g yeast extract (Bacto-Yeast extract from Difco) 20 g peptone (Difco Bacto-peptone) 20 g glucose mix the ingredients in water distilled. Fill the final volume to 1 liter with distilled water - Autoclave for 15 min at 1200C.

- YPG sorbitol medium use the formula of liquid YPG medium to which, after autoclaving, sorbitol is added at a concentration of 1 M.

- solid regeneration medium without leucine 6.7 g yeast nitrogen base without amino acids
(Difco Yeast Nitrogen Base Without Amino Acids) 20 mg adenine 20 mg uracil 20 mg L-tryptophan 20 mg L-histidine 20 mg L-arginine 20 mg L-methionine 30 mg L-tyrosine 30 mg of L-isoleucine 30 mg of L-lysine 50 mg of L-phenylalanine 100 mg of L-glutamic acid 150 mg of L-valine 20 g of glucose 30 g of agar 182 g of sorbitol mix all ingredients in 'distilled water. Complete the final volume at 11, with distilled water. Autoclave for 15 min at 1200C. After autoclaving, add 200 mg of L-threonine and 100 mg of L-aspartic acid.

SECTION 10: Erlenmeyer expression of the NC30 protein by the strain of
transformed yeast and demonstration of the protein in the
medium of culture on polyacrylamide gel in the presence of
SDS 1) Culture of the strain EMY761 pEMR673
A colony of EMY761 strain pEMR673 (obtained in section 9) was cultured in 50 ml of liquid medium without uracil.

This medium contains for 1 liter:
- 6.7 g yeast nitrogen base without amino acids
(Yeast nitrogen base with amino acids of Difco).

5.0 g of casein hydrolyzate (casaminoacids from
Difco)
- 10 g of glucose
After stirring overnight at 30 ° C., the culture was centrifuged for 10 min, the pellet was taken up in 10 ml of sterile water and again centrifuged for 10 min. The expression of the NC30 protein was induced by resuming the cells in 50 ml of medium of the following composition:
- 6.7 g / l of yeast nitrogen base without Difco's Aminoacids
- 5.0 g / l casein hydrolyzate (casaminoacids from Difco)
- 30.0 g / l of glycerol
- 30.0 g / l of galactose
- 10 ml of ethanol.

 The culture was returned to 300C with stirring for 24 h.

2) Analysis of the expressed protein
a) polyacrylamide gel in the presence of SDS
Sample preparation
A portion of the cells cultured overnight in a medium called liquid medium without uracil with glucose, the composition of which is specified in Table 2 below, was centrifuged: uninduced sample. Cells grown overnight in medium, called liquid medium without uracil with ethanol, glycerol and galactose (Table 2 below) were centrifuged induced sample. The supernatant was collected. To 10 ml of supernatant, 5 ml of 50% trichloroacetic acid containing 2 mg / ml of deoxycholate was added.

The mixture was put at + 40C for 30 min and then centrifuged for 30 min. The pellet was taken up in about 1 ml of cold acetone (+4 C) and again centrifuged for 30 min. The pellet, after having been dried, is taken up in approximately 20 liters of a buffer called loading buffer consisting of 0.125 M Tris-HCl pH 6.8, 4% SDS, 0.002% bromophenol blue, 20% glycerol, ss-mercaptoethanol 10% according to the protocol described by
Laemmli in 1970, well known to those skilled in the art. The pellet is solubilized by boiling for 15 min and then neutralized.

 The samples are deposited on a polyacrylamide gel in the presence of SDS and subjected to electrophoresis.

Results: The analysis of the gel (Coomassie blue revelation) shows for the induced sample the presence of several additional bands compared to the uninduced sample, the two main ones corresponding to an apparent molecular weight of 9 + 2 and 16 + 2 kDa. The other additional bands observed, which are quite numerous and diffuse, probably correspond to a variable degree of glycosylation.

N-glycosylation of a protein by yeast is known to involve simple N-glycosylation ("core glycosylation") in the endoplasmic reticulum and N-hyperglycosylation ("outer-chain glycosylation") in the Golgi apparatus. (RA Hitzeman et al, 1990, "Methods in Enzymology, No. 185," Academic Press, pp. 421-440). In general, simple N-glycosylation leads to a glyco-protein of homogeneous apparent molecular weight (one band) and N-hyperglycosylation to a glycoprotein of heterogeneous apparent molecular weight (plurality of diffuse bands).
b) Immunoblotting (Western blot) with eventual treatment with endoglycosidase H
Sample preparation
A portion of the cells cultured overnight in a liquid medium without uracil with glucose (Table 2) was centrifuged: uninduced sample. Cells grown overnight in a liquid medium without uracil with ethanol, with glycerol and galactose, (Table 2) were centrifuged: induced sample.

The supernatant was collected. To 10 ml of supernatant, 5 ml of 50% trichloroacetic acid containing 2 mg / ml of deoxycholate was added.

