[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

EP1242599A2 - Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators - Google Patents

Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators

Info

Publication number
EP1242599A2
EP1242599A2 EP00993418A EP00993418A EP1242599A2 EP 1242599 A2 EP1242599 A2 EP 1242599A2 EP 00993418 A EP00993418 A EP 00993418A EP 00993418 A EP00993418 A EP 00993418A EP 1242599 A2 EP1242599 A2 EP 1242599A2
Authority
EP
European Patent Office
Prior art keywords
protein
sequence seq
mbp
cdc25c
plasmid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00993418A
Other languages
German (de)
French (fr)
Inventor
Françoise GOUBIN-GRAMATICA
Bernard Ducommun
Grégoire Prevost
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ipsen Pharma SAS
Original Assignee
Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR9915722A external-priority patent/FR2815347A1/en
Priority claimed from FR0006883A external-priority patent/FR2812292A1/en
Application filed by Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS filed Critical Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS
Publication of EP1242599A2 publication Critical patent/EP1242599A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a method for obtaining human phosphatases Cdc25. It also relates to a method of identifying modulators of human phosphatases Cdc25.
  • the entry of the cell into the process of cell division is regulated by a set of kinases and phosphatases useful for synchronizing the different phases of the cell cycle and allowing the reorganization of the cell architecture.
  • Cyclin-dependent kinases play a major role in this control and already several inhibitors of this family of kinases have been identified.
  • One of these compounds (flavopiridol) is already in Phase II clinical (Senderowicz and Sausville, J. Natl. Cancer Inst. (2000), 92, 376-387).
  • CDKs are activated by a dephosphorylation carried out by the phosphatases Cdc25, on tyrosine and threonine residues.
  • Cdc25 proteins are coded by a family: Cdc25A, Cdc25B and Cdc25C (Cans et al., Medicine Sciences (1998), 3, 269-274).
  • Cdc25 Some inhibitors of Cdc25 are identified, but they have only a weak activity (cf. Baratte, B., Meijer, L., Gal forcingov, K., and Beach, D., Anticancer Res. (1992), 12, 873-880 ; Rice, RL et al., Biochemistry (1997), 36, 15965-15974; and Ham, SW, Park,., Lee, SJ, Kim, W., Kang, K., and Choi, KH, Bioorg. Med Chem. Lett. (1998), 8, 2507-2510).
  • Cdc25B2 is a tyrosine phosphatase protein close to the Cdc25B phosphatase (now called Cdc25Bl). It has been identified in a DNA library encoding Burkitt's lymphoma. Cdc25B2 differs from Cdc25Bl by an insertion of 14 amino acids and a deletion of 41 amino acids upstream of the catalytic domain. Cdc25Bl and Cdc25B2 are splicing variants of the same gene. A third variant, Cdc25B3, carrying the two sequences of 14 and 41 amino acids, was identified from from the same bank (Baldin et al., Oncogene (1997), 14, 2485-2495).
  • the three variants are detected in primocultures and cell lines. Analysis of the variants shows that Cdc25B2 is more weakly expressed than Cdc25B3 in all of the lines tested, but that the expression of the two variants increases during the G2 phase and mitosis (Forrest et al., Biochem. Biophys. Res. Commun . (1999), 260, 510-515). Hernandez et al. report that Cdc25A and -B2 but not Cdc25Bl, -B3 and -C, are over-expressed in a large number of lymphomas (35% and 39%) (Hernandez et al., Int. J. Cancer (2000), 89 ( 2), 148-52). Normal lymphocytes express the messengers of Cdc25Bl and -B3 and very weakly those of Cdc 25 A, -B2 and -C.
  • the Cdc25C phosphatase is itself regulated by phosphorylation on serine-216 by other enzymes Cdsl or Chkl and binds to very conserved members of the protein family 14-3-3 (Zeng, Y. et al. , Nature (1998), 395, 507-510).
  • microorganisms bacteria, yeasts
  • immunoprecipitation using a specific antibody from a mixture of proteins affinity chromatography, using a specific ligand (effectors, repressors, activators); two-dimensional electrophoresis, as a function of the molecular mass and the isoelectric point of the protein; capillary electrophoresis; enrichment by differential precipitation with different salts; "- etc.
  • the proteins Cdc25Bl, Cdc25B2, Cdc25B3 or Cdc25C could be fused with systems as varied as:
  • beta galactosidase protein affine for the amino-phenyl- ⁇ -D-thiogalactopyranoside (Germino, J. and Bastia, D., Proc. Natl. Acad. Sci. U.S.A. (1984), 81, 4692-4696);
  • biotin-carboxylase carrier affine for avidin (Germino, F.j. and Moskowitz, N.K., Methods Enzymol. (1999), 303, 422-450);
  • the protein intein refines for chitin (see Chong, S., et al., Gene (1997), 192, 271-281; Carr, S., et al., Vaccine (1999), 18, 153-159 ); 11.
  • the maltose-binding protein refines for amylose (Ahaded, A., et al., Prep. Biochem. Biotechnol. (1999), 29, 163-176).
  • the Applicant has however just developed a method which makes it possible to obtain the enzymes Cdc25Bl, Cdc25B2, Cdc25B3 and Cdc25C human in an active form and in unlimited quantity.
  • the present invention facilitates on the one hand the research and the study of the physiological or / and physiopathological activities of this protein and on the other hand facilitates the research of agents modulating these activities.
  • the invention makes it possible to obtain a human protein Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C recombined with the maltose-binding protein (MBP) which retains its phosphatase activity without requiring separation with the MBP part, thus preventing any contamination with proteases.
  • MBP maltose-binding protein
  • the high level of expression of the protein after induction allows an excellent purification yield and the preparation in unlimited quantity of the enzyme.
  • the invention therefore firstly relates to a fusion protein between the maltose-binding protein (MBP) and a protein chosen from the proteins Cdc25Bl, Cdc25B2, Cdc25B3 and Cdc25C.
  • the present invention relates in particular to a protein chosen from the following proteins:
  • a fusion protein between human Cdc25B3 phosphatase and MBP which is characterized in that it is coded by the sequence SEQ. ID No. 14 (shown below); or - a fusion protein between human Cdc25C phosphatase and MBP, which is characterized in that it is coded by the sequence SEQ. ID # 1 (shown below).
  • the subject of the invention is also the bacterial strain JM 109 transfected: - with the plasmid of sequence SEQ. ID No. 9 (shown below), said strain being useful for the preparation of the protein Cdc25Bl;
  • the invention further relates to a process for preparing said fusion proteins, characterized in that it comprises the following successive stages: - culture of the bacterial strain JM 109 transfected with the plasmid of sequence SEQ. ID No. 9, the plasmid with sequence SEQ. ID No. 10, the plasmid with sequence SEQ. ID No. 11 or the plasmid with sequence SEQ. ID No. 5, in LB medium supplemented with ampicillin;
  • the invention relates to an application of said fusion protein to a method of identifying modulators of the protein Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C, characterized in that it comprises the following successive steps:
  • the determination of the quantity of 3-O-methylfluorescein produced can be carried out, for example, by measuring the optical density of the solution, the absorbance linked to 3-O-methylfluorescein at the wavelength of 477 nm , or alternatively by fluorimetry using excitation at the wavelength of 475 nm and a reading at the wavelength of 510 nm.
  • the system used (New England Biolabs # 800 - pMAL TM protein fusion and purification system) is based on the production of a fusion protein between the protein of interest, here the human Cdc25C protein, and the bacterial protein MBP (Maltose - Binding Protein) X! Escherichia coli.
  • MBP Maltose - Binding Protein
  • the DNA coding for human Cdc25C phosphatase corresponds to accession number 4502706.
  • the DNA encoding Cdc25C was amplified by polymerase chain reaction (PCR) using the primers C-XBAI SENS and C-XBAI ANTI-SENS (of sequences SEQ. ID. Nos . 3 and 4 respectively) and introduced into the vector pCDNA3-HA at the Xbal site to give the vector pcDNA3-HA Cdc25C.
  • the Cdc25C / Xbal insert has been sequenced in its entirety at this stage.
  • the primer C-XBAI SENS has the sequence SEQ. ID. n ° 3 represented below:
  • the primer C-XBAI ANTI-SENS has the sequence SEQ. ID. n ° 4 represented below:
  • the vector pcDNA3-HA Cdc25C was digested with Xbal, the insert Cdc25C / Xbal (1456 base pairs) purified and introduced into the vector pMAL TM -c2X (New England Biolabs, # 800-76) at the site Xbal, to give the vector pMAL - Hs Cdc25C (of sequence SEQ. ID No. 2).
  • This vector allows the production of a fusion protein MBP-Cdc25C of 868 amino acids, from the bacterial promoter Ptac inducible by isopropylthiogalactoside (IPTG).
  • sequence SEQ. ID. n ° 2 of the protein Hs Cdc25C with its XBAI restriction ends is as follows:
  • the vector pMAL - Hs Cdc25C was introduced into the strain of Escherichia coli (E. coli) JM109 (Stratagene # 200271). An isolated colony was selected and the production of a protein of theoretical apparent molecular weight of 97 kDa after culture in the presence of IPTG was observed by the analysis of total bacterial proteins on denaturing polyacrylamide gel and staining with Coomassie blue. . The identity of the fusion protein was then confirmed by western blot and immunodetection with an anti-Cdc25C antibody.
  • the plasmid DNA isolated from this clone was sequenced in the region corresponding to Cdc25C to verify the absence of mutations or modifications of the sequence which could have been generated during the subcloning and / or transformation processes. DNA.
  • TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG
  • GCATGGTCCA ACATCGACAC CAGCAAAGTG AATTATGGTG TAACGGTACT GCCGACCTTC
  • GGT ⁇ A ⁇ CTGG ATTCTT ⁇ AGG ACTT ⁇ .
  • GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCCCAG ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT
  • AATAGCCCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA
  • AACCATCACC CAAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC
  • GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG
  • CTGCCTGTTC ATCCGCGTCC AGCTCGTTGA GTTTCTCCAG AAGCGTTAAT GTCTGGCTTC
  • the XBAI sites are underlined by a single solid line
  • the ORF of Cdc25C is underlined in dotted lines
  • the maltose-binding protein sequence (MBP) is underlined by a solid double line and the bold sequence corresponds to the region plasmid DNA isolated from the JM 109 strain and sequenced).
  • This clone can be stored at -80 ° C. in the form of a saturated culture added with glycerol (final concentration 25%) or "glycerol stock". This strain will be used for all subsequent production stages.
  • LB + ampicillin medium 100 ⁇ g / ml (LB amp.) are inoculated with 100 ⁇ l of glycerol stock from the clone JM109 / pMAL-Cdc25C and cultured for 14 to 16 hours at 37 ° C. with stirring (180 to 220 rpm).
  • This preculture is then diluted fifty times (20 ml per liter of medium) in an LB amp medium. + 2 g / 1 of glucose and cultured at 37 ° C / 180 rpm to reach an optical density at 600 nm of between 0.55 and 0.60.
  • the synthesis of the fusion protein is then induced by adding IPTG (0.3 mM) at 37 ° C for 3 hours.
  • the bacteria are harvested by centrifugation, washed once in 40 ml of cold PB S per liter of culture, and the bacterial pellet is then frozen in liquid nitrogen and stored at -80 ° C.
  • Induction is analyzed immediately by depositing 2.5 ⁇ 10 7 cells, taken before and after induction, on denaturing polyacrylamide gel and staining of proteins with Coomassie blue (FIG. 1, lines 1 and 2 respectively). 2.2 - Lysis and extraction.
  • a bacterial pellet corresponding to 1 liter of induced culture is thawed in ice, resuspended in 35 ml of lysis buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 ⁇ g / ml lysozyme, 1 ⁇ g / ml leupeptine, 2 ⁇ g / ml aprotinin, 1 mM PMSF) and incubated 45 min in ice.
  • the bacterial suspension is then sonicated (4 cycles of 1 min in 50% discontinuous mode, alternated with 1 min of pause), then centrifuged for 35 min. at 110,000 g.
  • the supernatant or soluble extract is kept for the purification of the protein MBP-Cdc25C ( Figure 1, line 3).
  • the soluble extract is passed through the affinity column at a flow rate of 0.15 ml / min; the eluate (that is to say the fraction not retained on the amylose-agarose) is collected for analysis (FIG. 1, line 4).
  • the column is washed with 20 ml (10 volumes) of column buffer.
  • a sample of the affinity matrix after passage of the soluble extract can optionally be analyzed (Figure 1, line 5).
  • the elution of the protein from the affinity matrix is carried out with a maltose buffer (20 mM Tris-HCl pH 7.4; 250 mM NaCl; 1 mM EDTA; 1 mM DTT; 10 mM maltose). 20 elution fractions of 0.5 ml are collected. For each fraction, the total protein concentration is evaluated by a Bradford type test and the fraction is analyzed by deposition on denaturing polyacrylamide gel and staining with Coomassie blue ( Figure 1, line 6). The fractions in which the complete MBP-Cdc25C fusion protein represents at least 90% of the total proteins are combined to form a batch, the activity of which is then tested. The batches are stored at -80 ° C.
  • CTGTCTGCGT .. GAAGAGGA ⁇ A GGTCTCTGAA C ⁇ AGTATCCT GCATTGTA ⁇ .T A ⁇ CCAGAGCT ATATATCCTT ... .AAAGGCG.GCT ACAGAGACTT CTTTCCAGAA. TATATGGAAC TGTGTGAACC ACAGAGCTAC TGCCCTATGC ATCATCAGGA CCACAAGACT GAGTTGCTGA GGTGTCGAAG ... CCAGAG ⁇ AAA GTG ⁇ AGGAAG ... . GGGAGC GC A. .. GC.TGCGGGAG .... CAGATTGC ⁇ C
  • sequence of the MBP-Cdc25C fusion protein obtained therefore corresponds to the sequence SEQ. ID # 1 shown below:
  • TCTCATCCAC AAGAGAGGAA GGAAGCTCTG GCTCAGGACC CAGTTTTAGG TCTAATCAAA
  • the phosphatase activity of. MBP-Cdc25C protein is evaluated by a 3-O-methylfluorescein phosphate dephosphorylation test (OMFP) with determination of the absorbance at 477 nm (OD 477 nm) of the reaction product (OMF).
  • OMFP 3-O-methylfluorescein phosphate dephosphorylation test
  • the MBP-Cdc25C protein stored in the elution buffer (the same as that described in paragraph 2.3), is diluted to the concentration of 20 nM in the phosphatase reaction buffer (50 mM Tris-HCl pH 8.2; 50 mM NaCl; 1 mM DTT; 20% glycerol), at room temperature, in a total reaction volume of 1 ml.
  • the reaction is initiated by the addition of a 0.3 mM solution of OMFP (prepared extemporaneously from a 7.5 mM stock solution in 100% DMSO (Sigma # M2629)) and takes place at 25 ° C.
  • the system used (New England Biolabs # 800 - pMAL TM protein fusion and purification system) is based on the production of a fusion protein between the protein of interest, here the human Cdc25B protein, and the bacterial protein MBP (Maltose - Binding Protein) to 'Escherichia coli.
  • MBP Maltose - Binding Protein
  • the DNA encoding the three splicing variants of human CDC25B phosphatase corresponds to the accession numbers: M81934 and Z68092.
  • the Cdc25B cDNAs in the form of Nde ⁇ (Klenow) / BamHI inserts were introduced into the vector pMAL TM -C2X (New England Biolabs, # 800-76) at the EcoRl (Klenow) / BamHI site to respectively give the pMAL vectors.
  • pMAL TM -C2X New England Biolabs, # 800-76
  • EcoRl EcoRl
  • CACGACCTCG CCGGGCTCGG CAGCCGCAGC CGCCTGACGC ACCTATCCCT GTCTCGACGG
  • CTGGCAGAGT GGGCCAGCCG CAGGGAAGCC TTTGCCCAGA GACCCAGCTC GGCCCCCGAC
  • AAGTTTGTGA TTGTAGACTG CAGATACCCC TATGAATATG AAGGCGGGCA CATCAAGACT GCGGTGAACT TGCCCCTGGA ACGCGACGCC GAGAGCTTCC TACTGAAGAG CCCCATCGCG
  • CTCTACTACC CTGAGATGTA TATCCTGAAA GGCGGCTACA AGGAGTTCTT CCCTCAGCAC
  • CTGTGTCATC CCATCATTTT CCATATCCTG GTGCCCCCCA CCCCTGGAAG AGCCCAGTCT GTTGAGTTAG TTAAGTTGGG TTAATACCAG CTTAAAGGCA GTATTTTGTG TCCTCCAGGA
  • sequence SEQ. ID. n ° 7 of the protein Hs Cdc25B2 with its Ndel and BamHI restriction ends is as follows:
  • CACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGGGAC CCTGCTCTTC
  • GTCATCCTCA TTTTCCACTG TGAATTCTCA TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC
  • sequence SEQ. ID. n ° 8 of the protein Hs Cdc25B3 with its Ndel and BamHI restriction ends is as follows:
  • CACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGGGAC CCTGCTCTTC
  • TTCCTCCTAC AGACAGTAGA CGGAAAGCAC
  • CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC
  • GGTTAATACC AGCTTAAAGG CAGTATTTTG TGTCCTCCAG GAGCTTCTTG TTTCCTTGTT AGGGTTAACC CTTCATCTTC CTGTGTCCTG AAACGCTCCT TTGTGTGTGT GTCAGCTGAG GATCC
  • the pMAL-Cdc25Bl-3 vectors were each introduced into the Escherichia coli JM109 strain (Stratagene # 200271). Colonies were selected based on their ability to produce a fusion protein after culture in the presence of IPTG. The identity of the proteins was verified by immunodetection with polyclonal antibodies directed against Cdc25B.