 The mixture was put at + 40C for 30 min and then centrifuged for 30 min. The pellet was taken up in about 1 ml of cold acetone (+4 C) and again centrifuged for 30 min. The pellet is taken up in 20 μl of a solubilization buffer (of composition Tris-HCl pH 6.8 10 mM, 13 mercaptoethanol 2%, SDS 1%). The pellet is brought to 1000C for 5 min.

 The sample is then divided into two parts - at the first part of 10 μl 10 μl of a 50 mM sodium citrate buffer pH5.5 containing endoglycosidase H (5 mIU: Boehringer ref.

1088726). The sample is placed at 37 ° C for about 1 night. 20 μl of loading buffer are then added to the second part of 10, and 10 μl of loading buffer are added. The samples are boiled for 10 minutes.

The samples are deposited on polyacrylamide gel in the presence of
SDS and electrophoresis is carried out according to the Laemmli protocol (reference already cited).

 The proteins contained in the gel are then transferred to nitrocellulose membrane (according to the technique of H. Towbin et al., 1979, Proc.

Natl. Acad. Sci. USA, 76, 4350-4354). Immunodetection, performed according to the protocol described in Bio-Rad's Immunoblot Assay Kit (ref 170-6450) involves the following steps:
saturation of the nitrocellulose membrane with a TBS buffer ("Tris Buffered Saline") which contains 3 g / 100 ml of gelatin for 30 min.

- rinsing the membrane with a buffer called T. TBS (TBS buffer which contains 0.05% Tween 20), twice for 5 min
contacting the membrane with the antiserum prepared in section 13 for 1 hour at room temperature
- rinsing the membrane with TBS buffer, twice for 5 min
the contacting of the membrane with the conjugated antibody of the kit
- rinsing the membrane with T.TBS buffer, twice for 5 min and once for 5 min with TBS buffer
the Antigen-Antibody complex is revealed by bringing the membrane into contact with a development buffer containing 4-chloro-naphthol-1 in diethylene and hydrogen peroxide
- rinsing the membrane with water.

Results: The immunoblot analysis shows for the untreated endoglycosidase H induced sample the presence of several additional bands compared to the uninduced sample, the two main ones of which correspond to an apparent molecular weight of 9+. 2 and 16 + 2 kDa. Other large and diffuse bands of higher molecular weight are also highlighted. All these bands are recognized by the immune system prepared in section 13.

In the sample induced band corresponding to an apparent molecular weight of 16 + 2 kDa tends to disappear after treatment with endoglycosidase
H while the band at 9 + 2kDa increases in intensity under the same conditions. These results show that the 16 + 2 kDa apparent molecular weight protein is N-glycosylated.

 The diffuse bands of higher molecular weight also disappear and there is the appearance of 2 bands corresponding to apparent molecular weights of approximately 18 + 2 and 20 + 2 kDa. The forms of NC30 protein resistant to endoglycosidase treatment H may correspond to the precursor having kept the pro pheromone sequence or to O-glycosylated forms of the NC30 protein.

Table 2
Composition and preparation of certain environments
used for the preparation of samples - Liquid medium without uracil with glucose - 6.7 g yeast nitrogen base without amino acids
(Difco Yeast nitrogen base without amino acids) - 5.0 g of casein hydrolyzate (Difco casaminoacids) - 10.0 g of glucose mix all ingredients in distilled water, and make up to 1 final with distilled water.

Autoclave for 10 min at 120il.

- Liquid medium without uracil with ethanol, glycerol and galactose: use the formula of the liquid medium without uracil described above but without glucose. After autoclaving add 10 ml of 100% ethanol, 30 g of glycerol and 30 g of galactose
SECTION 11: Production of the NC30 protein in a fermenter using the
strain EMY761 pEMR673
The culture of the EMY761 strain pEMR673 is made in fermentor as follows ::
a) Preculture phase in a conical flask with baffles
From 1 ml of suspension of culture containing 20% of glycerol of the above strain with a cell number corresponding to an OD of 3 for X = 600 nm (on Kontron spectro), a conical flask with beadlets of 500 ml containing 90 ml of autoclavable phase semisynthetic growth medium (MCHPA), supplemented with 1.28 g of 2- (N-morpholino) ethanesulfonic acid MES buffer (M 8250 of Sigma) and 10 ml of filtered phase semisynthetic growth medium ( MCHPF). The chemical compositions and the preparatory mode of the MCHPA and MCHPF media are specified below. After incubation for 24 h with stirring at 30in, the optical density of the culture (OD) at X = 600 nm is about 7.

b) Growth phase in fermenters
The above culture is used to seed a 2.5 1 fermentor, pre-filled with:
800 ml of MCHPA medium
100 ml of MCHPF medium
The pH of the culture is regulated by the fermenter at a set point of 5.5. Similarly, the oxygen pressure is maintained above 30 mmHg by regulating the stirring. Initially, the air flow rate is fixed at 1 Vmin, or about 1 VVM, and is then increased stepwise as needed.

 After 6 to 7 hours of culture at 30in, 72 ml of a 500 g / l glucose solution, in total 36 g of glucose, are added linearly and for 9 hours.

c) Expression phase in fermenter
To the mixture described above, 100 ml of autoclavable phase semisynthetic expression medium (MEHPA) and 100 ml of semisynthetic phase-filtered expression medium (MEHPF), the chemical compositions and the method of preparation of which are specified below, are added. The culture is then continued, for about 5 hours without addition. The concentrations of the three carbon sources (glycerol, galactose, ethanol) are monitored by
HPLC and supplemented with sterile injections so as to approach the following values: glycerol 15 g / l, galactose 15 g / l, ethanol 7.5 g / l.

 Between 23 and 24 h after and under induction a neighboring OD of 90 is reached and the culture is stopped.

 The culture suspension is then centrifuged at 11,500 xg for 30 min. The yeast cell pellet is removed, the conserved supernatant is frozen at -80 ° C.

CHEMICAL COMPOSITION OF GROWTH AND EXPRESSION MEDIA - Autoclavable phase growth medium "MCHPA"
For 800 mi final
(with ultrapure water)

<tb> NTA <SEP>(<SEP> nitrolotriacetic acid) <SEP> 1 <SEP> g
<tb> K2SO4 <SEP> Is <SEP>
<tb> NaCl <SEP> 0.5 <SEP> g
<tb> MgSO4, <SEP> 7H2O <SEP> 1.0 <SEP> g
<tb> CaCl2,7H2O <SEP> 700 <SEP> mg
<tb> acid <SEP> glutamic <SEP> 3,7 <SEP> g
<tb> HY-CASE <SE> SF <SEP> (Sheffield <SEP> Products) <SEP> 25 <SEP> g
<tb> Leucine <SEP> 1.8 <SEP> g
<tb> histidine <SEP> 500 <SEP> mg
<tb> methionine <SEP><SEP> Is <SEP>
<tb> oligoelements <SEP> of <SEP> type <SEP> iS <SEP> (see <SEP> below) <SEP> 5 <SEP> ml
<Tb>
Adjust the pH to 5.5 with concentrated H2SO4 or concentrated KOH.

 AutocIaver for 20 min at 1200C.

- List of trace elements of type I - S
For 1 liter final
(with ultrapure water '

<tb> sulfate <SEP> of <SEP> Copper <SEP> CuSO4, <SEP> 5H2O <SEP> 780 <SEP> mg
<tb> acid <SEP> boric acid <SEP> H3BO3 <SEP> 5g <SEP>
<tb> sulfate <SEP> of <SEP> zinc <SEP> ZuSO4, <SEP> 7H2O <SEP> 3 <SEP> g
<tb> iodide <SEP> of <SEP> potassium <SEP> KI <SEP> 1 <SEP> g
<tb> sulfate <SEP> of <SEP> manganese <SEP> MnSO4, <SEP> 2H2O <SEP> 3.5 <SEP> g
<tb> molybdate <SEP> of <SEP> sodium <SEP> Na2MO4, <SEP> 2H2O <SEP> 2 <SEP> g
<tb> chloride <SEP> ferric <SEP> FeXl3, <SEP> 6H2O <SEP> 4.8 <SEP> g
<Tb>
Add to the solution 100 ml concentrated hydrochloric acid
Complete at 1000 mi.

- Growth medium hemispherical phase filtered "MCHPF"
for the final 100 ml (with
ultrapurified water)

<tb> KH2P 4 <SEP> 4 <SEP> g <SEP>
<tb> tryptophan <SEP> 350 <SEP> mg
<tb> vitamins <SEP> of <SEP> type <SEP> IS <SEP> (cf below) <SEP> 1 <SEP> ml
<tb> glucose <SEP> 15 <SEP> g
<Tb>
Heat to dissolve, temperate, add type I - S vitamins and sterilize by 0.2 μm membrane filtration.

- List of vitamins type of type I - S
for the final 100 ml (with
ultrapurified water)

<tb> biotin <SEP> 5mg
<tb> acid <SEP> folic acid <SEP> 4 <SEP> mg
<tb> niacin <SEP> 6mg
<tb>(SEP> nicotinic acid <SEP> pyridoxine.HCl) <SEP>
<tb> thiamine. <SEP> HCl <SEP> Is <SEP>
<tb> Ca. <SEP> Pantothenate <SEP> 5g <SEP>
<tb> m. <SEP> inositol <SEP> 10 <SEP> g
<Tb>
Make up to 100 ml after dissolution
Filter cold with sterile 0.2 m membrane.