  • the plasmid DNAs isolated from these three clones were sequenced in the region corresponding to Cdc25B.
  • the following sequences were obtained (the doubly underlined part corresponds to the ORF of MBP and the underlined part in dotted lines corresponds to the ORF of Cdc25Bl, Cdc25B2 or Cdc25B3):
  • GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC
  • CTGGAGGCCT CAGGTGCTGT CCATGGGAAA GATGGTGTGG TGTCCTGCCT GTCTGCCCCA GCCCAGATTC CCCTGTGTCA TCCCATCATT TTCCATATCC TGGTGCCCCC CACCCCTGGA
  • TTGTGTCCTC CAGGAGCTTC TTGTTTCCTT GTTAGGGTTA ACCCTTCATC TTCCTGTGTC
  • the recombinant proteins MBP-Cdc25Bl, B2 and B3 are produced exactly as described for MBP-Cdc25C.
  • SEQ sequences. ID No. 12, SEQ. ID # 13 and SEQ. ID No. 14 are obtained respectively for the fusion proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3:
  • GCCTTTGCCC AGAGACCCAG CTCGGCCCCC GACCTGATGT GTCTCAGTCC TGACCGGAAG ATGGAAGTGG AGGAGCTCAG CCCCCTGGCC CTAGGTCGCT TCTCTCTGAC CCCTGCAGAG
  • CTATTGACGG GCAAGTTCAG CAACATCGTG GATAAGTTTG TGATTGTAGA CTGCAGATAC CCCTATGAAT ATGAAGGCGG GCACATCAAG ACTGCGGTGA ACTTGCCCCT GGAACGCGAC GCCGAGAGCT TCCTACTGAA GAGCCCCATC GCGCCCTGTA GCC
  • FIG. 1 (FIG 1) represents the analysis chromatography relating to the expression induction of the fusion protein MBP-Cdc25C
  • the lines 1 and 2 of FIG. 1 correspond respectively to the total extract of JM109 / pMAL- Cdc25C with or without addition of IPTG.
  • Line 3 corresponds to the soluble extract.
  • Lines 4 and 5 correspond respectively to the fractions not retained and retained on amylose-agarose.
  • line 6 of FIG. 1 corresponds to the elution fraction No. 12 which contains practically only fusion protein
  • FIG. 2 represents the results of the measurement of the activity of the recombinant protein MBP-Cdc25C (a "+” signifying that menadione was added to the sample, a "-" that the sample was not treated with menadione)
  • FIG. 3 represents the results of the measurement of the activity of the recombinant proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3.
  • the reaction is carried out with 300 ng of enzyme per test. MBP at the same concentration is used in the control.
  • the measured fluorescence values allow the slopes to be calculated: 0.0025 ⁇ fluo / sec for the control, 0.0361 ⁇ fluo / sec for Cdc25Bl, 0.0350 ⁇ fluo / sec for Cdc25B2 and 0.0372 ⁇ fluo / sec for Cdc25B3.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention concerns a method for obtaining human cdc 25B1, cdc25B2, cdcB3 and cdc25C phosphatases. More particularly, the invention concerns a protein for fusion between human cdc25B1, cdc25B2, cdcB3 or cdc25C phosphatase and the maltose binding protein (MBP) of Escherichia coli, the DNA sequence coding for said fusion protein, a method for preparing said fusion protein and a method for identifying human cdc25B1, cdc25B2, cdcB3 or cdc25C protein modulators.

Description

Méthode d'obtention de phosphatases humaines Cdc25 et méthode d'identification de modulateurs de phosphatases humaines Cdc25 Method for obtaining human phosphatases Cdc25 and method for identifying modulators of human phosphatases Cdc25
La présente invention concerne une méthode d'obtention de phosphatases humaines Cdc25. Elle concerne également une méthode d'identification de modulateurs de phosphatases humaines Cdc25.The present invention relates to a method for obtaining human phosphatases Cdc25. It also relates to a method of identifying modulators of human phosphatases Cdc25.
L'entrée de la cellule dans le processus de la division cellulaire est régulée par un ensemble de kinases et de phosphatases utiles pour synchroniser les différentes phases du cycle cellulaire et permettre la réorganisation de l'architecture cellulaire.The entry of the cell into the process of cell division is regulated by a set of kinases and phosphatases useful for synchronizing the different phases of the cell cycle and allowing the reorganization of the cell architecture.
Les kinases dépendantes des cyclines (CDKs) jouent un rôle majeur dans ce contrôle et déjà plusieurs inhibiteurs de cette famille de kinases sont identifiés. Un de ces composés (flavopiridol) est déjà en Phase II clinique (Senderowicz et Sausville, J. Natl. Cancer Inst. (2000), 92, 376-387).Cyclin-dependent kinases (CDKs) play a major role in this control and already several inhibitors of this family of kinases have been identified. One of these compounds (flavopiridol) is already in Phase II clinical (Senderowicz and Sausville, J. Natl. Cancer Inst. (2000), 92, 376-387).
Ces CDKs sont activées par une déphosphorylation effectuée par les phosphatases Cdc25, sur des résidus tyrosine et thréonine. Dans les cellules humaines, les protéines Cdc25 sont codées par une famille : Cdc25A, Cdc25B et Cdc25C (Cans et coll., Medicine Sciences (1998), 3, 269-274).These CDKs are activated by a dephosphorylation carried out by the phosphatases Cdc25, on tyrosine and threonine residues. In human cells, the Cdc25 proteins are coded by a family: Cdc25A, Cdc25B and Cdc25C (Cans et al., Medicine Sciences (1998), 3, 269-274).
Quelques inhibiteurs de Cdc25 sont identifiés, mais ils possèdent seulement une faible activité (cf. Baratte, B., Meijer, L., Galaktionov, K., et Beach, D., Anticancer Res. (1992), 12, 873-880 ; Rice, R.L. et coll., Biochemistry (1997), 36, 15965-15974 ; et Ham, S.W., Park, ., Lee, S.J., Kim, W., Kang, K., et Choi, K.H., Bioorg. Med. Chem. Lett. (1998), 8, 2507-2510).Some inhibitors of Cdc25 are identified, but they have only a weak activity (cf. Baratte, B., Meijer, L., Galaktionov, K., and Beach, D., Anticancer Res. (1992), 12, 873-880 ; Rice, RL et al., Biochemistry (1997), 36, 15965-15974; and Ham, SW, Park,., Lee, SJ, Kim, W., Kang, K., and Choi, KH, Bioorg. Med Chem. Lett. (1998), 8, 2507-2510).
Cdc25B2 est une protéine tyrosine phosphatase proche de la phosphatase Cdc25B (appelée maintenant Cdc25Bl). Elle a été identifiée dans une banque d'ADN codant de lymphome de Burkitt. Cdc25B2 diffère de Cdc25Bl par une insertion de 14 acides aminés et une délétion de 41 acides aminés en amont du domaine catalytique. Cdc25Bl et Cdc25B2 sont des variants d'épissage d'un même gêne. Un troisième variant, Cdc25B3, portant les deux séquences de 14 et 41 acides aminés, a été identifié à partir de la même banque (Baldin et coll., Oncogene (1997), 14, 2485-2495). Les trois variants sont détectés dans les primocultures et les lignées cellulaires. L'analyse des variants montre que Cdc25B2 est plus faiblement exprimé que Cdc25B3 dans l'ensemble des lignées testées mais que l'expression des deux variants augmente durant la phase G2 et la mitose (Forrest et coll., Biochem. Biophys. Res. Commun. (1999), 260, 510-515). Hernandez et coll. rapportent que Cdc25A et -B2 mais pas Cdc25Bl, -B3 et -C, sont sur-exprimés dans un grand nombre de lymphomes (35% et 39%) (Hernandez et coll., Int. J. Cancer (2000), 89(2), 148-52). Les lymphocytes normaux expriment les messagers de Cdc25Bl et -B3 et très faiblement ceux de Cdc 25 A, -B2 et -C.Cdc25B2 is a tyrosine phosphatase protein close to the Cdc25B phosphatase (now called Cdc25Bl). It has been identified in a DNA library encoding Burkitt's lymphoma. Cdc25B2 differs from Cdc25Bl by an insertion of 14 amino acids and a deletion of 41 amino acids upstream of the catalytic domain. Cdc25Bl and Cdc25B2 are splicing variants of the same gene. A third variant, Cdc25B3, carrying the two sequences of 14 and 41 amino acids, was identified from from the same bank (Baldin et al., Oncogene (1997), 14, 2485-2495). The three variants are detected in primocultures and cell lines. Analysis of the variants shows that Cdc25B2 is more weakly expressed than Cdc25B3 in all of the lines tested, but that the expression of the two variants increases during the G2 phase and mitosis (Forrest et al., Biochem. Biophys. Res. Commun . (1999), 260, 510-515). Hernandez et al. report that Cdc25A and -B2 but not Cdc25Bl, -B3 and -C, are over-expressed in a large number of lymphomas (35% and 39%) (Hernandez et al., Int. J. Cancer (2000), 89 ( 2), 148-52). Normal lymphocytes express the messengers of Cdc25Bl and -B3 and very weakly those of Cdc 25 A, -B2 and -C.
La surexpression des trois Cdc25B variants dans la levure montre que Cdc25B2 semble plus active que Bl ou B3 (B2>B3>B1). L'épissage alternatif de Cdc25B peut donc jouer un rôle dans le contrôle de la prolifération cellulaire.The overexpression of the three variant Cdc25B in yeast shows that Cdc25B2 seems more active than Bl or B3 (B2> B3> B1). The alternative splicing of Cdc25B can therefore play a role in the control of cell proliferation.
La phosphatase Cdc25C est par ailleurs elle même régulée par phosphorylation sur la sérine-216 par d'autres enzymes Cdsl ou Chkl et se lie à des membres très conservés de la famille des protéines 14-3-3 (Zeng, Y. et coll., Nature (1998), 395, 507-510).The Cdc25C phosphatase is itself regulated by phosphorylation on serine-216 by other enzymes Cdsl or Chkl and binds to very conserved members of the protein family 14-3-3 (Zeng, Y. et al. , Nature (1998), 395, 507-510).
La recherche d'inhibiteurs plus efficaces des phosphatases passe par la nécessité d'avoir une protéine qui conserve son activité phosphatase, en quantité non limitante pour autoriser des criblages à grande échelle.The search for more effective inhibitors of phosphatases requires the need for a protein which retains its phosphatase activity, in non-limiting amounts to allow large-scale screening.
L'étude d'une protéine nécessite de grandes quantités de cette dernière pour satisfaire à toutes les caractéristiques qui peuvent être analysées notamment dans les domaines tels que la biophysique (taille, séquence, structure...), de la biochimie (activité, stabilité, régulation...) ou de la pharmacologie (activateurs, inhibiteurs,...).The study of a protein requires large quantities of the latter to satisfy all the characteristics which can be analyzed in particular in fields such as biophysics (size, sequence, structure ...), biochemistry (activity, stability , regulation ...) or pharmacology (activators, inhibitors, ...).
La production et la purification de cette protéine en grande quantité se heurte à plusieurs obstacles qui peuvent être :The production and purification of this protein in large quantities faces several obstacles which may be:
- expression absente ou trop faible de la protéine ;- absent or too low expression of the protein;
- expression d'une protéine tronquée ; obtention d'une protéine sans activité biologique ;- expression of a truncated protein; obtaining a protein without biological activity;
- perte de l'activité biologique de la protéine durant la purification ;- loss of the biological activity of the protein during the purification;
- rendement de purification très faible ; - perte de l'activité biologique de la protéine durant la conservation ;- very low purification yield; - loss of the biological activity of the protein during storage;
- perte de la source de production.- loss of the source of production.
Seul le succès à chacune de ces étapes permet finalement de produire à long terme une protéine biologiquement active en quantité illimitée. Face à de telles exigences, la purification de nombreuses protéines reste un processus très délicat voire même impossible dans certains cas.Only success at each of these stages ultimately produces an unlimited quantity of biologically active protein in the long term. Faced with such demands, the purification of many proteins remains a very delicate process, even impossible in some cases.
Cependant, de nombreux efforts sont faits pour développer de nouvelles stratégies de production et de purification telles que :However, many efforts are made to develop new production and purification strategies such as:
- culture de cellules (animales ou végétales) à grande échelle ;- large-scale cell (animal or plant) culture;
- culture de microorganismes (bactéries, levures) à grande échelle ; immunoprécipitation à l'aide d'un anticorps spécifique à partir d'un mélange de protéines ; chromatographie d'affinité, à l'aide d'un ligand spécifique (effecteurs, répresseurs, activateurs) ; électrophorèse bidimensionnelle, en fonction de la masse moléculaire et du point isoélectrique de la protéine ; électrophorèse capillaire ; enrichissement par précipitation différentielle avec différents sels ;" - etc.- large-scale culture of microorganisms (bacteria, yeasts); immunoprecipitation using a specific antibody from a mixture of proteins; affinity chromatography, using a specific ligand (effectors, repressors, activators); two-dimensional electrophoresis, as a function of the molecular mass and the isoelectric point of the protein; capillary electrophoresis; enrichment by differential precipitation with different salts; "- etc.
Par ailleurs, de nouveaux systèmes essaient maintenant de combiner la production et la purification des protéines. Ces systèmes permettent une expression souvent inductible de la production d'une protéine recombinante fusionnée avec une protéine permettant une chromatographie d'affinité (appelée tag). Cette dernière partie peut être éliminée par l'addition d'une protease qui reconnaît spécifiquement l'endroit de la fusion (Sheibani, N., Prep. Biochem. Biotechnol. (1999), 29, 77-90). Le nombre de systèmes proposés augmentent mais le succès de ces différentes approches reste très variable en fonction des protéines à purifier. La conformation et la solubilité des protéines restent des paramètres impossibles à contrôler dans ces nouveaux systèmes (voir Guise, A.D., West, S.M. et Chaudhuri, J.B., Mol. Biotechnol. (1996), 6, 53-64 ; Kelley, R.F. et Winkler, M.E., Genêt. Eng. (N.Y., 1990), 12, 1-19).In addition, new systems are now trying to combine the production and purification of proteins. These systems allow an often inducible expression of the production of a recombinant protein fused with a protein allowing affinity chromatography (called tag). This last part can be eliminated by the addition of a protease which specifically recognizes the location of the fusion (Sheibani, N., Prep. Biochem. Biotechnol. (1999), 29, 77-90). The number of systems offered is increasing, but the success of these different approaches remains highly variable depending on the proteins to be purified. The conformation and solubility of proteins remain parameters that cannot be controlled in these new systems (see Guise, AD, West, SM and Chaudhuri, JB, Mol. Biotechnol. (1996), 6, 53-64; Kelley, RF and Winkler , ME, Genet Eng. (NY, 1990), 12, 1-19).
Les protéines Cdc25Bl, Cdc25B2, Cdc25B3 ou Cdc25C pourraient être fusionnées avec des systèmes aussi variés que :The proteins Cdc25Bl, Cdc25B2, Cdc25B3 or Cdc25C could be fused with systems as varied as:
1. Le résidu de six histidines reconnu par l'anticorps anti-motif six histidine (Katsafanas, G.C. et Moss, B., Virology (1999), 258, 469-479) ;1. The residue of six histidines recognized by the anti-motif motif six histidine (Katsafanas, G.C. and Moss, B., Virology (1999), 258, 469-479);
2. Le résidu de neuf acides aminés de Hemagglutinine d'Influenza reconnu par l'anticorps 3F10 (Robert, I. et Quirin-Stricker, C, J. Mol. Neurosci. (1998), 11, 243-251) ; 3. Le résidu de 11 acides aminés du virus de stomatite vésiculaire reconnu par l'anticorps P5D4 (Le Maout, S., et coll., Proc. Natl. Acad. Sci. U.S.A. (1997), 94, 13329-13334) ;2. The residue of nine amino acids of Hemagglutinin from Influenza recognized by the antibody 3F10 (Robert, I. and Quirin-Stricker, C, J. Mol. Neurosci. (1998), 11, 243-251); 3. The residue of 11 amino acids from the vesicular stomatitis virus recognized by the antibody P5D4 (Le Maout, S., et al., Proc. Natl. Acad. Sci. USA (1997), 94, 13329-13334);
4. Le résidu de 6 acides aminés de la protéine de capside du virus bovin papilloma (AU1) reconnu par l'anticorps anti-AUl (Le Maout, S., et coll., Proc. Natl. Acad.4. The 6 amino acid residue of the bovine papilloma virus (AU1) capsid protein recognized by the anti-AU1 antibody (Le Maout, S., et al., Proc. Natl. Acad.