Store at a temperature of + 4 C.

for 400 ml (with
ultrapurified water)

<tb> NTA <SEP> 1g
<tb> K2SO4 <SEP> 1.74 <SEP> g
<tb> acid <SEP> glutamic <SEP> 5 <SEP> g
<tb> HY-CASE <SE> SF <SEP> (Sheffield <SEP> Products) <SEP> 20 <SEP> g
<tb> Leucine <SEP> 1.8 <SEP> g
<tb> histidine <SEP> 500 <SEP> mg
<tb> methionine <SEP> 1g
<tb> tryptophan
<tb> MgSO4, <SEP> 2H2O <SEP> 600 <SEP> mg
<tb> oligoelements <SEP> of <SEP> type <SEP> IS <SEP> (cf <SEP> above) <SEP> 5 <SEP> mi
<Tb>
Adjust the pH to 5.5 with concentrated H2SO4 or KOH
Autoclave for 20 min at a temperature of 120il.

- Medium of expression semisynthetic phase filtered "MEHPF"
for the final 100 ml (with
purified water)

<tb> KH2PO4 <SEP> 2g
<tb> tryptophan <SEP> 350 <SEP> mg
<tb> vitamins <SEP> of <SEP> type <SEP> IS <SEP> (cf <SEP> above)
<tb> glycrol <SEP> 15 <SEP> g
<tb> ethanol <SEP> 15 <SEP> g
<tb> galactose <SEP> 7.5 <SEP> g
<Tb>
Heat to dissolve, temper, add vitamins and sterilize by 0.2 μm membrane filtration.

d) Analysis of the protein produced
Samples were prepared analogously to that described in Section 10 and subjected to SDS polyacrylamide gel electrophoresis. A band distribution on the gel identical to that observed in section 10 for the induced sample is observed.

SECTION 12: Purification of the NC30 protein produced in yeast and
determination of its aminoterminal sequence 1) Purification of the two major forms of NC30 protein
The two major forms of the recombinant yeast protein, those of bands corresponding to apparent molecular masses after polyacrylamide gel electrophoresis in the presence of 9 + 2 kDa and 16 + 2 kDa SDS were isolated and purified from 500 ml. the supernatant obtained in section II according to the following method:
Several steps were successively carried out: ion exchange chromatography on a Q fast flow column (Pharmacia) (5 × 5 cm), previously equilibrated with a 50 mM sodium acetate solution pH 4.0. Flow rate: 1 ml / min.The pH of the supernatant is previously adjusted to 4.0
Under these operating conditions, the protein does not bind to the gel.

ion exchange chromatography on a fast flow S column (Pharmacia) (5 × 4 cm), previously equilibrated with a 50 mM sodium acetate solution pH 4.0, with a 1M NaCl solution in eluent 50 mM sodium acetate buffer pH 4.0, Flow rate: 1 ml / min.

The eluate is concentrated on a YM5 membrane (Amicon) to a volume of approximately 2 ml and then deposited on an ACA 54 (IBF) filtration gel column (100 × 1.5 cm) equilibrated in a phosphate buffer. 1 M containing 0.14 M NaCl, flow rate: 0.2 ml / min. The fractions containing the recombinant protein (determined by SDS polyacrylamide gel electrophoretic analysis) are pooled.

 The solution thus obtained was subjected to an electrophoretic analysis on polyacrylamide gel in the presence of SDS and revelation with silver nitrate.

The two major forms of the NC30 protein corresponding to apparent molecular masses of 9 + 2 kDa and 16 + 2 KDa are observed, with a purity level greater than 70%. This solution was used in the biological activity tests described below.

In another experiment, a reversed column HPLC step
C4 (Brownlee) eluting with a linear gradient of acetonitrile / 0.1% TFA (trifluoroacetic acid) in H 2 O, 30% to 70% was introduced into this purification protocol, making it possible to obtain a product having a purity higher than 90% (estimated by electrophoretic analysis on polyacrylamide gel in the presence of SDS and revelation with silver nitrate).

2) Determination of the aminoterminal sequence of the two major forms of NC30 protein
The purified protein was subjected to 16% polyacrylamide gel electrophoresis in the presence of SDS. The proteins of the gel are transferred to an Immobilon membrane (Millipore) at 0.8 mA / cm 2 for 1 h in a buffer of 25 mM tris-borate composition pH 9.0 and 10% methanol.

The two bands corresponding to the apparent molecular weights of 9 + 2 kDa and 16 + 2 kDa were cut and deposited on a sequencer
Applied Biosystems Model 470 A, coupled with an Applied Biosystems Phenylthiohidantoic Analyzer, Model 120A

These two bands have the same amino terminal sequence (trl):
Ser Pro Gly Pro Pro Pro Pro Ser Thr Ala Leu,
Which is the expected aminoterminal sequence: that of the mature protein of 114 amino acids (see Figure 2) described in section 5, whose coding sequence is introduced into the vector pEMR673 described in section 9.

SECTION 13: Cytoplasmic expression of the NC30 protein in E. coli and
antiserum preparation 1) Cytoplasmic expression of the NC3O protein in E. coli:
An expression vector of the mature methionylated 112 amino acid NC30 protein in E. coli, designated pSE714.12 was constructed by insertion into the pE13a vector open to the NdeI and BamH1 sites of a DNA fragment carrying a portion NC30 cDNA. This expression vector comprises from 5 'to 3': the T7 phage RNA polymerase promoter, contained in the plasmid
pET3a, described by Rosenberg et al, Gene, 56, 125-135 - the part of NC30 cDNA that encodes the mature 112 acid protein
amino acids (see section 5), preceded by a translational ATG terminator - the T7 phage gene 10 terminator (Studier et al., 1986, 189, 113-130).

This expression cassette only functions in the presence of phage T7 specific RNA polymerase. It is therefore necessary to synthesize this
RNA polymerase at the host E. Coli strain. An expression cassette of this
RNA polymerase was constructed by placing the coding sequence of this enzyme (cloned into CE6 lambda phage DNA by Studier et al., 1986, J. Mol Biol, 125-135) under the control of the lambda PR promoter. This expression cassette also comprises a CI allele (CI857) encoding a thermosensitive form of the repressor of this PR promoter (P. Leplatois et al., 1983, Biochemistry, 65, 317-324). Therefore, at low temperature, the expression cassette of the DNA polymerase is repressed, at high temperature, the expression is derepressed. This expression cassette was cloned into an integration vector pEJL407 derived from N plasmids. Kleckner (1984,
Gene, 32, 369-379). The vector obtained is the plasmid pEMR648. This vector is maintained in the episomal state in the cell but it causes the integration of one or more copies of the polymerase expression cassette (including the IC repressor) when transposition is induced with IPTG (isopropyl-ss-thio-galactose). The transposase responsible for this integration is under control of the lacI gene but is not itself transposed, which makes it possible to obtain stable integrants after dissection of the plasmid. By transforming E. coli strain K12 HB101 (Gibco BRL - ref 8260 SA) with pEMR648, and then causing the integration events on the transformants, an HB101 derivative, called VG112, which comprises 2 cassettes was obtained. expression of the T7 phase polymerase under the control of the thermosensitive PL-CI system, integrated into the chromosome. The E. coli VG112 strain was removed from the plasmid pEMR648 and transformed at low temperature (300C) with the plasmid pEMR714.

The selected transformation, called strain VG112 pSE714.12, and deposited with the
CNCM on December 20, 1991 under No. 1-1162 was cultured on LB medium containing ampicillin at 100.1g / ml at 300C until OD at 600 nm of 1.

Then the expression of the polymerase gene was induced using IPTG at 410C for 2 h. Analysis of a total cellular extract on denaturing polyacrylamide gel made it possible to demonstrate a 9 kDa protein corresponding to a supernumerary band with respect to the pretreated and untransformed VG112 strain (control strain). Lysis of the cells by sonication followed by centrifugation makes it possible to separate the cell extract in 2 fractions: a soluble fraction (supernatant) and an insoluble fraction (pellet). The protein NC30 is found with the proteins of the insoluble fraction and represents approximately 50% (in mass) of the proteins of this fraction.

2) Preparation of an antiserum recognizing the NC3O protein
This insoluble fraction was used to immunize a rabbit (male of about 2 kg, New Zealand). Immunizations were performed every 15 days according to the protocol described by Vaitukaitis, 1981, Methods in Enzymology, 73, 46. For the first injection, a volume of antigenic solution is emulsified by a volume of complete adjuvant of Freud (Sigma- 4258). Six recalls were given as incomplete Freud's adjuvant (Sigma-ref 5506).

 The antiserum obtained is capable of recognizing the NC30 protein produced by yeast and COS cells by immunodetection after polyacrylamide gel electrophoresis in the presence of SDS.

SECTION 14: Demonstration for the NC30 protein of an activity
inhibitory effect on IL-1b messenger RNA production and
IL-6 by peripheral blood monocytes stimulated by
LPS 1) - Method used
a) Preparation of the cells
From a pocket of peripheral blood (taken from a healthy volunteer in a blood transfusion center) most of the red blood cells are removed by sedimentation at 1 g for 30 min in a medium containing 0.6% dextran, 0.09% NaCl. Subsequently the cells are deposited over a layer of Ficoll-Paque (Pharmacia) and centrifuged at 400 g for 30 min.

Peripheral blood mononuclear cells (PBMNC), which are present at the interface between Ficoll and the supernatant, are removed. The PBMNCs are placed in RPMI medium (RPMI 1640-Gibco BRL medium) containing 10% fetal calf serum (FCS), on culture dishes 15 cm in diameter, at a rate of 1 to 5 × 10 7 cells per dish. After 30 min the medium is aspirated and the adherent cells on the dish (mainly consisting of monocytes) are incubated as described above.

b) Incubation of cells with LPS and NC30 protein
The adherent PBMNC are incubated in 20 ml of RPMI / 10% FCS for 4 hours at 37 ° C. in an atmosphere containing 5% CO 2 in the presence of 5 μg per ml of Lipopolysaccharide LPS (ref M391-Sigma) and increasing concentrations of the NC30 protein from purified yeast (0.1 to 10 ng / ml), or COS cell supernatants either transfected with plasmid pSE1 NC30 and cultured as described in section 7, or transfected with plasmid pSE1 and cultured in the same conditions (witness).