Sci. U.S.A. (1997), 94, 13329-13334) ;Sci. U.S.A. (1997), 94, 13329-13334);
5. Le résidu de 12 acides aminés de la chaîne lourde de la protéine C reconnu par l'anticorps HPC4 (Rezaie, A.R., et coll., Protein Expr. Purif. (1992), 3, 453-460) ;5. The residue of 12 amino acids of the heavy chain of protein C recognized by the HPC4 antibody (Rezaie, A.R., et al., Protein Expr. Purif. (1992), 3, 453-460);
6. La protéine C-myc reconnue par l'anticorps 9E10 (Bae, S.H., et coll., J. Biol. Chem. (1999), 274, 14624-14631) ;6. The protein C-myc recognized by the antibody 9E10 (Bae, S.H., et al., J. Biol. Chem. (1999), 274, 14624-14631);
7. La protéine bêta galactosidase affine pour l'amino-phényl- β-D-thiogalactopyranoside (Germino, J. et Bastia, D., Proc. Natl. Acad. Sci. U.S.A. (1984), 81, 4692-4696) ;7. The beta galactosidase protein affine for the amino-phenyl-β-D-thiogalactopyranoside (Germino, J. and Bastia, D., Proc. Natl. Acad. Sci. U.S.A. (1984), 81, 4692-4696);
8. La protéine gluthation S-transférase reconnue par l'anticorps anti-GST (Carr, S., et coll., Vaccine (1999), 18, 153-159) ;8. The protein gluthation S-transferase recognized by the anti-GST antibody (Carr, S., et al., Vaccine (1999), 18, 153-159);
9. La biotin-carboxylase carrier affine pour l'avidine (Germino, F.j. et Moskowitz, N.K., Methods Enzymol. (1999), 303, 422-450) ;9. The biotin-carboxylase carrier affine for avidin (Germino, F.j. and Moskowitz, N.K., Methods Enzymol. (1999), 303, 422-450);
10. La protéine intéine affine pour la chitine (voir Chong, S., et coll., Gène (1997), 192, 271-281 ; Carr, S., et coll., Vaccine (1999), 18, 153-159) ; 11. La protéine liant le maltose affine pour l'amylose (Ahaded, A., et coll., Prep. Biochem. Biotechnol. (1999), 29, 163-176).10. The protein intein refines for chitin (see Chong, S., et al., Gene (1997), 192, 271-281; Carr, S., et al., Vaccine (1999), 18, 153-159 ); 11. The maltose-binding protein refines for amylose (Ahaded, A., et al., Prep. Biochem. Biotechnol. (1999), 29, 163-176).
Pour autant, rien ne permet de prédire si l'opération sera effectivement couronnée de succès.However, there is nothing to predict whether the operation will actually be successful.
La demanderesse vient cependant de mettre au point une méthode qui permet l'obtention des enzymes Cdc25Bl, Cdc25B2, Cdc25B3 et Cdc25C humaines sous une forme active et en quantité illimitée. La présente invention facilite d'une part la recherche et l'étude des activités physiologiques ou/et physio-pathologiques de cette protéine et d'autre part facilite la recherche d'agents modulateurs de ces activités.The Applicant has however just developed a method which makes it possible to obtain the enzymes Cdc25Bl, Cdc25B2, Cdc25B3 and Cdc25C human in an active form and in unlimited quantity. The present invention facilitates on the one hand the research and the study of the physiological or / and physiopathological activities of this protein and on the other hand facilitates the research of agents modulating these activities.
L'invention permet d'obtenir une protéine humaine Cdc25Bl, Cdc25B2, Cdc25B3 ou Cdc25C recombmée avec la protéine liant le maltose (MBP) qui conserve son activité phosphatase sans nécessiter la séparation avec la partie MBP, prévenant ainsi toute contamination avec des protéases. Le haut niveau du taux d'expression de la protéine après induction permet un rendement de purification excellent et la préparation en quantité illimitée de l'enzyme. L'invention a donc tout d'abord pour objet une protéine de fusion entre la protéine liant le maltose (MBP) et une protéine choisie parmi les protéines Cdc25Bl, Cdc25B2, Cdc25B3 et Cdc25C.The invention makes it possible to obtain a human protein Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C recombined with the maltose-binding protein (MBP) which retains its phosphatase activity without requiring separation with the MBP part, thus preventing any contamination with proteases. The high level of expression of the protein after induction allows an excellent purification yield and the preparation in unlimited quantity of the enzyme. The invention therefore firstly relates to a fusion protein between the maltose-binding protein (MBP) and a protein chosen from the proteins Cdc25Bl, Cdc25B2, Cdc25B3 and Cdc25C.
La présente invention concerne en particulier une protéine choisie parmi les protéines suivantes :The present invention relates in particular to a protein chosen from the following proteins:
- une protéine de fusion entre la phosphatase Cdc25Bl humaine et la MBP, laquelle est caractérisée en ce qu'elle est codée par la séquence SEQ. ID n° 12 (représentée plus loin) ;- A fusion protein between human Cdc25Bl phosphatase and MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 12 (shown below);
- une protéine de fusion entre la phosphatase Cdc25B2 humaine et la MBP, laquelle est caractérisée en ce qu'elle est codée par la séquence SEQ. ID n° 13 (représentée plus loin) ;- a fusion protein between human Cdc25B2 phosphatase and MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 13 (shown below);
- une protéine de fusion entre la phosphatase Cdc25B3 humaine et la MBP, laquelle est caractérisée en ce qu'elle est codée par la séquence SEQ. ID n° 14 (représentée plus loin) ; ou - une protéine de fusion entre la phosphatase Cdc25C humaine et la MBP, laquelle est caractérisée en ce qu'elle est codée par la séquence SEQ. ID n° 1 (représentée plus loin).- a fusion protein between human Cdc25B3 phosphatase and MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 14 (shown below); or - a fusion protein between human Cdc25C phosphatase and MBP, which is characterized in that it is coded by the sequence SEQ. ID # 1 (shown below).
Elle a également pour objet l'ADN codant pour lesdites protéines de fusion, ainsi que l'ADN complémentaire à l'ADN codant pour lesdites protéine de fusion.It also relates to DNA coding for said fusion proteins, as well as DNA complementary to DNA coding for said fusion proteins.
L'invention a de plus pour objet la souche bactérienne JM 109 transfectée : - par le plasmide de séquence SEQ. ID n° 9 (représentée plus loin), ladite souche étant utile à la préparation de la protéine Cdc25Bl ;The subject of the invention is also the bacterial strain JM 109 transfected: - with the plasmid of sequence SEQ. ID No. 9 (shown below), said strain being useful for the preparation of the protein Cdc25Bl;
- par le plasmide de séquence SEQ. ID n° 10 (représentée plus loin), ladite souche étant utile à la préparation de la protéine Cdc25B2 ;- by the plasmid of sequence SEQ. ID No. 10 (shown below), said strain being useful for the preparation of the protein Cdc25B2;
- par le plasmide de séquence SEQ. ID n° 11 (représentée plus loin), ladite souche étant utile à la préparation de la protéine Cdc25B3 ; ou- by the plasmid of sequence SEQ. ID No. 11 (shown below), said strain being useful for the preparation of the protein Cdc25B3; or
- par le plasmide de séquence SEQ. ID n° 5 (représentée plus loin), ladite souche étant utile à la préparation de la protéine Cdc25C.- by the plasmid of sequence SEQ. ID No. 5 (shown below), said strain being useful for the preparation of the protein Cdc25C.
L'invention concerne de plus un procédé de préparation desdites protéines de fusion, caractérisé en ce qu'il comporte les étapes successives suivantes : - culture de la souche bactérienne JM 109 transfectée par le plasmide de séquence SEQ. ID n° 9, le plasmide de séquence SEQ. ID n° 10, le plasmide de séquence SEQ. ID n° 11 ou le plasmide de séquence SEQ. ID n° 5, dans un milieu LB additionné d'ampicilline ;The invention further relates to a process for preparing said fusion proteins, characterized in that it comprises the following successive stages: - culture of the bacterial strain JM 109 transfected with the plasmid of sequence SEQ. ID No. 9, the plasmid with sequence SEQ. ID No. 10, the plasmid with sequence SEQ. ID No. 11 or the plasmid with sequence SEQ. ID No. 5, in LB medium supplemented with ampicillin;
- induction de la synthèse de la protéine de fusion par ajout d'isopropylthiogalactoside ;- induction of the synthesis of the fusion protein by addition of isopropylthiogalactoside;
- lyse des bactéries ; - purification de la protéine de fusion obtenue par chromatographie sur résine d'amylose-agarose et récupération des fractions contenant la protéine purifiée.- lysis of bacteria; - Purification of the fusion protein obtained by chromatography on amylose-agarose resin and recovery of the fractions containing the purified protein.
L'invention concerne enfin une application de ladite protéine de fusion à une méthode d'identification de modulateurs de la protéine Cdc25Bl, Cdc25B2, Cdc25B3 ou Cdc25C, caractérisée en ce qu'elle comporte les étapes successives suivantes :Finally, the invention relates to an application of said fusion protein to a method of identifying modulators of the protein Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C, characterized in that it comprises the following successive steps:
- ajout, à une solution de phosphate de 3-O-méthylfluorescéine, de la protéine de fusion telle qu'obtenue par le procédé de préparation décrit précédemment et d'un composé présumé être un modulateur de la protéine Cdc25Bl, Cdc25B2, Cdc25B3 ou Cdc25C ;- addition, to a 3-O-methylfluorescein phosphate solution, of the fusion protein as obtained by the preparation process described above and of a compound presumed to be a modulator of the protein Cdc25Bl, Cdc25B2, Cdc25B3 or Cdc25C ;
- détermination de la quantité de 3-O-méthylfluorescéine produite rapportée à la quantité initiale de phosphate de 3-O-méthylfluorescéine.- Determination of the amount of 3-O-methylfluorescein produced compared to the initial amount of 3-O-methylfluorescein phosphate.
La détermination de la quantité de 3-O-méthylfluorescéine produite peut être réalisée, par exemple, par mesure de la densité optique de la solution, de l'absorbance liée à la 3-O-méthylfluorescéine à la longueur d'onde de 477 nm, ou bien encore par fluorimétrie en utilisant une excitation à la longueur d'onde de 475 nm et une lecture à la longueur d'onde de 510 nm.The determination of the quantity of 3-O-methylfluorescein produced can be carried out, for example, by measuring the optical density of the solution, the absorbance linked to 3-O-methylfluorescein at the wavelength of 477 nm , or alternatively by fluorimetry using excitation at the wavelength of 475 nm and a reading at the wavelength of 510 nm.
A moins qu'ils ne soient définis d'une autre manière, tous les termes techniques et scientifiques utilisés ici ont la même signification que celle couramment comprise par un spécialiste ordinaire du domaine auquel appartient cette invention. De même, toutes les publications, demandes de brevets, tous les brevets et toutes autres références mentionnées ici sont incorporées par référence. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by an ordinary specialist in the field to which this invention belongs. Likewise, all publications, patent applications, all patents and all other references mentioned herein are incorporated by reference.
PARTIE EXPERIMENTALEEXPERIMENTAL PART
A / Protéine de fusion ..MBP-Cdç25Ç.:A / Fusion protein .. MBP-Cdç25Ç . :
1 - Construction du vecteur d'expression pour MBP-Cdc25 C.1 - Construction of the expression vector for MBP-Cdc25 C.
1. 1 - Principe du système utilisé.1. 1 - Principle of the system used.
Le système utilisé (New England Biolabs #800 - pMAL™ protein fusion et système de purification) est basé sur la production d'une protéine de fusion entre la protéine d'intérêt, ici la protéine Cdc25C humaine, et la protéine bactérienne MBP (Maltose- Binding Protein) X! Escherichia coli. Cette méthode permet de purifier en une étape la protéine de fusion grâce à l'affinité de la MBP pour le maltose.The system used (New England Biolabs # 800 - pMAL ™ protein fusion and purification system) is based on the production of a fusion protein between the protein of interest, here the human Cdc25C protein, and the bacterial protein MBP (Maltose - Binding Protein) X! Escherichia coli. This method makes it possible to purify the fusion protein in one step thanks to the affinity of MBP for maltose.
1.2 - Origine de l'ADNc de Cdc25C1.2 - Origin of the Cdc25C cDNA
L'ADN codant pour la phosphatase Cdc25C humaine correspond au numéro d'accession 4502706.The DNA coding for human Cdc25C phosphatase corresponds to accession number 4502706.
A partir de ce plasmide, l'ADN codant pour Cdc25C a été amplifié par réaction de polymérisation en chaîne (PCR) en utilisant les amorces C-XBAI SENS et C-XBAI ANTI-SENS (de séquences SEQ. ID. nos 3 et 4 respectivement) et introduit dans le vecteur pCDNA3-HA au site Xbal pour donner le vecteur pcDNA3-HA Cdc25C. L'insert Cdc25C / Xbal a été, à ce stade, séquence dans son intégralité.From this plasmid, the DNA encoding Cdc25C was amplified by polymerase chain reaction (PCR) using the primers C-XBAI SENS and C-XBAI ANTI-SENS (of sequences SEQ. ID. Nos . 3 and 4 respectively) and introduced into the vector pCDNA3-HA at the Xbal site to give the vector pcDNA3-HA Cdc25C. The Cdc25C / Xbal insert has been sequenced in its entirety at this stage.
L'amorce C-XBAI SENS a pour séquence la séquence SEQ. ID. n° 3 représentée ci-après :The primer C-XBAI SENS has the sequence SEQ. ID. n ° 3 represented below:
5 ' - GTTCTAGAAT GTCTAGAA CTCTTC - 3 '5 '- GTTCTAGAAT GTCTAGAA CTCTTC - 3'
L'amorce C-XBAI ANTI-SENS a pour séquence la séquence SEQ. ID. n° 4 représentée ci-après :The primer C-XBAI ANTI-SENS has the sequence SEQ. ID. n ° 4 represented below:
5' -GGCTCTGA GTTGCGC CGG - 3' 1.3 - Construction du vecteur pMAL - Hs Cdc25C5 '-GGCTCTGA GTTGCGC CGG - 3' 1.3 - Construction of the vector pMAL - Hs Cdc25C
Le vecteur pcDNA3-HA Cdc25C a été digéré par Xbal, l'insert Cdc25C / Xbal (1456 paires de bases) purifié et introduit dans le vecteur pMAL™-c2X (New England Biolabs, #800-76) au site Xbal, pour donner le vecteur pMAL - Hs Cdc25C (de séquence SEQ. ID n° 2). Ce vecteur permet la production d'une protéine de fusion MBP-Cdc25C de 868 acides aminés, à partir du promoteur bactérien Ptac inductible par l'isopropylthiogalactoside (IPTG).The vector pcDNA3-HA Cdc25C was digested with Xbal, the insert Cdc25C / Xbal (1456 base pairs) purified and introduced into the vector pMAL ™ -c2X (New England Biolabs, # 800-76) at the site Xbal, to give the vector pMAL - Hs Cdc25C (of sequence SEQ. ID No. 2). This vector allows the production of a fusion protein MBP-Cdc25C of 868 amino acids, from the bacterial promoter Ptac inducible by isopropylthiogalactoside (IPTG).