c) RNA preparation and analysis
The cells are washed with PBS and then scraped directly into 1 ml of buffer D (of composition: 4M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M t3-mercaptoethanol: Chomczynski P.

and Sacchi N. (1987), Anal. Biochem., 162, 156-159). RNA is prepared by the phenolic extraction method at acidic pH described by these authors. Between 1 and 5 μg of RNA on 1% agarose gel are deposited in the presence of formaldehyde (Sambrook et al, op cit). After migration, the RNAs are transferred to a reinforced nitrocellulose membrane (Schleicher and Schuell) and hybridized with radiolabeled cDNA probes as in section 4. The hybridization intensities of each RNA with the different probes are quantified by phosphorescence analysis on a Phosphorimager device (Molecular
Dynamics, 800E. Arques Avenue, Sunnyvale, CA, 94086- USA).

2) - Results
Mean and standard deviations of the results obtained with the protein
NC30 purified in four experiments, are summarized in Table 3 below, in which the amounts of messenger RNA measured by phosphorescence analysis are expressed as a percentage relative to the amount of messenger RNA measured for the sample from the cells treated with
LPS alone.

Table 3
Quantity of messenger ARNs
IL-1ss and IL-6 measured for
different concentrations of NC30 protein

<tb><SEP> Quantity <SEP> of RNA <SEP> messengers
<tb><SEP> IL-i <SEP> (3 <SEP> IL-6
<tb> condition <SEP> of <SEP> stimulation <SEP> of <SEP> cells
<tb> LPS <SEP> 100 <SEP> 100
<tb> LPS <SEP> + <SEP> protein <SEP> NC30 <SEP> to <SEP><SEP> 97 <SEP> + <SEP> 36 <SEP> 77 <SEP> + <SEP> 18
<tb> concentration <SEP> of <SEP> 0.1 <SEP> ng / ml
<tb> LPS <SEP> + <SEP> protein <SEP> NC30 <SEP> to <SEP><SEP> 54 <SEP> + <SEP> 39 <SEP> 24 <SEP> + <SEP> 11
<tb> concentration <SEP> of <SEP> 1 <SEP> ng / ml
<tb> LPS <SEP> + <SEP> protein <SEP> NC30 <SEP> to <SEP><SEP> 30 <SEP> + <SEP> 5 <SEP> 13 <SEP> +4
<tb><SEP> concentration <SEP> of <SEP> 10 <SEP> ng / ml
<Tb>
It can be seen from the table above that the accumulation of
Messenger RNAs of IL-1ss and IL-6 in LPS-treated monocytes is inhibited by NC30 protein. The most important inhibition is observed for a concentration of 10 ng / ml of the NC30 protein, and the dose to obtain a 50% inhibition (IC 50) is is of the order of 1 ng / ml.

 Likewise, inhibition of the production of the messenger RNAs of FL-1β and of IL-6 is observed in the presence of COS cell supernatants transfected with the plasmid pSE1-NC30 and no inhibition in the presence of control COS cell supernatants. .

In other experiments, inhibition of the production of IL-1P and IL-6 proteins in LPS-treated monocyte culture media was also observed in the presence of NC30 protein. IL-6 was assayed by its effect on the proliferation of hybridoma line B9 according to the method described by LA Aarden, 1987, Eur. J. Immun., 17, 1411-1416. The IL-1b assay was performed on EL4 cells according to the method described by
EW Palaszynski, 1987, Biochem and Biophys. Res. Comm., 147, p. 204-211, which measures the competition of the season with radiolabelled IL-1ss.

SECTION 15: Demonstration for the NC30 protein of the modulation of
amount of CD23 surface antigen by B cells
of tonsils 1) Method used
a) cell preparation
The human tonsils were removed after surgery on a 6-year-old girl. Tonsils were scalpeled in RPMI medium cooled to 4 ° C. The cells released into the medium after this operation are filtered on gauze so as to form a homogeneous cell suspension. After two washes, the cells are digitized and taken up in fetal calf serum containing 10% DMSO (Merck).

7.5.106 cells in a volume of 1 ml are distributed in each freezing tube. The cells are placed in a thermos at -80 ° C for 24 h and then stored in liquid nitrogen.

b) Incubation of cells with NC30 protein
On the day of the experiment, an aliquot of the cells is thawed at 370 ° C. and then diluted slowly in 50 ml of RPMI medium containing 10% fetal calf serum. The cells are centrifuged to remove DMSO.

After counting the cells, 100 μl of a cell suspension readjusted to 4 × 10 6 cells / ml are distributed in 96-well microtiter plates (NUNC).

 The purified NC30 protein is added at different concentrations in RPMI medium containing 10% fetal calf serum. 100 l of the different concentrations are added to the cells in the microculture wells.

Incubation is continued for 48 h at 37 ° C in an atmosphere containing 5% CO 2.

c) Labeling of cells for immunofluorescence
After incubation, the cells are transferred into micronics tubes (Labsystem). For single-immunofluorescence analysis, 10 μl of anti-CD23 antibody coupled to FITC (fluorescein isothiocyanate) (Immunotech) are added to the cell suspension. For the double immunofluorescence assay, 10 μl of phycoerythrin-coupled anti-CD23 antibody and 10 μg of FITC-coupled anti-CD20 antibody (Becton Dickinson) are added simultaneously to the cell suspension.

 Incubation is continued for 30 min at 4 ° C., then the cells are centrifuged and the pellet taken up in 250 μl of cold PBS buffer. 50 μl of a solution of propidium iodide (Sigma) at 20 μUml are added in order to distinguish the dead cells during the analysis.

d) Flow cytometry analysis
The samples are analyzed by fluorescence assay on a FacStar Plus cell sorter (Becton Dickinson) with a fluorescence excitation laser wavelength of 488 nm. In the single immunofluorescence assay, FITC and propidium iodide emissions are collected using 530 nm and 630 nm interference filters, respectively. In the double-immunofluorescence assay, the additional emission due to phycoerythrin is collected through a 575 nm interference filter. For this analysis, an electronic compensation system is used to avoid contamination of the FITC fluorescence. in the phycoerythrin channel, fluorescence of phycoerythrin in the propidium iodide channel and fluorescence of propidium iodide in the phycoerythrin channel. The results are collected and processed using the LysIS II software (Becton Dickinson).

2) Results
a) Modulation of the amount of the CD23 antigen on tonsillar cells by the CN30 protein
The means and standard deviations of the results obtained are collated in Table 4 below, for an incubation of the tonsillar cells for 48 h and a range of the NC30 protein concentrations of 10-3 to 102 ng / ml.
Table 4
Variation of the percentage of tonsil cells
expressing the CD23 antigen in the presence of
different concentrations of NC30 protein

<tb> Concentration <SEP> in <SEP>% <SEP> of <SEP> cells
<tb><SEP> NC3O <SEP> (ngbml) <SEP> expressing <SEP> CD23
<tb><SEP> O <SEP> 4,8 + 1,2 <SEP>
<tb><SEP> 10-3 <SEP> 1 <SEP> 4,8 + 08 <SEP>
<tb><SEP> 10-2 <SEP> 4.9 + 04 <SEP>
<tb><SEP> 10-1 <SEP> 12.0 + 24 <SEP>
<tb><SEP> 1 <SEP> 17.0 <SEP> + <SEP> 2.5
<tb><SEP> 10 <SEP> 27.3 + 16 <SEP>
<tb><SEP> 102 <SEP> 20.4 + 2.5 <SEP>
<Tb>
It is found that the percentage of tonsil cells expressing the CD23 antigen (low affinity IgE receptor) is greater for a concentration of NC30 protein greater than 10-1 ng / ml than the percentage obtained in the absence of the NC30 protein. The most important effect of this protein is found at a concentration of 10 ng / ml.

b) Characterization of the cells on which the NC30 protein modulates
the expression of CD23 antigen
The characterization of cells expressing the CD23 antigen has been
performed in double-immunofluorescence using an anti-CD23 antibody
coupled with phycoerythrin and an anti-CD20 antibody coupled to FITC. The latter is directed against a receptor present only on the B cells.

The flow cytometry analysis shows that only a fraction of B cells express the CD23 antigen under the action of the NC30 protein.

 The NC30 protein is therefore a new human lymphokine with activities towards at least two key cells of the immune system.

It inhibits the synthesis of IL-1p and IL-6 by LPS-activated peripheral blood monocytes and increases the expression of the CD23 antigen on B tons lymphocytes. It is therefore a new interleukin.

Claims (24)