La séquence SEQ. ID. n° 2 de la protéine Hs Cdc25C avec ses extrémités de restriction XBAI est la suivante :The sequence SEQ. ID. n ° 2 of the protein Hs Cdc25C with its XBAI restriction ends is as follows:
TCTAGAATGT CTACGGAACT CTTCTCATCC ACAAGAGAGG AAGGAAGCTC TGGCTCAGGATCTAGAATGT CTACGGAACT CTTCTCATCC ACAAGAGAGG AAGGAAGCTC TGGCTCAGGA
CCCAGTTTTA GGTCTAATCA AAGGAAAATG TTAAACCTGC TCCTGGAGAG AGACACTTCCCCCAGTTTTA GGTCTAATCA AAGGAAAATG TTAAACCTGC TCCTGGAGAG AGACACTTCC
TTTACCGTCT GTCCAGATGT CCCTAGAACT CCAGTGGGC AATTTCTTGG TGATTCTGCATTTACCGTCT GTCCAGATGT CCCTAGAACT CCAGTGGGC AATTTCTTGG TGATTCTGCA
AACCTAAGCA TTTTGTCTGG AGGAACCCCA AAATGTTGCC TCGATCTTTC GAATCTTAGCAACCTAAGCA TTTTGTCTGG AGGAACCCCA AAATGTTGCC TCGATCTTTC GAATCTTAGC
AGTGGGGAGA TAACTGCCAC TCAGCTTACC ACTTCTGCAG ACCTTGATGA AACTGGTCACAGTGGGGAGA TAACTGCCAC TCAGCTTACC ACTTCTGCAG ACCTTGATGA AACTGGTCAC
CTGGATTCTT CAGGACTTCA GGAAGTGCAT TTAGCTGGGA TGAATCATGA CCAGCACCTACTGGATTCTT CAGGACTTCA GGAAGTGCAT TTAGCTGGGA TGAATCATGA CCAGCACCTA
ATGAAATGTA GCCCAGCACA GCTTCTTTGT AGCACTCCGA ATGGTTTGGA CCGTGGCCATATGAAATGTA GCCCAGCACA GCTTCTTTGT AGCACTCCGA ATGGTTTGGA CCGTGGCCAT
AGAAAGAGAG ATGCAATGTG TAGTTCATCT GCAAATAAAG AAAATGACAA TGGAAACTTGAGAAAGAGAG ATGCAATGTG TAGTTCATCT GCAAATAAAG AAAATGACAA TGGAAACTTG
GTGGACAGTG AAATGAAATA TTTGGGCAGT CCCATTACTA CTGTTCCAAA ATTGGATAAAGTGGACAGTG AAATGAAATA TTTGGGCAGT CCCATTACTA CTGTTCCAAA ATTGGATAAA
AATCCAAACC TAGGAGAAGA CCAGGCAGAA GAGATTTCAG ATGAATTAAT GGAGTTTTCCAATCCAAACC TAGGAGAAGA CCAGGCAGAA GAGATTTCAG ATGAATTAAT GGAGTTTTCC
CTGAAAGATC AAGAAGCAAA GGTGAGCAGA AGTGGCCTAT ATCGCTCCCC GTCGATGCCACTGAAAGATC AAGAAGCAAA GGTGAGCAGA AGTGGCCTAT ATCGCTCCCC GTCGATGCCA
GAGAACTTGA ACAGGCCAAG ACTGAAGCAG GTGGAAAAAT TCAAGGACAA CACAATACCAGAGAACTTGA ACAGGCCAAG ACTGAAGCAG GTGGAAAAAT TCAAGGACAA CACAATACCA
GATAAAGTTA AAAAAAAGTA TTTTTCTGGC CAAGGAAAGC TCAGGAAGGG CTTATGTTTAGATAAAGTTA AAAAAAAGTA TTTTTCTGGC CAAGGAAAGC TCAGGAAGGG CTTATGTTTA
AAGAAGACAG TCTCTCTGTG TGACATTACT ATCACTCAGA TGCTGGAGGA AGATTCTAACAAGAAGACAG TCTCTCTGTG TGACATTACT ATCACTCAGA TGCTGGAGGA AGATTCTAAC
CAGGGGCACC TGATTGGTGA TTTTTCCAAG GTATGTGCGC TGCCAACCGT GTCAGGGAAACAGGGGCACC TGATTGGTGA TTTTTCCAAG GTATGTGCGC TGCCAACCGT GTCAGGGAAA
CACCAAGATC TGAAGTATGT CAACCCAGAA ACAGTGGCTG CCTTACTGTC GGGGAAGTTCCACCAAGATC TGAAGTATGT CAACCCAGAA ACAGTGGCTG CCTTACTGTC GGGGAAGTTC
CAGGGTCTGA TTGAGAAGTT TTATGTCATT GATTGTCGCT ATCCATATGA GTATCTGGGACAGGGTCTGA TTGAGAAGTT TTATGTCATT GATTGTCGCT ATCCATATGA GTATCTGGGA
GGACACATCC AGGGAGCCTT AAACTTATAT AGTCAGGAAG AACTGTTTAA CTTCTTTCTGGGACACATCC AGGGAGCCTT AAACTTATAT AGTCAGGAAG AACTGTTTAA CTTCTTTCTG
AAGAAGCCCA TCGTCCCTTT GGACACCCAG AAGAGAATAA TCATCGTGTT CCACTGTGAAAAGAAGCCCA TCGTCCCTTT GGACACCCAG AAGAGAATAA TCATCGTGTT CCACTGTGAA
TTCTCCTCAG AGAGGGGCCC CCGAATGTGC CGCTGTCTGC GTGAAGAGGA CAGGTCTCTGTTCTCCTCAG AGAGGGGCCC CCGAATGTGC CGCTGTCTGC GTGAAGAGGA CAGGTCTCTG
AACCAGTATC CTGCATTGTA CTACCCAGAG CTATATATCC TTAAAGGCGG CTACAGAGACAACCAGTATC CTGCATTGTA CTACCCAGAG CTATATATCC TTAAAGGCGG CTACAGAGAC
TTCTTTCCAG AATATATGGA ACTGTGTGAA CCACAGAGCT ACTGCCCTAT GCATCATCAGTTCTTTCCAG AATATATGGA ACTGTGTGAA CCACAGAGCT ACTGCCCTAT GCATCATCAG
GACCACAAGA CTGAGTTGCT GAGGTGTCGA AGCCAGAGCA AAGTGCAGGA AGGGGAGCGGGACCACAAGA CTGAGTTGCT GAGGTGTCGA AGCCAGAGCA AAGTGCAGGA AGGGGAGCGG
.ÇAGÇTGÇGGG ... AGCAGATTGC ..ÇÇTTÇTGGTG....AAGGAÇATGA ... GCCÇATGATA ACATTCCAGC CACTGGCTGC TAACATCTAG A (les sites XBAI sont soulignés en continu, le cadre de lecture ouverte (ou ORF pour Open Reading Frame) de Cdc25C est souligné en pointillé) . ÇAGÇTGÇGGG ... AGCAGATTGC .. ÇÇTTÇTGGTG .... AAGGAÇATGA ... GCCÇATGATA ACATTCCAGC CACTGGCTGC TAACATCTAG A (the XBAI sites are underlined continuously, the open reading frame (or ORF for Open Reading Frame) of Cdc25C is underlined in dotted lines)
1.4 - Création de la souche JM109 / pMAL - Hs Cdc25C1.4 - Creation of the strain JM109 / pMAL - Hs Cdc25C
Le vecteur pMAL - Hs Cdc25C a été introduit dans la souche d 'Escherichia coli (E. coli) JM109 (Stratagene #200271). Une colonie isolée a été sélectionnée et la production d'une protéine de poids moléculaire apparent théorique de 97 kDa après culture en présence d'IPTG a été observée par l'analyse des protéines bactériennes totales sur gel de polyacrylamide dénaturant et coloration au bleu de Coomassie. L'identité de la protéine de fusion a été ensuite confirmée par western blot et immunodétection avec un anticorps anti-Cdc25C.The vector pMAL - Hs Cdc25C was introduced into the strain of Escherichia coli (E. coli) JM109 (Stratagene # 200271). An isolated colony was selected and the production of a protein of theoretical apparent molecular weight of 97 kDa after culture in the presence of IPTG was observed by the analysis of total bacterial proteins on denaturing polyacrylamide gel and staining with Coomassie blue. . The identity of the fusion protein was then confirmed by western blot and immunodetection with an anti-Cdc25C antibody.
Finalement, l'ADN plasmidique isolé de ce clone a été séquence dans la région correspondant à Cdc25C pour vérifier l'absence de mutations ou de modifications de la séquence qui auraient pu être générées au cours des processus de sous-clonage et/ou de transformation de l'ADN.Finally, the plasmid DNA isolated from this clone was sequenced in the region corresponding to Cdc25C to verify the absence of mutations or modifications of the sequence which could have been generated during the subcloning and / or transformation processes. DNA.
La séquence suivante (désignée par SΕQ. ID. n° 5) a été obtenue pour la souche JM109 / pMAL - Hs Cdc25C :The following sequence (designated by SΕQ. ID. N ° 5) was obtained for the strain JM109 / pMAL - Hs Cdc25C:
CCGACACCAT CGAATGGTGC AAAACCTTTC GCGGTATGGC ATGATAGCGC CCGGAAGAGACCGACACCAT CGAATGGTGC AAAACCTTTC GCGGTATGGC ATGATAGCGC CCGGAAGAGA
GTCAATTCAG GGTGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCGGTCAATTCAG GGTGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG
GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCACGTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCAC
AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGCAACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGC
ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCGACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCG
TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATCTGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATC
TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGATTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGA
CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATCCACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATC
TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGGTGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGG
CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAGCGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAG
CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAACGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAA
TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACGTGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG
ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTCACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTC
GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC CAGGCGGTGAGCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC CAGGCGGTGA
AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG GCGCCCAATA WO 01/44467 . ιo - PCTFR00/03496AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG GCGCCCAATA WO 01/44467. ιo - PCTFR00 / 03496
CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTTCGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTT
CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT CACTCATTAGCCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT CACTCATTAG
GCACAATTCT CATGTTTGAC AGCTTATCAT CGACTGCACG GTGCACCAAT GCTTCTGGCGGCACAATTCT CATGTTTGAC AGCTTATCAT CGACTGCACG GTGCACCAAT GCTTCTGGCG
TCAGGCAGCC ATCGGAAGCT GTGGTATGGC TGTGCAGGTC GTAAATCACT GCATAATTCGTCAGGCAGCC ATCGGAAGCT GTGGTATGGC TGTGCAGGTC GTAAATCACT GCATAATTCG
TGTCGCTCAA GGCGCACTCC CGTTCTGGAT AATGTTTTTT GCGCCGACAT CATAACGGTTTGTCGCTCAA GGCGCACTCC CGTTCTGGAT AATGTTTTTT GCGCCGACAT CATAACGGTT
CTGGCAAATA TTCTGAAATG AGCTGTTGAC AATTAATCAT CGGCTCGTAT AATGTGTGGACTGGCAAATA TTCTGAAATG AGCTGTTGAC AATTAATCAT CGGCTCGTAT AATGTGTGGA
ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCCAGT CCGTTTAGGT GTTTTCACGAATTGTGAGCG GATAACAATT TCACACAGGA AACAGCCAGT CCGTTTAGGT GTTTTCACGA
GCACTTCACC AACAAGGACC ATAGATTATG AAAATCGAAG AAGGTAAACT GGTAATCTGGGCACTTCACC AACAAGGACC ATAGATTATG AAAATCGAAG AAGGTAAACT GGTAATCTGG
ATTAACGGCG ATAAAGGCTA TAACGGTCTC GCTGAAGTCG GTAAGAAATT CGAGAAAGATATTAACGGCG ATAAAGGCTA TAACGGTCTC GCTGAAGTCG GTAAGAAATT CGAGAAAGAT
ACCGGAATTA AAGTCACCGT TGAGCATCCG GATAAACTGG AAGAGAAATT CCCACAGGTTACCGGAATTA AAGTCACCGT TGAGCATCCG GATAAACTGG AAGAGAAATT CCCACAGGTT
GCGGCAACTG GCGATGGCCC TGACATTATC TTCTGGGCAC ACGACCGCTT TGGTGGCTACGCGGCAACTG GCGATGGCCC TGACATTATC TTCTGGGCAC ACGACCGCTT TGGTGGCTAC
GCTCAATCTG GCCTGTTGGC TGAAATCACC CCGGACAAAG CGTTCCAGGA CAAGCTGTATGCTCAATCTG GCCTGTTGGC TGAAATCACC CCGGACAAAG CGTTCCAGGA CAAGCTGTAT
CCGTTTACCT GGGATGCCGT ACGTTACAAC GGCAAGCTGA TTGCTTACCC GATCGCTGTTCCGTTTACCT GGGATGCCGT ACGTTACAAC GGCAAGCTGA TTGCTTACCC GATCGCTGTT
GAAGCGTTAT CGCTGATTTA TAACAAAGAT CTGCTGCCGA ACCCGCCAAA AACCTGGGAAGAAGCGTTAT CGCTGATTTA TAACAAAGAT CTGCTGCCGA ACCCGCCAAA AACCTGGGAA
GAGATCCCGG CGCTGGATAA AGAACTGAAA GCGAAAGGTA AGAGCGCGCT GATGTTCAACGAGATCCCGG CGCTGGATAA AGAACTGAAA GCGAAAGGTA AGAGCGCGCT GATGTTCAAC
CTGCAAGAAC CGTACTTCAC CTGGCCGCTG ATTGCTGCTG ACGGGGGTTA TGCGTTCAAGCTGCAAGAAC CGTACTTCAC CTGGCCGCTG ATTGCTGCTG ACGGGGGTTA TGCGTTCAAG
TATGAAAACG GCAAGTACGA CATTAAAGAC GTGGGCGTGG ATAACGCTGG CGCGAAAGCGTATGAAAACG GCAAGTACGA CATTAAAGAC GTGGGCGTGG ATAACGCTGG CGCGAAAGCG
GGTCTGACCT TCCTGGTTGA CCTGATTAAA AACAAACACA TGAATGCAGA CACCGATTACGGTCTGACCT TCCTGGTTGA CCTGATTAAA AACAAACACA TGAATGCAGA CACCGATTAC
TCCATCGCAG AAGCTGCCTT TAATAAAGGC GAAACAGCGA TGACCATCAA CGGCCCGTGGTCCATCGCAG AAGCTGCCTT TAATAAAGGC GAAACAGCGA TGACCATCAA CGGCCCGTGG
GCATGGTCCA ACATCGACAC CAGCAAAGTG AATTATGGTG TAACGGTACT GCCGACCTTCGCATGGTCCA ACATCGACAC CAGCAAAGTG AATTATGGTG TAACGGTACT GCCGACCTTC
AAGGGTCAAC CATCCAAACC GTTCGTTGGC GTGCTGAGCG CAGGTATTAA CGCCGCCAGTAAGGGTCAAC CATCCAAACC GTTCGTTGGC GTGCTGAGCG CAGGTATTAA CGCCGCCAGT
CCGAACAAAG AGCTGGCAAA AGAGTTCCTC GAAAACTATC TGCTGACTGA TGAAGGTCTGCCGAACAAAG AGCTGGCAAA AGAGTTCCTC GAAAACTATC TGCTGACTGA TGAAGGTCTG
GAAGCGGTTA ATAAAGACAA ACCGCTGGGT GCCGTAGCGC TGAAGTCTTA CGAGGAAGAGGAAGCGGTTA ATAAAGACAA ACCGCTGGGT GCCGTAGCGC TGAAGTCTTA CGAGGAAGAG
TTGGCGAAAG ATCCACGTAT TGCCGCCACC ATGGAAAACG CCCAGAAAGG TGAAATCATGTTGGCGAAAG ATCCACGTAT TGCCGCCACC ATGGAAAACG CCCAGAAAGG TGAAATCATG
CCGAACATCC CGCAGATGTC CGCTTTCTGG TATGCCGTGC GTACTGCGGT GATCAACGCCCCGAACATCC CGCAGATGTC CGCTTTCTGG TATGCCGTGC GTACTGCGGT GATCAACGCC
GCCAGCGGTC GTCAGACTGT CGATGAAGCC CTGAAAGACG CGCAGACTAA TTCGAGCTCGGCCAGCGGTC GTCAGACTGT CGATGAAGCC CTGAAAGACG CGCAGACTAA TTCGAGCTCG
AACAACAACA ACAATAACAA TAACAACAAC CTCGGGATCG AGGGAAGGAT TTCAGAATTCAACAACAACA ACAATAACAA TAACAACAAC CTCGGGATCG AGGGAAGGAT TTCAGAATTC
GGATCCTCTA GAATGTCTAC GGAAÇTÇTTC TCATÇÇACA .. GAGAGGAAGG ... AAGCTCTGGCGGATCCTCTA GAATGTCTAC GGAAÇTÇTTC TCATÇÇACA .. GAGAGGAAGG ... AAGCTCTGGC
TÇAGGAÇCCA.. GTTTTAGGTÇ TAATÇAAAGG AAAATGTTAA... ACCTGÇTÇÇT ... GGAGAGAG.AC ACT ÇCTTT . ÇÇGTÇTGTÇÇ AGATGTÇÇÇT AGAACTCCAG... TGGGCAAATT.... TCTTGGTG T CTGCAAACC TAAGCATTTT. GTÇ.TGGAGGA AÇCÇÇ AAAT . GTTGÇÇTCGA ... ÇTTTCGAATTÇAGGAÇCCA .. GTTTTAGGTÇ TAATÇAAAGG AAAATGTTAA ... ACCTGÇTÇÇT ... GGAGAGAG . AC ACT ÇCTTT . ÇÇGTÇTGTÇÇ AGATGTÇÇÇT AGAACTCCAG ... TGGGCAAATT .... TCTTGGTG T CTGCAAACC TAAGCATTTT . GTÇ . TGGAGGA AÇCÇÇ AAAT . GTTGÇÇTCGA ... ÇTTTCGAAT
ÇTTAGÇAGTG ... GGGAGATAAÇ....TGCCACTÇAG CTTAÇÇAÇTT ÇTGCAGAÇCT . TGATGAAAÇTÇTTAGÇAGTG ... GGGAGATAAÇ .... TGCCACTÇAG CTTAÇÇAÇTT ÇTGCAGAÇCT . TGATGAAAÇT
GGTÇAÇCTGG . ATTCTTÇAGG ACTTÇ.AGGAA GTGÇATTTAG... CTGGGATGAA . TCATGAÇCAGGGTÇAÇCTGG . ATTCTTÇAGG ACTTÇ . AGGAA GTGÇATTTAG ... CTGGGATGAA . TCATGAÇCAG
ÇAÇCTAATG AATGTAGÇ Ç... AGCACAGCTT.. CTTTGTAGÇA .. ÇTCÇGAATGG .. TTTGGACCGT GGÇÇATAGAA AGAGAGATGÇ ... AATGTG.TAG TCATCTGCAA .. AT AAGA^_AA.....TGACAATGGAÇAÇCTAATG AATGTAGÇ Ç ... AGCACAGCTT .. CTTTGTAGÇA .. ÇTCÇGAATGG .. TTTGGACCGT GGÇÇATAGAA AGAGAGATGÇ ... AATGTG . TAG TCATCTGCAA .. AT AAGA ^ _AA ..... TGACAATGGA
AAÇTTGGTGG ACAGTGAAAT . GAAATATTTG GGCAGTCCCA .. T.TACTAÇTGT..... CCAAAAT.TGAAÇTTGGTGG ACAGTGAAAT . GAAATATTTG GGCAGTCCCA .. T. TACTAÇTGT ..... CCAAAAT . TG
GATAAAAATC CAAAC.CTAGG AGAAGAÇÇAG GCAGAAGAGA... .TTCAGATGA....ATTAATGGAGGATAAAAATC CAAAC . CTAGG AGAAGAÇÇAG GCAGAAGAGA .... TTCAGATGA .... ATTAATGGAG
TTTTCCCTGA AAGATCAAGA AGCAAAGGTG AGCAGAAGTG GCCTATATCG CTCCCCGTCG ATGCCAGAGA ACTTGAACAG GCCAAGACTG AAGCAGGTGG AAAAATTCAA GGACAACACATTTTCCCTGA AAGATCAAGA AGCAAAGGTG AGCAGAAGTG GCCTATATCG CTCCCCGTCG ATGCCAGAGA ACTTGAACAG GCCAAGACTG AAGCAGGTGG AAAAATTCAA GGACAACACA
ATACCAGATA AAGTTAAAAA AAAGTATTTT TCTGGCCAAG GAAAGCTCAG GAAGGGCTTAATACCAGATA AAGTTAAAAA AAAGTATTTT TCTGGCCAAG GAAAGCTCAG GAAGGGCTTA
TGTTTAAAGA AGACAGTCTC TCTGTGTGAC ATTACTATCA CTCAGATGCT GGAGGAAGATTGTTTAAAGA AGACAGTCTC TCTGTGTGAC ATTACTATCA CTCAGATGCT GGAGGAAGAT
TCTAACCAGG GGCACCTGAT TGGTGATTTT TCCAAGGTAT GTGCGCTGCC AACCGTGTCA GGGAAACACC AAGATCTGAA GTATGTCAAC CCAGAAACAG TGGCTGCCTT ACTGTCGGGGTCTAACCAGG GGCACCTGAT TGGTGATTTT TCCAAGGTAT GTGCGCTGCC AACCGTGTCA GGGAAACACC AAGATCTGAA GTATGTCAAC CCAGAAACAG TGGCTGCCTT ACTGTCGGGG
AAGTTCCAGG GTCTGATTGA GAAGTTTTAT GTCATTGATT GTCGCTATCC ATATGAGTATAAGTTCCAGG GTCTGATTGA GAAGTTTTAT GTCATTGATT GTCGCTATCC ATATGAGTAT
CTGGGAGGAC ACATCCAGGG AGCCTTAAAC TTATATAGTC AGGAAGAACT GTTTAACTTCCTGGGAGGAC ACATCCAGGG AGCCTTAAAC TTATATAGTC AGGAAGAACT GTTTAACTTC
TTTCTGAAGA AGCCCATCGT CCCTTTGGAC ACCCAGAAGA GAATAATCAT CGTGTTCCACTTTCTGAAGA AGCCCATCGT CCCTTTGGAC ACCCAGAAGA GAATAATCAT CGTGTTCCAC
TGTGAATTCT CCTCAGAGAG GGGCCCCCGA ATGTGCCGCT GTCTGCGTGA AGAGGACAGG TCTCTGAACC AGTATCCTGC ATTGTACTAC CCAGAGCTAT ATATCCTTAA AGGCGGCTACTGTGAATTCT CCTCAGAGAG GGGCCCCCGA ATGTGCCGCT GTCTGCGTGA AGAGGACAGG TCTCTGAACC AGTATCCTGC ATTGTACTAC CCAGAGCTAT ATATCCTTAA AGGCGGCTAC
AGAGACTTCT TTCCAGAATA TATGGAACTG TGTGAACCAC AGAGCTACTG CCCTATGCATAGAGACTTCT TTCCAGAATA TATGGAACTG TGTGAACCAC AGAGCTACTG CCCTATGCAT
CATCAGGACC ACAAGACTGA GTTGCTGAGG TGTCGAAGCC AGAGCAAAGT GCAGGAAGGGCATCAGGACC ACAAGACTGA GTTGCTGAGG TGTCGAAGCC AGAGCAAAGT GCAGGAAGGG
GAGCGGCAGC TGCGGGAGCA GATTGCCCTT CTGGTGAAGG ACATGAGCCC ATGATAACATGAGCGGCAGC TGCGGGAGCA GATTGCCCTT CTGGTGAAGG ACATGAGCCC ATGATAACAT
TCCAGCCACT GGCTGCTAAC ATCTAGAGTC GACCTGCAGG CAAGCTTGGC ACTGGCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCCT GGCGTTACCC AACTTAATCG CCTTGCAGCATCCAGCCACT GGCTGCTAAC ATCTAGAGTC GACCTGCAGG CAAGCTTGGC ACTGGCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCCT GGCGTTACCC AACTTAATCG CCTTGCAGCA
CATCCCCCTT TCGCCAGCTG GCGTAATAGC GAAGAGGCCC GCACCGATCG CCCTTCCCAACATCCCCCTT TCGCCAGCTG GCGTAATAGC GAAGAGGCCC GCACCGATCG CCCTTCCCAA
CAGTTGCGCA GCCTGAATGG CGAATGGCAG CTTGGCTGTT TTGGCGGATG AGATAAGATTCAGTTGCGCA GCCTGAATGG CGAATGGCAG CTTGGCTGTT TTGGCGGATG AGATAAGATT
TTCAGCCTGA TACAGATTAA ATCAGAACGC AGAAGCGGTC TGATAAAACA GAATTTGCCTTTCAGCCTGA TACAGATTAA ATCAGAACGC AGAAGCGGTC TGATAAAACA GAATTTGCCT
GGCGGCAGTA GCGCGGTGGT CCCACCTGAC CCCATGCCGA ACTCAGAAGT GAAACGCCGT AGCGCCGATG GTAGTGTGGG GTCTCCCCAT GCGAGAGTAG GGAACTGCCA GGCATCAAATGGCGGCAGTA GCGCGGTGGT CCCACCTGAC CCCATGCCGA ACTCAGAAGT GAAACGCCGT AGCGCCGATG GTAGTGTGGG GTCTCCCCAT GCGAGAGTAG GGAACTGCCA GGCATCAAAT
AAAACGAAAG GCTCAGTCGA AAGACTGGGC CTTTCGTTTT ATCTGTTGTT TGTCGGTGAAAAAACGAAAG GCTCAGTCGA AAGACTGGGC CTTTCGTTTT ATCTGTTGTT TGTCGGTGAA
CGCTCTCCTG AGTAGGACAA ATCCGCCGGG AGCGGATTTG AACGTTGCGA AGCAACGGCCCGCTCTCCTG AGTAGGACAA ATCCGCCGGG AGCGGATTTG AACGTTGCGA AGCAACGGCC
CGGAGGGTGG CGGGCAGGAC GCCCGCCATA AACTGCCAGG CATCAAATTA AGCAGAAGGCCGGAGGGTGG CGGGCAGGAC GCCCGCCATA AACTGCCAGG CATCAAATTA AGCAGAAGGC
CATCCTGACG GATGGCCTTT TTGCGTTTCT ACAAACTCTT TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGACATCCTGACG GATGGCCTTT TTGCGTTTCT ACAAACTCTT TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA
AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCAAAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA
TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGATTTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT
CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAGCAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG
AGTTTTCGCC CCGAAGAACG TTCTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC GCGGTATTAT CCCGTGTTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCTAGTTTTCGCC CCGAAGAACG TTCTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC GCGGTATTAT CCCGTGTTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT
CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACACAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA
GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTTGTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT
CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCATCTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT
GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTAGTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA
CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGACTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGA
CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGTCCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT
GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCTGAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCCCAG ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT
GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG ACCAAGTTTA CTCATATATAGAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG ACCAAGTTTA CTCATATATA
CTTTAGATTG ATTTACCCCG GTTGATAATC AGAAAAGCCC CAAAAACAGG AAGATTGTATCTTTAGATTG ATTTACCCCG GTTGATAATC AGAAAAGCCC CAAAAACAGG AAGATTGTAT
AAGCAAATAT TTAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAGAAGCAAATAT TTAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG
AATAGCCCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGAAATAGCCCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA
ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTGACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG
AACCATCACC CAAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACCAACCATCACC CAAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC
CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGCCTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC
GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTAA AAGGATCTAGGCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTAA AAGGATCTAG
GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCACGTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC
TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGCTGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC
GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAATGTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT
ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCTACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT
ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGTACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT
CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACGCTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG
GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCGGGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG
GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGGGTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG
TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGCTATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC
TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTGTCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG
GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC GCAGCCGAAC GACCGAGCGCGCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC GCAGCCGAAC GACCGAGCGC
AGCGAGTCAG TGAGCGAGGA AGCGGAAGAG CGCCTGATGC GGTATTTTCT CCTTACGCATAGCGAGTCAG TGAGCGAGGA AGCGGAAGAG CGCCTGATGC GGTATTTTCT CCTTACGCAT
CTGTGCGGTA TTTCACACCG CATATGGTGC ACTCTCAGTA CAATCTGCTC TGATGCCGCACTGTGCGGTA TTTCACACCG CATATGGTGC ACTCTCAGTA CAATCTGCTC TGATGCCGCA
TAGTTAAGCC AGTATACACT CCGCTATCGC TACGTGACTG GGTCATGGCT GCGCCCCGACTAGTTAAGCC AGTATACACT CCGCTATCGC TACGTGACTG GGTCATGGCT GCGCCCCGAC
ACCCGCCAAC ACCCGCTGAC GCGCCCTGAC GGGCTTGTCT GCTCCCGGCA TCCGCTTACA GACAAGCTGT GACCGTCTCC GGGAGCTGCA TGTGTCAGAG GTTTTCACCG TCATCACCGAACCCGCCAAC ACCCGCTGAC GCGCCCTGAC GGGCTTGTCT GCTCCCGGCA TCCGCTTACA GACAAGCTGT GACCGTCTCC GGGAGCTGCA TGTGTCAGAG GTTTTCACCG TCATCACCGA
AACGCGCGAG GCAGCTGCGG TAAAGCTCAT CAGCGTGGTC GTGCAGCGAT TCACAGATGTAACGCGCGAG GCAGCTGCGG TAAAGCTCAT CAGCGTGGTC GTGCAGCGAT TCACAGATGT
CTGCCTGTTC ATCCGCGTCC AGCTCGTTGA GTTTCTCCAG AAGCGTTAAT GTCTGGCTTCCTGCCTGTTC ATCCGCGTCC AGCTCGTTGA GTTTCTCCAG AAGCGTTAAT GTCTGGCTTC
TGATAAAGCG GGCCATGTTA AGGGCGGTTT TTTCCTGTTT GGTCACTTGA TGCCTCCGTGTGATAAAGCG GGCCATGTTA AGGGCGGTTT TTTCCTGTTT GGTCACTTGA TGCCTCCGTG
TAAGGGGGAA TTTCTGTTCA TGGGGGTAAT GATACCGATG AAACGAGAGA GGATGCTCAC GATACGGGTT ACTGATGATG AACATGCCCG GTTACTGGAA CGTTGTGAGG GTAAACAACTTAAGGGGGAA TTTCTGTTCA TGGGGGTAAT GATACCGATG AAACGAGAGA GGATGCTCAC GATACGGGTT ACTGATGATG AACATGCCCG GTTACTGGAA CGTTGTGAGG GTAAACAACT
GGCGGTATGG ATGCGGCGGG ACCAGAGAAA AATCACTCAG GGTCAATGCC AGCGCTTCGTGGCGGTATGG ATGCGGCGGG ACCAGAGAAA AATCACTCAG GGTCAATGCC AGCGCTTCGT
TAATACAGAT GTAGGTGTTC CACAGGGTAG CCAGCAGCAT CCTGCGATGC AGATCCGGAATAATACAGAT GTAGGTGTTC CACAGGGTAG CCAGCAGCAT CCTGCGATGC AGATCCGGAA
CATAATGGTG CAGGGCGCTG ACTTCCGCGT TTCCAGACTT TACGAAACAC GGAAACCGAA GACCATTCAT GTTGTTGCTC AGGTCGCAGA CGTTTTGCAG CAGCAGTCGC TTCACGTTCGCATAATGGTG CAGGGCGCTG ACTTCCGCGT TTCCAGACTT TACGAAACAC GGAAACCGAA GACCATTCAT GTTGTTGCTC AGGTCGCAGA CGTTTTGCAG CAGCAGTCGC TTCACGTTCG
CTCGCGTATC GGTGATTCAT TCTGCTAACC AGTAAGGCAA CCCCGCCAGC CTAGCCGGGTCTCGCGTATC GGTGATTCAT TCTGCTAACC AGTAAGGCAA CCCCGCCAGC CTAGCCGGGT
CCTCAACGAC AGGAGCACGA TCATGCGCAC CCGTGGCCAG GACCCAACGC TGCCCGAAAT TCCTCAACGAC AGGAGCACGA TCATGCGCAC CCGTGGCCAG GACCCAACGC TGCCCGAAAT T
(les sites XBAI sont soulignés par une simple ligne continue, l'ORF de Cdc25C est souligné en pointillé, la séquence de la protéine liant le maltose (MBP) est soulignée par une double ligne continue et la séquence en gras correspond à la région de l'ADN plasmidique isolé à partir de la souche JM 109 et séquencée).(the XBAI sites are underlined by a single solid line, the ORF of Cdc25C is underlined in dotted lines, the maltose-binding protein sequence (MBP) is underlined by a solid double line and the bold sequence corresponds to the region plasmid DNA isolated from the JM 109 strain and sequenced).
Ce clone peut être conservé à -80 °C sous forme de culture saturée additionnée de glycérol (concentration finale 25%) ou " stock glycérol " . Cette souche sera utilisée pour toutes les étapes de production ultérieures.This clone can be stored at -80 ° C. in the form of a saturated culture added with glycerol (final concentration 25%) or "glycerol stock". This strain will be used for all subsequent production stages.
2 - Production et purification de la protéine recombinante MBP-Cdc25C :2 - Production and purification of the recombinant protein MBP-Cdc25C:
NB : sauf indication contraire, tous les réactifs chimiques proviennent de SIGMA-ALDRICH.NB: unless otherwise indicated, all chemical reagents come from SIGMA-ALDRICH.
2.1 - Culture bactérienne et induction d'expression de la protéine de fusion2.1 - Bacterial culture and induction of expression of the fusion protein
50 ml de milieu LB + ampicilline 100 μg/ml (LB amp.) sont inoculés avec 100 μl de stock glycérol du clone JM109/pMAL-Cdc25C et cultivés 14 à 16 heures à 37 °C sous agitation (180 à 220 rpm). Cette pré-culture est ensuite diluée cinquante fois (20 ml par litre de milieu) dans un milieu LB amp. + 2 g/1 de glucose et cultivée à 37 °C / 180 rpm pour atteindre une densité optique à 600 nm comprise entre 0,55 et 0,60. La synthèse de la protéine de fusion est alors induite par ajout IPTG (0,3 mM) à 37 °C pendant 3 heures. Les bactéries sont récoltées par centrifugation, lavées une fois dans 40 ml de PB S froid par litre de culture, et le culot bactérien est ensuite congelé dans l'azote liquide et conservé à -80 °C.50 ml of LB + ampicillin medium 100 μg / ml (LB amp.) Are inoculated with 100 μl of glycerol stock from the clone JM109 / pMAL-Cdc25C and cultured for 14 to 16 hours at 37 ° C. with stirring (180 to 220 rpm). This preculture is then diluted fifty times (20 ml per liter of medium) in an LB amp medium. + 2 g / 1 of glucose and cultured at 37 ° C / 180 rpm to reach an optical density at 600 nm of between 0.55 and 0.60. The synthesis of the fusion protein is then induced by adding IPTG (0.3 mM) at 37 ° C for 3 hours. The bacteria are harvested by centrifugation, washed once in 40 ml of cold PB S per liter of culture, and the bacterial pellet is then frozen in liquid nitrogen and stored at -80 ° C.
L'induction est analysée immédiatement par dépôt de 2,5.107 cellules, prélevées avant et après induction, sur gel de polyacrylamide dénaturant et coloration des protéines au bleu de Coomassie (Figure 1, lignes 1 et 2 respectivement). 2.2 - Lyse et extraction.Induction is analyzed immediately by depositing 2.5 × 10 7 cells, taken before and after induction, on denaturing polyacrylamide gel and staining of proteins with Coomassie blue (FIG. 1, lines 1 and 2 respectively). 2.2 - Lysis and extraction.