1. A cytokine-type activity protein, characterized in that it comprises the following sequence (a1):  Gly Pro Pro Pro Pro Ser Thr Ala Leu Arg Glu Leu Glu Glu Leu  1 5 10 15  Val Asn Thr Thrn Gln Asn Gln Lys Ala Pro Asr Cys Asn Gly Ser Met  20 25 30  Val Trp Ser Asn Island Leu Thr Ala Asp Met Tyr Cys Ala Ala Leu Glu  35 40 45  Ser Leu Asn Island Val Ser Gly Cys Ser Ala Island Glu Lys Thr Gln Arg  50 55 60  Met Leu Ser Gly Phe Cys Pro His Lys Ser Val Ala Gly Gln Phe Ser  65 70 75 80  Ser Leu His Val Arg Asp Thr Lys Glu Val Ala Gln Phe Val Lys  85 90 95  Asp Leu Leu Leu Leu Leu Lys Leu Phe Arg Glu Gly Arg Phe Asn  100 105 110  or a sequence having a high degree of homology with the sequence (a1) 2. Protein according to claim 1, characterized in that it comprises immediately upstream of the sequence (a1) the sequence: Ser Pro. 3. Protein according to one of claims 1 and 2, characterized in that it has an apparent molecular mass of 9.0 ± 2 kDa. 4. Protein according to one of claims 1 and 2, characterized in that it has an apparent molecular mass of 16.0 ± 2 kDa. 5. Protein according to one of claims 1 and 2, characterized in that it is N-glycosylated. 6. Protein according to one of claims 1 and 2, characterized in that it has a degree of purity, determined by electrophoresis on polyacrylamide gel in the presence of SDS and silver revelation, greater than 70%. 7. Protein according to one of claims 1 and 2, characterized in that it has a degree of purity, determined by electrophoresis on polyacrylamide gel in the presence of SDS and revelation with silver, greater than 90%. 8. recombinant DNA, characterized in that it encodes a protein according to one of claims 1 and 2 or for a precursor of a protein according to one of claims 1 and 2. 9. Recombinant DNA, characterized in that it codes for a precursor of a protein according to one of claims 1 and 2, which comprises a signal sequence. 10. Recombinant DNA according to claim 9, characterized in that the coding sequence for the mature protein comprises the following sequence:  GGCCCTGTGC CTCCCTCTAC AGCCCTCAGG GAGCTCATTG  AGGAGCTGGT CAACATCACC CAGAACCAGA AG GCTCCGCT  CTGCAATGGC AGCATGGTAT GGAGCATCAA CCTGACAGCT  GACATGTACT GTGCAGCCCT GGAATCCCTG ATCAACGTGT  CAGGCTGCAG TGCCATCGAG AAGACCCAGA G GATG CTGAG  CGGATTCTGC CCGCACAAGG TCTCAGCTGG GCAGTnTCC  AGCTTGCATG TCCGAGACAC CAAAATCGAG GTGGCCCAGT TTGTAAAG GA C CTG CTCTTA CATTTAAAGA AAGIT'i'CG  CGAGGGACGG TTCAAC Recombinant DNA according to one of Claims 9 and 10, characterized in that the signal sequence is a sequence chosen from the following sequences (b1), (b2), (b3) and (b4): (b1)  Met His Pro Leu Leu Asn Leu Leu Leu Al Leu Leu Leu Met Ala  1 5 10 15  Leu Leu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly Phe Ala  20 25 30 (b2)  Met His Pro Leu Leu Asn Leu Leu Leu Al Leu Leu Leu Met Ala  1 5 10 15  Leu Leu Leu Thr Thr Val They went to Leu Thr Cys Leu Gly Gly Phe Ala  20 25 30  Ser Pro (b3)  Met Ala Leu Leu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly  1 5 10 15  Phe Ala (b4)  Met Ala Leu Leu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly  1 5 10 15  Phe Ala Ser Pro  20 12. Recombinant DNA according to claim 11, characterized in that the nucleotide sequence coding for the signal peptide is chosen from the following sequences (Nbl), (Nb2), (Nb3) and (Nb4): (Nbl)  ATGCATCCGC TCCTCAATCC TCTCCTGTTG GCACTGGGCC  TCATGGCGCT TITGTTGACC ACGGTCATTG CTCTCACTTG  CCTTGGCGGC TTTGCC (Nb2)  ATGCATCCGC TCCTCAATCC TCTCCTGTTG GCACTGGGCC  TCATGGCGCT TTTGTTGACC ACGGTCATTG CTCTCACTTG  CaTGGCGGC TTTGCCTCCC CA (Nb3)  ATGGCGCTTT TGTTGACCAC GGTCATTGCT CTCACITGCC  TTGGCGGCTT TGCC (Nb4)  ATGGCGCTTT TGTTGACCAC GGTCATTGCT CTCACTTGCC  TTGGCGG (l l TGCCTCCCCA 13.Vector expression, characterized in that it contains, with the means necessary for its expression, a recombinant DNA according to one of claims 8 to 12. 14. Eukaryotic cells, characterized in that they contain, with the means necessary for their expression, a recombinant DNA according to one of claims 8 to 10. 15. Eukaryotic cells according to claim 14, characterized in that they are animal cells. Animal cells according to claim 15, characterized in that they contain an expression vector according to claim 13. Animal cells according to claim 16, characterized in that they are CHO cells. 18. Animal cells according to claim 16, characterized in that they are COS cells. 19. Eukaryotic cells according to claim 14, characterized in that they are yeast cells. 20. Prokaryotic microorganism characterized in that it is transformed by an expression vector according to claim 13. 21. Prokaryotic microorganism, characterized in that it belongs to the species E. coli. 22. Process for preparing a protein according to one of claims 1 to 7, characterized in that it comprises a step of culturing animal cells according to one of claims 15 to 18, followed by isolation and purification of the recombinant protein. 23. Process for the preparation of a protein according to one of claims 1 to 7, characterized in that it comprises a yeast cell culture step according to claim 19 followed by the isolation and purification of the protein. recombinant. 24. Medicinal product characterized in that it contains a protein according to one of claims 1 to 7.