Un culot bactérien correspondant à 1 litre de culture induite est décongelé dans la glace, re-suspendu dans 35 ml de tampon de lyse (20 mM Tris-HCl pH 7,4, 250 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 μg/ml lysozyme, 1 μg/ml leupeptine, 2 μg/ml aprotinine, 1 mM PMSF) et incubé 45 min dans la glace. La suspension bactérienne est ensuite soniquée (4 cycles de 1 min en mode discontinu 50%, alternés avec 1 min de pause), puis centrifugée pendant 35 min. à 110 000 g. Le surnageant ou extrait soluble est conservé pour la purification de la protéine MBP-Cdc25C (Figure 1, ligne 3).A bacterial pellet corresponding to 1 liter of induced culture is thawed in ice, resuspended in 35 ml of lysis buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 μg / ml lysozyme, 1 μg / ml leupeptine, 2 μg / ml aprotinin, 1 mM PMSF) and incubated 45 min in ice. The bacterial suspension is then sonicated (4 cycles of 1 min in 50% discontinuous mode, alternated with 1 min of pause), then centrifuged for 35 min. at 110,000 g. The supernatant or soluble extract is kept for the purification of the protein MBP-Cdc25C (Figure 1, line 3).
2.3 - Purification par affinité sur résine d'amylose-agarose.2.3 - Affinity purification on amylose-agarose resin.
Pour un extrait soluble correspondant à 1 litre de culture bactérienne induite, 2 ml de résine d'amylose-agarose (New England Biolabs #800-21) sont déposés sur une colonne chromatographique HR 5/10 (Pharmacia) et lavés par 20 ml (10 volumes) de tampon de colonne (20 mM Tris-HCl pH 7,4 ; 250 mM NaCl ; 1 mM EDTA ; 1 mM DTT ; 1 μg/ml leupeptine ; 2 μg/ml aprotinine). L'extrait soluble est passé sur la colonne d'affinité à un débit de 0,15 ml/min ; l'éluat (c'est-à-dire la fraction non retenue sur l'amylose-agarose) est récolté pour analyse (Figure 1, ligne 4). La colonne est lavée par 20 ml (10 volumes) de tampon de colonne. Un échantillon de la matrice d'affinité après passage de l'extrait soluble peut être éventuellement analysé (Figure 1, ligne 5).For a soluble extract corresponding to 1 liter of induced bacterial culture, 2 ml of amylose-agarose resin (New England Biolabs # 800-21) are deposited on a chromatographic column HR 5/10 (Pharmacia) and washed with 20 ml ( 10 volumes) of column buffer (20 mM Tris-HCl pH 7.4; 250 mM NaCl; 1 mM EDTA; 1 mM DTT; 1 μg / ml leupeptin; 2 μg / ml aprotinin). The soluble extract is passed through the affinity column at a flow rate of 0.15 ml / min; the eluate (that is to say the fraction not retained on the amylose-agarose) is collected for analysis (FIG. 1, line 4). The column is washed with 20 ml (10 volumes) of column buffer. A sample of the affinity matrix after passage of the soluble extract can optionally be analyzed (Figure 1, line 5).
L'élution de la protéine de la matrice d'affinité est effectuée avec un tampon maltose (20 mM Tris-HCl pH 7,4 ; 250 mM NaCl ; 1 mM EDTA ; 1 mM DTT ; 10 mM maltose). 20 fractions d'élution de 0,5 ml sont recueillies. Pour chaque fraction, la concentration protéique totale est évaluée par un essai de type Bradford et la fraction est analysée par dépôt sur gel de polyacrylamide dénaturant et coloration au bleu de Coomassie (Figure 1, ligne 6). Les fractions dans lesquelles la protéine de fusion MBP-Cdc25C complète représente au moins 90 % des protéines totales sont rassemblées pour former un lot dont l'activité est ensuite testée. Les lots sont conservés à -80°C.The elution of the protein from the affinity matrix is carried out with a maltose buffer (20 mM Tris-HCl pH 7.4; 250 mM NaCl; 1 mM EDTA; 1 mM DTT; 10 mM maltose). 20 elution fractions of 0.5 ml are collected. For each fraction, the total protein concentration is evaluated by a Bradford type test and the fraction is analyzed by deposition on denaturing polyacrylamide gel and staining with Coomassie blue (Figure 1, line 6). The fractions in which the complete MBP-Cdc25C fusion protein represents at least 90% of the total proteins are combined to form a batch, the activity of which is then tested. The batches are stored at -80 ° C.
Le séquençage de l'insert compris entre les sites Xbal donne le résultat suivant :The sequencing of the insert included between the Xbal sites gives the following result:
ATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCATATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT
CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC WO 01/44467 . 15 . PCT/FRO0/O3496ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC WO 01/44467. 15. PCT / FRO0 / O3496
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAGG ATTTCAGAAT TCGGATCCTC TAGAATGTCT ACGGAACTCT TÇTCATÇCAÇ AAGAGAGGAA _ .GGAAGCTCTG. ..GCTÇAGGAÇC .. CAGTTTTAGG... TÇTAATCAAA GGAAAATGTT AAACCTGÇTC .. CTGGAGAGAG ACAÇTTCCTT. .. TACÇGTCTGT.... CC AG ATGTCÇ ÇTAGAACTCÇ.. AGTGGGÇAAA TTTÇTTGGTG... ATTCTGCAAA... ÇCTAAGCATT.....TTGTC.TGGAG GAACCCÇAAA .... ATGTTGÇCTC ... GATCTTTÇGA... ATCTTAGCAG.. TGGGGAGATA... AÇTGCCAÇTC AGCTTACÇAÇ ... TTÇTGC-^ . GGAÇTTCAGGACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAGG ATTTCAGAAT TCGGATCCTC TAGAATGTCT ACGGAACTCT TÇTCATÇCAÇ AAGAGAGGAA _ .GGAAGCTCTG. ..GCTÇAGGAÇC .. CAGTTTTAGG ... TÇTAATCAAA GGAAAATGTT AAACCTGÇTC .. CTGGAGAGAG ACAÇTTCCTT. .. TACÇGTCTGT .... CC AG ATGTCÇ ÇTAGAACTCÇ .. AGTGGGÇAAA TTTÇTTGGTG ... ATTCTGCAAA ... ÇCTAAGCATT ..... TTGTC.TGGAG GAACCCÇAAA .... ATGTTGÇCTC ... GATCTTTÇGA ... ATCTTAGCAG .. TGGGAG .. AÇTGCCAÇTC AGCTTACÇAÇ ... TTÇTGC- ^. GGAÇTTCAGG
AAGTGÇATTT . _ AGÇTGGGATG .... AATÇ. AT.GAÇ C .... AGÇ AÇCTAAT.... GAAATGTAGC C C AGÇAÇAGÇ TTCTTTGTAG C ACTCÇGAAT ... GG^TT.GGACÇ ... GT.GGÇÇ ATAG... AAAGAG AGAT.... GÇ AATGTGTA GTTCATÇTGÇ ..AAAT^^GAA..._AAT.GAÇ.AATG ..GAAACTTGGT.... GGAC.AGTGAA. ...ATGAAATATT TGGGCAGTÇC CATTACTACT ....GTTCCA^_AAT....TGGATAA^^ GGAGAAGACCAAGTGÇATTT. _ AGÇTGGGATG .... AATÇ. AT.GAÇ C .... AGÇ AÇCTAAT .... GAAATGTAGC CC AGÇAÇAGÇ TTCTTTGTAG C ACTCÇGAAT ... GG ^ TT.GGACÇ ... GT.GGÇÇ ATAG ... AAAGAG AGAT .... GÇ AATGTGTA GTTCATÇTGÇ ..AAAT ^^ GAA ..._ AAT.GAÇ.AATG ..GAAACTTGGT .... GGAC.AGTGAA. ... ATGAAATATT TGGGCAGTÇC CATTACTACT .... GTTCCA ^ _AAT .... TGGATAA ^^ GGAGAAGACC
AGGC AGAAGA. . GATTTCAG AT ....G^TTAATGG .... AGTTTTCÇÇT.... GAAAGATÇAA ... GAAGC AAAGG TGAGCAGAAG ....TGGCÇTAT.AT ....ÇGCT .GAAÇTTGAAC. . .AGG^CAAGAÇAGGC AGAAGA. . GATTTCAG AT .... G ^ TTAATGG .... AGTTTTCÇÇT .... GAAAGATÇAA ... GAAGC AAAGG TGAGCAGAAG .... TGGCÇTAT.AT .... ÇGCT .GAAÇTTGAAC. . .AGG ^ CAAGAÇ
TGi^GÇAG^T.....GG; ^AAAT.TC ....^GGACAACA CAA ACCAGA . TAAAGTTAAA AAAAA.σTATT TT.TCTGGÇCA.....AGGAAA.GÇT.C AGGAAG.GGCT TATGT.TTAAA ... GAAGAÇAGTC ... TCTÇTGTGTG AÇATTACTAT. .. CAÇTÇAGATG ... CTGGAGGAAG....ATTCTAACCA GGGGCAC.CT.G ATTGGT.GATT TTTÇCAAGG.T _ ATGTGCGCTG ....CCAACCGTGT..... CAGGGAAAÇA CCAAGATCTG. AAGTATG.TÇA AÇ.ÇCAGAAAC _AGTGG^TGCÇ....TTACTGTCGG.... GGAAGTTCCA.. GGGTÇTGATT. ...G GAAGTTTT ATGTÇATTGA.....TTGTCGÇ.TAT CCATATGAGT ATC.TGGGAGG ... AÇAC TCCAG. ...GGAGÇÇTTAA ACTTATATAG ....TCAGGAAGAA CTGTTTAACT TCTTTÇTGAA ... GAAGCCÇATC GTÇCCTTTGG ACACCCAGAA GAGAATAATÇ. . .ATCGTGTTCC . ACTGTGAATT CTCCTCAGAG AGGGGÇCÇ.C.C GAATGTGCCG. CTGTCTGCGT .. GAAGAGGAÇA GGTCTCTGAA CÇAGTATCCT GCATTGTAÇ.T AÇCCAGAGCT ATATATCCTT... .AAAGGCG.GCT ACAGAGACTT CTTTCCAGAA . TATATGGAAC TGTGTGAACC ACAGAGCTAC TGCCCTATGC ATCATCAGGA CCACAAGACT GAGTTGCTGA GGTGTCGAAG ... CCAGAGÇAAA GTGÇAGGAAG .... GGGAGC GC A. .. GC.TGCGGGAG ....CAGATTGCÇ CTGi GCAG ^ ^ T ..... GG; ^ AAAT.TC .... ^ GGACAACA CAA ACCAGA. TAAAGTTAAA AAAAA.σTATT TT.TCTGGÇCA ..... AGGAAA.GÇT.C AGGAAG.GGCT TATGT.TTAAA ... GAAGAÇAGTC ... TCTÇTGTGTG AÇATTACTAT. .. CAÇTÇAGATG ... CTGGAGGAAG .... ATTCTAACCA GGGGCAC.CT.G ATTGGT.GATT TTTÇCAAGG.T _ ATGTGCGCTG .... CCAACCGTGT ..... CAGGGAAAÇA CCAAGATCTG. AAGTATG.TÇA AÇ.ÇCAGAAAC _AGTGG ^ TGCÇ .... TTACTGTCGG .... GGAAGTTCCA .. GGGTÇTGATT. ... G GAAGTTTT ATGTÇATTGA ..... TTGTCGÇ.TAT CCATATGAGT ATC.TGGGAGG ... AÇAC TCCAG. ... GGAGÇÇTTAA ACTTATATAG .... TCAGGAAGAA CTGTTTAACT TCTTTÇTGAA ... GAAGCCÇATC GTÇCCTTTGG ACACCCAGAA GAGAATAATÇ. . .ATCGTGTTCC. ACTGTGAATT CTCCTCAGAG AGGGGÇCÇ.CC GAATGTGCCG. CTGTCTGCGT .. GAAGAGGAÇA GGTCTCTGAA CÇAGTATCCT GCATTGTAÇ.T AÇCCAGAGCT ATATATCCTT ... .AAAGGCG.GCT ACAGAGACTT CTTTCCAGAA. TATATGGAAC TGTGTGAACC ACAGAGCTAC TGCCCTATGC ATCATCAGGA CCACAAGACT GAGTTGCTGA GGTGTCGAAG ... CCAGAGÇAAA GTGÇAGGAAG ... . GGGAGC GC A. .. GC.TGCGGGAG .... CAGATTGCÇ C
T.T_ÇTGGTGAA.._ ^ ATTCCAGCCA CTGGCTGCTA ACATCTAGAT.T_ÇTGGTGAA .._ ^ ATTCCAGCCA CTGGCTGCTA ACATCTAGA
(les sites XBAI sont soulignés par une simple ligne continue, l'ORF de Cdc25C est souligné en pointillé et la séquence en italique correspond à l'ORF de la protéine liant le maltose (MBP))(the XBAI sites are underlined by a single solid line, the ORF of Cdc25C is underlined in dotted lines and the sequence in italics corresponds to the ORF of the maltose-binding protein (MBP))
La séquence de la protéine de fusion MBP-Cdc25C obtenue correspond donc à la séquence SEQ. ID n° 1 représentée ci-dessous :The sequence of the MBP-Cdc25C fusion protein obtained therefore corresponds to the sequence SEQ. ID # 1 shown below:
ATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGTATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT
CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATCATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTACACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC
AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAAAACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA
GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCGGATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG
CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAACTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA
GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATTGACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT
AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAAAAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA
GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTTGGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT
GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTCGGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC
CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTGCTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG
GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCCGGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC
ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAAACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA
GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAACGCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC
AACCTCGGGA TCGAGGGAAGG ATTTCAGAAT TCGGATCCTC TAGAATGTCT ACGGAACTCTAACCTCGGGA TCGAGGGAAGG ATTTCAGAAT TCGGATCCTC TAGAATGTCT ACGGAACTCT
TCTCATCCAC AAGAGAGGAA GGAAGCTCTG GCTCAGGACC CAGTTTTAGG TCTAATCAAATCTCATCCAC AAGAGAGGAA GGAAGCTCTG GCTCAGGACC CAGTTTTAGG TCTAATCAAA
GGAAAATGTT AAACCTGCTC CTGGAGAGAG ACACTTCCTT TACCGTCTGT CCAGATGTCC CTAGAACTCC AGTGGGCAAA TTTCTTGGTG ATTCTGCAAA CCTAAGCATT TTGTCTGGAGGGAAAATGTT AAACCTGCTC CTGGAGAGAG ACACTTCCTT TACCGTCTGT CCAGATGTCC CTAGAACTCC AGTGGGCAAA TTTCTTGGTG ATTCTGCAAA CCTAAGCATT TTGTCTGGAG
GAACCCCAAA ATGTTGCCTC GATCTTTCGA ATCTTAGCAG TGGGGAGATA ACTGCCACTCGAACCCCAAA ATGTTGCCTC GATCTTTCGA ATCTTAGCAG TGGGGAGATA ACTGCCACTC
AGCTTACCAC TTCTGCAGAC CTTGATGAAA CTGGTCACCT GGATTCTTCA GGACTTCAGGAGCTTACCAC TTCTGCAGAC CTTGATGAAA CTGGTCACCT GGATTCTTCA GGACTTCAGG
AAGTGCATTT AGCTGGGATG AATCATGACC AGCACCTAAT GAAATGTAGC CCAGCACAGCAAGTGCATTT AGCTGGGATG AATCATGACC AGCACCTAAT GAAATGTAGC CCAGCACAGC
TTCTTTGTAG CACTCCGAAT GGTTTGGACC GTGGCCATAG AAAGAGAGAT GCAATGTGTA GTTCATCTGC AAATAAAGAA AATGACAATG GAAACTTGGT GGACAGTGAA ATGAAATATTTTCTTTGTAG CACTCCGAAT GGTTTGGACC GTGGCCATAG AAAGAGAGAT GCAATGTGTA GTTCATCTGC AAATAAAGAA AATGACAATG GAAACTTGGT GGACAGTGAA ATGAAATATT
TGGGCAGTCC CATTACTACT GTTCCAAAAT TGGATAAAAA TCCAAACCTA GGAGAAGACC AGGCAGAAGA GATTTCAGAT GAATTAATGG AGTTTTCCCT GAAAGATCAA GAAGCAAAGGTGGGCAGTCC CATTACTACT GTTCCAAAAT TGGATAAAAA TCCAAACCTA GGAGAAGACC AGGCAGAAGA GATTTCAGAT GAATTAATGG AGTTTTCCCT GAAAGATCAA GAAGCAAAGG
TGAGCAGAAG TGGCCTATAT CGCTCCCCGT CGATGCCAGA GAACTTGAAC AGGCCAAGACTGAGCAGAAG TGGCCTATAT CGCTCCCCGT CGATGCCAGA GAACTTGAAC AGGCCAAGAC
TGAAGCAGGT GGAAAAATTC AAGGACAACA CAATACCAGA TAAAGTTAAA AAAAAGTATTTGAAGCAGGT GGAAAAATTC AAGGACAACA CAATACCAGA TAAAGTTAAA AAAAAGTATT
TTTCTGGCCA AGGAAAGCTC AGGAAGGGCT TATGTTTAAA GAAGACAGTC TCTCTGTGTG ACATTACTAT CACTCAGATG CTGGAGGAAG ATTCTAACCA GGGGCACCTG ATTGGTGATTTTTCTGGCCA AGGAAAGCTC AGGAAGGGCT TATGTTTAAA GAAGACAGTC TCTCTGTGTG ACATTACTAT CACTCAGATG CTGGAGGAAG ATTCTAACCA GGGGCACCTG ATTGGTGATT
TTTCCAAGGT ATGTGCGCTG CCAACCGTGT CAGGGAAACA CCAAGATCTG AAGTATGTCATTTCCAAGGT ATGTGCGCTG CCAACCGTGT CAGGGAAACA CCAAGATCTG AAGTATGTCA
ACCCAGAAAC AGTGGCTGCC TTACTGTCGG GGAAGTTCCA GGGTCTGATT GAGAAGTTTTACCCAGAAAC AGTGGCTGCC TTACTGTCGG GGAAGTTCCA GGGTCTGATT GAGAAGTTTT
ATGTCATTGA TTGTCGCTAT CCATATGAGT ATCTGGGAGG ACACATCCAG GGAGCCTTAAATGTCATTGA TTGTCGCTAT CCATATGAGT ATCTGGGAGG ACACATCCAG GGAGCCTTAA
ACTTATATAG TCAGGAAGAA CTGTTTAACT TCTTTCTGAA GAAGCCCATC GTCCCTTTGG ACACCCAGAA GAGAATAATC ATCGTGTTCC ACTGTGAATT CTCCTCAGAG AGGGGCCCCCACTTATATAG TCAGGAAGAA CTGTTTAACT TCTTTCTGAA GAAGCCCATC GTCCCTTTGG ACACCCAGAA GAGAATAATC ATCGTGTTCC ACTGTGAATT CTCCTCAGAG AGGGGCCCCC
GAATGTGCCG CTGTCTGCGT GAAGAGGACA GGTCTCTGAA CCAGTATCCT GCATTGTACTGAATGTGCCG CTGTCTGCGT GAAGAGGACA GGTCTCTGAA CCAGTATCCT GCATTGTACT
ACCCAGAGCT ATATATCCTT AAAGGCGGCT ACAGAGACTT CTTTCCAGAA TATATGGAACACCCAGAGCT ATATATCCTT AAAGGCGGCT ACAGAGACTT CTTTCCAGAA TATATGGAAC
TGTGTGAACC ACAGAGCTAC TGCCCTATGC ATCATCAGGA CCACAAGACT GAGTTGCTGATGTGTGAACC ACAGAGCTAC TGCCCTATGC ATCATCAGGA CCACAAGACT GAGTTGCTGA
GGTGTCGAAG CCAGAGCAAA GTGCAGGAAG GGGAGCGGCA GCTGCGGGAG CAGATTGCCC TTCTGGTGAA GGACATGAGC CCATGAGGTGTCGAAG CCAGAGCAAA GTGCAGGAAG GGGAGCGGCA GCTGCGGGAG CAGATTGCCC TTCTGGTGAA GGACATGAGC CCATGA
3 - Détermination de l'activité de la protéine de fusion MBP-Cdc25C :3 - Determination of the activity of the fusion protein MBP-Cdc25C:
L'activité phosphatase de la. protéine MBP-Cdc25C est évaluée par un essai de déphosphorylation du phosphate de 3-O-méthylfluorescéine (OMFP) avec détermination de l'absorbance à 477 nm (DO 477 nm) du produit de la réaction (OMF).The phosphatase activity of. MBP-Cdc25C protein is evaluated by a 3-O-methylfluorescein phosphate dephosphorylation test (OMFP) with determination of the absorbance at 477 nm (OD 477 nm) of the reaction product (OMF).
La protéine MBP-Cdc25C, conservée dans le tampon d'élution (le même que celui décrit au paragraphe 2.3), est diluée à la concentration de 20 nM dans le tampon de réaction phosphatase (50 mM Tris-HCl pH 8,2 ; 50 mM NaCl ; 1 mM DTT ; 20% glycérol), à température ambiante, dans un volume réactionnel total de 1 ml. La réaction est initiée par l'ajout d'une solution 0,3 mM de OMFP (préparée extemporanément à partir d'une solution stock 7,5 mM dans du DMSO 100% (Sigma #M2629)) et se déroule à 25 °C dans une cuve pour spectrophotométrie à usage unique en polystyrène (Fisher Scientific #A12- 103-056). La DO 477 nm est mesurée après 90 min. La référence pour la mesure d'absorbance est constituée par le tampon de réaction contenant 0,3 mM d'OMFP, sans protéine MBP-Cdc25C, au temps to de la réaction. Un exemple représentatif des résultats d'une telle détermination d'activité est illustré par la Figure 2. WO 01/44467 - 18 - PCT/FRO0/O3496The MBP-Cdc25C protein, stored in the elution buffer (the same as that described in paragraph 2.3), is diluted to the concentration of 20 nM in the phosphatase reaction buffer (50 mM Tris-HCl pH 8.2; 50 mM NaCl; 1 mM DTT; 20% glycerol), at room temperature, in a total reaction volume of 1 ml. The reaction is initiated by the addition of a 0.3 mM solution of OMFP (prepared extemporaneously from a 7.5 mM stock solution in 100% DMSO (Sigma # M2629)) and takes place at 25 ° C. in a single-use polystyrene spectrophotometry tank (Fisher Scientific # A12-103-056). The OD 477 nm is measured after 90 min. The reference for the measurement of absorbance is constituted by the reaction buffer containing 0.3 mM of OMFP, without protein MBP-Cdc25C, at time to of the reaction. A representative example of the results of such an activity determination is illustrated in Figure 2. WO 01/44467 - 18 - PCT / FRO0 / O3496
B / .Protéines ; de ... fusion....MBP-Çdç25B.lJ MBP-Çdc25B2 etB / .Proteins ; de ... fusion .... MBP-Çdç25B.l J MBP-Çdc25B2 and
MBP-Cdç25B3 :MBP-Cdç25B3:
1 - Construction du vecteur d'expression pour MBP-Cdc25Bl, MBP-Cdc25B2 et MBP-Cdc25B31 - Construction of the expression vector for MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3
1. 1 - Principe du système utilisé.1. 1 - Principle of the system used.
Le système utilisé (New England Biolabs #800 - pMAL™ protein fusion et système de purification) est basé sur la production d'une protéine de fusion entre la protéine d'intérêt, ici la protéine Cdc25B humaine, et la protéine bactérienne MBP (Maltose- Binding Protein) à' Escherichia coli. Cette méthode permet de purifier en une étape la protéine de fusion grâce à l'affinité de la MBP pour le maltose.The system used (New England Biolabs # 800 - pMAL ™ protein fusion and purification system) is based on the production of a fusion protein between the protein of interest, here the human Cdc25B protein, and the bacterial protein MBP (Maltose - Binding Protein) to 'Escherichia coli. This method makes it possible to purify the fusion protein in one step thanks to the affinity of MBP for maltose.
1.2 - Origine de l'ADNc de Cdc25B1.2 - Origin of the Cdc25B cDNA
L'ADN codant pour les trois variants d'épissage de la phosphatase CDC25B humaine correspond aux numéros d'accession : M81934 et Z68092.The DNA encoding the three splicing variants of human CDC25B phosphatase corresponds to the accession numbers: M81934 and Z68092.
1.3 - Construction des vecteurs pMAL - Hs Cdc25B1.3 - Construction of the pMAL vectors - Hs Cdc25B
Les différences entre les ADN des 3 variants de Cdc25B concernent deux exons codant des domaines de 14 et 42 acides aminés localisés dans la région régulatrice de la molécule (Baldin et coll., Oncogene (1997), 14, 2485-2490) et n'affectant pas ses régions 5' et 3'. La stratégie de clonage utilisée pour ces trois variants est donc identique.The differences between the DNAs of the 3 variants of Cdc25B relate to two exons encoding domains of 14 and 42 amino acids located in the regulatory region of the molecule (Baldin et al., Oncogene (1997), 14, 2485-2490) and n ' not affecting its 5 'and 3' regions. The cloning strategy used for these three variants is therefore identical.
Les ADNc de Cdc25B sous la forme d'inserts NdeΙ(Klenow) / BamHl ont été introduits dans le vecteur pMAL™-C2X (New England Biolabs, #800-76) au site EcoRl (Klenow) / BamHl pour donner respectivement les vecteurs pMAL-Hs Cdc25Bl, pMAL-Hs Cdc25B2 et pMAL-Hs Cdc25B3. Ces vecteurs permettent la production des protéines de fusion MBP-Cdc25Bl (962 acides aminés), MPB-Cdc25B2 (933 acides aminés) et MBP-Cdc25B3 (974 acides aminés) à partir du promoteur bactérien Ptac inductible par l'isopropylthiogalactoside (IPTG). La séquence SEQ. ID. n° 6 de la protéine Hs Cdc25Bl avec ses extrémités de restriction Ndel et BamHl est la suivante :The Cdc25B cDNAs in the form of NdeΙ (Klenow) / BamHI inserts were introduced into the vector pMAL ™ -C2X (New England Biolabs, # 800-76) at the EcoRl (Klenow) / BamHI site to respectively give the pMAL vectors. -Hs Cdc25Bl, pMAL-Hs Cdc25B2 and pMAL-Hs Cdc25B3. These vectors allow the production of the fusion proteins MBP-Cdc25Bl (962 amino acids), MPB-Cdc25B2 (933 amino acids) and MBP-Cdc25B3 (974 amino acids) from the bacterial promoter Ptac inducible by isopropylthiogalactoside (IPTG). The sequence SEQ. ID. n ° 6 of the protein Hs Cdc25Bl with its Ndel and BamHI restriction ends is as follows:
CATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTGCATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG
TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCTC CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATGTGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCT CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATG
CACGACCTCG CCGGGCTCGG CAGCCGCAGC CGCCTGACGC ACCTATCCCT GTCTCGACGGCACGACCTCG CCGGGCTCGG CAGCCGCAGC CGCCTGACGC ACCTATCCCT GTCTCGACGG
GCATCCGAAT CCTCCCTGTC GTCTGAATCC TCCGAATCTT CTGATGCAGG TCTCTGCATGGCATCCGAAT CCTCCCTGTC GTCTGAATCC TCCGAATCTT CTGATGCAGG TCTCTGCATG
GATTCCCCCA GCCCTATGGA CCCCCACATG GCGGAGCAGA CGTTTGAACA GGCCATCCAGGATTCCCCCA GCCCTATGGA CCCCCACATG GCGGAGCAGA CGTTTGAACA GGCCATCCAG
GCAGCCAGCC GGATCATTCG AAACGAGCAG TTTGCCATCA GACGCTTCCA GTCTATGCCG GTGAGGCTGC TGGGCCACAG CCCCGTGCTT CGGAACATCA CCAACTCCCA GGCGCCCGACGCAGCCAGCC GGATCATTCG AAACGAGCAG TTTGCCATCA GACGCTTCCA GTCTATGCCG GTGAGGCTGC TGGGCCACAG CCCCGTGCTT CGGAACATCA CCAACTCCCA GGCGCCCGAC
GGCCGGAGGA AGAGCGAGGC GGGCAGTGGA GCTGCCAGCA GCTCTGGGGA AGACAAGGAGGGCCGGAGGA AGAGCGAGGC GGGCAGTGGA GCTGCCAGCA GCTCTGGGGA AGACAAGGAG
AATGATGGAT TTGTCTTCAA GATGCCATGG AAGCCCACAC ATCCCAGCTC CACCCATGCTAATGATGGAT TTGTCTTCAA GATGCCATGG AAGCCCACAC ATCCCAGCTC CACCCATGCT
CTGGCAGAGT GGGCCAGCCG CAGGGAAGCC TTTGCCCAGA GACCCAGCTC GGCCCCCGACCTGGCAGAGT GGGCCAGCCG CAGGGAAGCC TTTGCCCAGA GACCCAGCTC GGCCCCCGAC
CTGATGTGTC TCAGTCCTGA CCGGAAGATG GAAGTGGAGG AGCTCAGCCC CCTGGCCCTA GGTCGCTTCT CTCTGACCCC TGCAGAGGGG GATACTGAGG AAGATGATGG ATTTGTGGACCTGATGTGTC TCAGTCCTGA CCGGAAGATG GAAGTGGAGG AGCTCAGCCC CCTGGCCCTA GGTCGCTTCT CTCTGACCCC TGCAGAGGGG GATACTGAGG AAGATGATGG ATTTGTGGAC
ATCCTAGAGA GTGACTTAAA GGATGATGAT GCAGTTCCCC CAGGCATGGA GAGTCTCATTATCCTAGAGA GTGACTTAAA GGATGATGAT GCAGTTCCCC CAGGCATGGA GAGTCTCATT
AGTGCCCCAC TGGTCAAGAC CTTGGAAAAG GAAGAGGAAA AGGACCTCGT CATGTACAGCAGTGCCCCAC TGGTCAAGAC CTTGGAAAAG GAAGAGGAAA AGGACCTCGT CATGTACAGC
AAGTGCCAGC GGCTCTTCCG CTCTCCGTCC ATGCCCTGCA GCGTGATCCG GCCCATCCTCAAGTGCCAGC GGCTCTTCCG CTCTCCGTCC ATGCCCTGCA GCGTGATCCG GCCCATCCTC
AAGAGGCTGG AGCGGCCCCA GGACAGGGAC ACGCCCGTGC AGAATAAGCG GAGGCGGAGC GTGACCCCTC CTGAGGAGCA GCAGGAGGCT GAGGAACCTA AAGCCCGCGT CCTCCGCTCAAAGAGGCTGG AGCGGCCCCA GGACAGGGAC ACGCCCGTGC AGAATAAGCG GAGGCGGAGC GTGACCCCTC CTGAGGAGCA GCAGGAGGCT GAGGAACCTA AAGCCCGCGT CCTCCGCTCA
AAATCACTGT GTCACGATGA GATCGAGAAC CTCCTGGACA GTGACCACCG AGAGCTGATTAAATCACTGT GTCACGATGA GATCGAGAAC CTCCTGGACA GTGACCACCG AGAGCTGATT
GGAGATTACT CTAAGGCCTT CCTCCTACAG ACAGTAGACG GAAAGCACCA AGACCTCAAGGGAGATTACT CTAAGGCCTT CCTCCTACAG ACAGTAGACG GAAAGCACCA AGACCTCAAG
TACATCTCAC CAGAAACGAT GGTGGCCCTA TTGACGGGCA AGTTCAGCAA CATCGTGGATTACATCTCAC CAGAAACGAT GGTGGCCCTA TTGACGGGCA AGTTCAGCAA CATCGTGGAT
AAGTTTGTGA TTGTAGACTG CAGATACCCC TATGAATATG AAGGCGGGCA CATCAAGACT GCGGTGAACT TGCCCCTGGA ACGCGACGCC GAGAGCTTCC TACTGAAGAG CCCCATCGCGAAGTTTGTGA TTGTAGACTG CAGATACCCC TATGAATATG AAGGCGGGCA CATCAAGACT GCGGTGAACT TGCCCCTGGA ACGCGACGCC GAGAGCTTCC TACTGAAGAG CCCCATCGCG
CCCTGTAGCC TGGACAAGAG AGTCATCCTC ATTTTCCACT GTGAATTCTC ATCTGAGCGTCCCTGTAGCC TGGACAAGAG AGTCATCCTC ATTTTCCACT GTGAATTCTC ATCTGAGCGT
GGGCCCCGCA TGTGCCGTTT CATCAGGGAA CGAGACCGTG CTGTCAACGA CTACCCCAGCGGGCCCCGCA TGTGCCGTTT CATCAGGGAA CGAGACCGTG CTGTCAACGA CTACCCCAGC
CTCTACTACC CTGAGATGTA TATCCTGAAA GGCGGCTACA AGGAGTTCTT CCCTCAGCACCTCTACTACC CTGAGATGTA TATCCTGAAA GGCGGCTACA AGGAGTTCTT CCCTCAGCAC
CCGAACTTCT GTGAACCCCA GGACTACCGG CCCATGAACC ACGAGGCCTT CAAGGATGAG CTAAAGACCT TCCGCCTCAA GACTCGCAGC TGGGCTGGGG AGCGGAGCCG GCGGGAGCTCCCGAACTTCT GTGAACCCCA GGACTACCGG CCCATGAACC ACGAGGCCTT CAAGGATGAG CTAAAGACCT TCCGCCTCAA GACTCGCAGC TGGGCTGGGG AGCGGAGCCG GCGGGAGCTC
TGTAGCCGGC TGCAGGACCA GTGAGGGGCC TGCGCCAGTC CTGCTACCTC CCTTGCCTTTTGTAGCCGGC TGCAGGACCA GTGAGGGGCC TGCGCCAGTC CTGCTACCTC CCTTGCCTTT
CGAGGCCTGA AGCCAGCTGC CCTATGGGCC TGCCGGGCTG AGGGCCTGCT GGAGGCCTCACGAGGCCTGA AGCCAGCTGC CCTATGGGCC TGCCGGGCTG AGGGCCTGCT GGAGGCCTCA
GGTGCTGTCC ATGGGAAAGA TGGTGTGGTG TCCTGCCTGT CTGCCCCAGC CCAGATTCCCGGTGCTGTCC ATGGGAAAGA TGGTGTGGTG TCCTGCCTGT CTGCCCCAGC CCAGATTCCC
CTGTGTCATC CCATCATTTT CCATATCCTG GTGCCCCCCA CCCCTGGAAG AGCCCAGTCT GTTGAGTTAG TTAAGTTGGG TTAATACCAG CTTAAAGGCA GTATTTTGTG TCCTCCAGGACTGTGTCATC CCATCATTTT CCATATCCTG GTGCCCCCCA CCCCTGGAAG AGCCCAGTCT GTTGAGTTAG TTAAGTTGGG TTAATACCAG CTTAAAGGCA GTATTTTGTG TCCTCCAGGA
GCTTCTTGTT TCCTTGTTAG GGTTAACCCT TCATCTTCCT GTGTCCTGAA ACGCTCCTTTGCTTCTTGTT TCCTTGTTAG GGTTAACCCT TCATCTTCCT GTGTCCTGAA ACGCTCCTTT
GTGTGTGTGT CAGCTGAGGA TCC (les sites Ndel et BamHl sont soulignés en continu)GTGTGTGTGT CAGCTGAGGA TCC (the Ndel and BamHl sites are underlined continuously)
La séquence SEQ. ID. n° 7 de la protéine Hs Cdc25B2 avec ses extrémités de restriction Ndel et BamHl est la suivante :The sequence SEQ. ID. n ° 7 of the protein Hs Cdc25B2 with its Ndel and BamHI restriction ends is as follows:
CATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCTCCATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGCCATCC TCATGGCCTC
CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATGCTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATG
CACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGGGAC CCTGCTCTTCCACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGGGAC CCTGCTCTTC
CGCAGCCGCA GCCGCCTGAC GCACCTATCC CTGTCTCGAC GGGCATCCGA ATCCTCCCTGCGCAGCCGCA GCCGCCTGAC GCACCTATCC CTGTCTCGAC GGGCATCCGA ATCCTCCCTG
TCGTCTGAAT CCTCCGAATC TTCTGATGCA GGTCTCTGCA TGGATTCCCC CAGCCCTATG GACCCCCACA TGGCGGAGCA GACGTTTGAA CAGGCCATCC AGGCAGCCAG CCGGATCATTTCGTCTGAAT CCTCCGAATC TTCTGATGCA GGTCTCTGCA TGGATTCCCC CAGCCCTATG GACCCCCACA TGGCGGAGCA GACGTTTGAA CAGGCCATCC AGGCAGCCAG CCGGATCATT
CGAAACGAGC AGTTTGCCAT CAGACGCTTC CAGTCTATGC CGGATGGATT TGTCTTCAAGCGAAACGAGC AGTTTGCCAT CAGACGCTTC CAGTCTATGC CGGATGGATT TGTCTTCAAG
ATGCCATGGA AGCCCACACA TCCCAGCTCC ACCCATGCTC TGGCAGAGTG GGCCAGCCGCATGCCATGGA AGCCCACACA TCCCAGCTCC ACCCATGCTC TGGCAGAGTG GGCCAGCCGC
AGGGAAGCCT TTGCCCAGAG ACCCAGCTCG GCCCCCGACC TGATGTGTCT CAGTCCTGACAGGGAAGCCT TTGCCCAGAG ACCCAGCTCG GCCCCCGACC TGATGTGTCT CAGTCCTGAC
CGGAAGATGG AAGTGGAGGA GCTCAGCCCC CTGGCCCTAG GTCGCTTCTC TCTGACCCCT GCAGAGGGGG ATACTGAGGA AGATGATGGA TTTGTGGACA TCCTAGAGAG TGACTTAAAGCGGAAGATGG AAGTGGAGGA GCTCAGCCCC CTGGCCCTAG GTCGCTTCTC TCTGACCCCT GCAGAGGGGG ATACTGAGGA AGATGATGGA TTTGTGGACA TCCTAGAGAG TGACTTAAAG
GATGATGATG CAGTTCCCCC AGGCATGGAG AGTCTCATTA GTGCCCCACT GGTCAAGACCGATGATGATG CAGTTCCCCC AGGCATGGAG AGTCTCATTA GTGCCCCACT GGTCAAGACC
TTGGAAAAGG AAGAGGAAAA GGACCTCGTC ATGTACAGCA AGTGCCAGCG GCTCTTCCGCTTGGAAAAGG AAGAGGAAAA GGACCTCGTC ATGTACAGCA AGTGCCAGCG GCTCTTCCGC
TCTCCGTCCA TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAGTCTCCGTCCA TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG
GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA AATCACTGTG TCACGATGAGGACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA AATCACTGTG TCACGATGAG
ATCGAGAACC TCCTGGACAG TGACCACCGA GAGCTGATTG GAGATTACTC TAAGGCCTTCATCGAGAACC TCCTGGACAG TGACCACCGA GAGCTGATTG GAGATTACTC TAAGGCCTTC
CTCCTACAGA CAGTAGACGG AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATGCTCCTACAGA CAGTAGACGG AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG
GTGGCCCTAT TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGCGTGGCCCTAT TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC CCTGTAGCCT GGACAAGAGAAGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC CCTGTAGCCT GGACAAGAGA
GTCATCCTCA TTTTCCACTG TGAATTCTCA TCTGAGCGTG GGCCCCGCAT GTGCCGTTTCGTCATCCTCA TTTTCCACTG TGAATTCTCA TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC
ATCAGGGAAC GAGACCGTGC TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTATATCAGGGAAC GAGACCGTGC TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT
ATCCTGAAAG GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAGATCCTGAAAG GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT GTAGCCGGCT GCAGGACCAGGACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT GTAGCCGGCT GCAGGACCAG
TGAGGGGCCT GCGCCAGTCC TGCTACCTCC CTTGCCTTTC GAGGCCTGAA GCCAGCTGCCTGAGGGGCCT GCGCCAGTCC TGCTACCTCC CTTGCCTTTC GAGGCCTGAA GCCAGCTGCC
CTATGGGCCT GCCGGGCTGA GGGCCTGCTG GAGGCCTCAG GTGCTGTCCA TGGGAAAGATCTATGGGCCT GCCGGGCTGA GGGCCTGCTG GAGGCCTCAG GTGCTGTCCA TGGGAAAGAT
GGTGTGGTGT CCTGCCTGTC TGCCCCAGCC CAGATTCCCC TGTGTCATCC CATCATTTTCGGTGTGGTGT CCTGCCTGTC TGCCCCAGCC CAGATTCCCC TGTGTCATCC CATCATTTTC
CATATCCTGG TGCCCCCCAC CCCTGGAAGA GCCCAGTCTG TTGAGTTAGT TAAGTTGGGT TAATACCAGC TTAAAGGCAG TATTTTGTGT CCTCCAGGAG CTTCTTGTTT CCTTGTTAGGCATATCCTGG TGCCCCCCAC CCCTGGAAGA GCCCAGTCTG TTGAGTTAGT TAAGTTGGGT TAATACCAGC TTAAAGGCAG TATTTTGTGT CCTCCAGGAG CTTCTTGTTT CCTTGTTAGG
GTTAACCCTT CATCTTCCTG TGTCCTGAAA CGCTCCTTTG TGTGTGTGTC AGCTGAGGAT CC WO 01/44467 - 21 - PCT/FROO/03496GTTAACCCTT CATCTTCCTG TGTCCTGAAA CGCTCCTTTG TGTGTGTGTC AGCTGAGGAT CC WO 01/44467 - 21 - PCT / FROO / 03496
(les sites Ndel et BamHl sont soulignés en continu)(the Ndel and BamHl sites are underlined continuously)
La séquence SEQ. ID. n° 8 de la protéine Hs Cdc25B3 avec ses extrémités de restriction Ndel et BamHl est la suivante :The sequence SEQ. ID. n ° 8 of the protein Hs Cdc25B3 with its Ndel and BamHI restriction ends is as follows:
CATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCTCCATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGCCATCC TCATGGCCTC
CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATGCTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATG
CACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGGGAC CCTGCTCTTCCACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGGGAC CCTGCTCTTC
CGCAGCCGCA GCCGCCTGAC GCACCTATCC CTGTCTCGAC GGGCATCCGA ATCCTCCCTGCGCAGCCGCA GCCGCCTGAC GCACCTATCC CTGTCTCGAC GGGCATCCGA ATCCTCCCTG
TCGTCTGAAT CCTCCGAATC TTCTGATGCA GGTCTCTGCA TGGATTCCCC CAGCCCTATG GACCCCCACA TGGCGGAGCA GACGTTTGAA CAGGCCATCC AGGCAGCCAG CCGGATCATTTCGTCTGAAT CCTCCGAATC TTCTGATGCA GGTCTCTGCA TGGATTCCCC CAGCCCTATG GACCCCCACA TGGCGGAGCA GACGTTTGAA CAGGCCATCC AGGCAGCCAG CCGGATCATT
CGAAACGAGC AGTTTGCCAT CAGACGCTTC CAGTCTATGC CGGTGAGGCT GCTGGGCCACCGAAACGAGC AGTTTGCCAT CAGACGCTTC CAGTCTATGC CGGTGAGGCT GCTGGGCCAC
AGCCCCGTGC TTCGGAACAT CACCAACTCC CAGGCGCCCG ACGGCCGGAG GAAGAGCGAGAGCCCCGTGC TTCGGAACAT CACCAACTCC CAGGCGCCCG ACGGCCGGAG GAAGAGCGAG
GCGGGCAGTG GAGCTGCCAG CAGCTCTGGG GAAGACAAGG AGAATGATGG ATTTGTCTTCGCGGGCAGTG GAGCTGCCAG CAGCTCTGGG GAAGACAAGG AGAATGATGG ATTTGTCTTC
AAGATGCCAT GGAAGCCCAC ACATCCCAGC TCCACCCATG CTCTGGCAGA GTGGGCCAGC CGCAGGGAAG CCTTTGCCCA GAGACCCAGC TCGGCCCCCG ACCTGATGTG TCTCAGTCCTAAGATGCCAT GGAAGCCCAC ACATCCCAGC TCCACCCATG CTCTGGCAGA GTGGGCCAGC CGCAGGGAAG CCTTTGCCCA GAGACCCAGC TCGGCCCCCG ACCTGATGTG TCTCAGTCCT
GACCGGAAGA TGGAAGTGGA GGAGCTCAGC CCCCTGGCCC TAGGTCGCTT CTCTCTGACCGACCGGAAGA TGGAAGTGGA GGAGCTCAGC CCCCTGGCCC TAGGTCGCTT CTCTCTGACC
CCTGCAGAGG GGGATACTGA GGAAGATGAT GGATTTGTGG ACATCCTAGA GAGTGACTTACCTGCAGAGG GGGATACTGA GGAAGATGAT GGATTTGTGG ACATCCTAGA GAGTGACTTA
AAGGATGATG ATGCAGTTCC CCCAGGCATG GAGAGTCTCA TTAGTGCCCC ACTGGTCAAGAAGGATGATG ATGCAGTTCC CCCAGAGGCATG GAGAGTCTCA TTAGTGCCCC ACTGGTCAAG
ACCTTGGAAA AGGAAGAGGA AAAGGACCTC GTCATGTACA GCAAGTGCCA GCGGCTCTTC CGCTCTCCGT CCATGCCCTG CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCCACCTTGGAAA AGGAAGAGGA AAAGGACCTC GTCATGTACA GCAAGTGCCA GCGGCTCTTC CGCTCTCCGT CCATGCCCTG CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCC
CAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAGCAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAG
CAGCAGGAGG CTGAGGAACC TAAAGCCCGC GTCCTCCGCT CAAAATCACT GTGTCACGATCAGCAGGAGG CTGAGGAACC TAAAGCCCGC GTCCTCCGCT CAAAATCACT GTGTCACGAT
GAGATCGAGA ACCTCCTGGA CAGTGACCAC CGAGAGCTGA TTGGAGATTA CTCTAAGGCCGAGATCGAGA ACCTCCTGGA CAGTGACCAC CGAGAGCTGA TTGGAGATTA CTCTAAGGCC
TTCCTCCTAC AGACAGTAGA CGGAAAGCAC CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC AACATCGTGG ATAAGTTTGT GATTGTAGACTTCCTCCTAC AGACAGTAGA CGGAAAGCAC CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC AACATCGTGG ATAAGTTTGT GATTGTAGAC
TGCAGATACC CCTATGAATA TGAAGGCGGG CACATCAAGA CTGCGGTGAA CTTGCCCCTGTGCAGATACC CCTATGAATA TGAAGGCGGG CACATCAAGA CTGCGGTGAA CTTGCCCCTG
GAACGCGACG CCGAGAGCTT CCTACTGAAG AGCCCCATCG CGCCCTGTAG CCTGGACAAGGAACGCGACG CCGAGAGCTT CCTACTGAAG AGCCCCATCG CGCCCTGTAG CCTGGACAAG
AGAGTCATCC TCATTTTCCA CTGTGAATTC TCATCTGAGC GTGGGCCCCG CATGTGCCGTAGAGTCATCC TCATTTTCCA CTGTGAATTC TCATCTGAGC GTGGGCCCCG CATGTGCCGT
TTCATCAGGG AACGAGACCG TGCTGTCAAC GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC TTCCCTCAGC ACCCGAACTT CTGTGAACCCTTCATCAGGG AACGAGACCG TGCTGTCAAC GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC TTCCCTCAGC ACCCGAACTT CTGTGAACCC
CAGGACTACC GGCCCATGAA CCACGAGGCC TTCAAGGATG AGCTAAAGAC CTTCCGCCTCCAGGACTACC GGCCCATGAA CCACGAGGCC TTCAAGGATG AGCTAAAGAC CTTCCGCCTC
AAGACTCGCA GCTGGGCTGG GGAGCGGAGC CGGCGGGAGC TCTGTAGCCG GCTGCAGGACAAGACTCGCA GCTGGGCTGG GGAGCGGAGC CGGCGGGAGC TCTGTAGCCG GCTGCAGGAC
CAGTGAGGGG CCTGCGCCAG TCCTGCTACC TCCCTTGCCT TTCGAGGCCT GAAGCCAGCTCAGTGAGGGG CCTGCGCCAG TCCTGCTACC TCCCTTGCCT TTCGAGGCCT GAAGCCAGCT
GCCCTATGGG CCTGCCGGGC TGAGGGCCTG CTGGAGGCCT CAGGTGCTGT CCATGGGAAA GATGGTGTGG TGTCCTGCCT GTCTGCCCCA GCCCAGATTC CCCTGTGTCA TCCCATCATTGCCCTATGGG CCTGCCGGGC TGAGGGCCTG CTGGAGGCCCC CAGGTGCTGT CCATGGGAAA GATGGTGTGG TGTCCTGCCT GTCTGCCCCA GCCCAGATTC CCCTGTGTCA TCCCATCATT
TTCCATATCC TGGTGCCCCC CACCCCTGGA AGAGCCCAGT CTGTTGAGTT AGTTAAGTTGTTCCATATCC TGGTGCCCCC CACCCCTGGA AGAGCCCAGT CTGTTGAGTT AGTTAAGTTG
GGTTAATACC AGCTTAAAGG CAGTATTTTG TGTCCTCCAG GAGCTTCTTG TTTCCTTGTT AGGGTTAACC CTTCATCTTC CTGTGTCCTG AAACGCTCCT TTGTGTGTGT GTCAGCTGAG GATCCGGTTAATACC AGCTTAAAGG CAGTATTTTG TGTCCTCCAG GAGCTTCTTG TTTCCTTGTT AGGGTTAACC CTTCATCTTC CTGTGTCCTG AAACGCTCCT TTGTGTGTGT GTCAGCTGAG GATCC
(les sites Ndel et BamHl sont soulignés en continu)(the Ndel and BamHl sites are underlined continuously)
1.4 - Création des souches JM109/pMAL-Cdc25Bl, B2 et B31.4 - Creation of strains JM109 / pMAL-Cdc25Bl, B2 and B3
Les vecteurs pMAL-Cdc25Bl-3 ont chacun été introduits dans la souche à' Escherichia coli JM109 (Stratagene #200271). Les colonies ont été sélectionnées sur la base de leur capacité à produire une protéine de fusion après culture en présence d'IPTG. L'identité des protéines a été vérifiée par immunodétection avec des anticorps polyclonaux dirigés contre Cdc25B.The pMAL-Cdc25Bl-3 vectors were each introduced into the Escherichia coli JM109 strain (Stratagene # 200271). Colonies were selected based on their ability to produce a fusion protein after culture in the presence of IPTG. The identity of the proteins was verified by immunodetection with polyclonal antibodies directed against Cdc25B.
Les ADN plasmidiques isolés de ces trois clones ont été séquences dans la région correspondant à Cdc25B. Les séquences suivantes ont été obtenues (la partie doublement soulignée correspond à l'ORF de MBP et la partie soulignée en pointillés à l'ORF de Cdc25Bl, Cdc25B2 ou Cdc25B3) :The plasmid DNAs isolated from these three clones were sequenced in the region corresponding to Cdc25B. The following sequences were obtained (the doubly underlined part corresponds to the ORF of MBP and the underlined part in dotted lines corresponds to the ORF of Cdc25Bl, Cdc25B2 or Cdc25B3):
• SEQ. ID n° 9 (pMAL-HsCdc25Bl) :• SEQ. ID # 9 (pMAL-HsCdc25Bl):
ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGTATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT
CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCATCTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT
CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATCATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTACACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC
AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAAAACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA
GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTGGATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG
AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCGAAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG
CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTGCTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG
GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCCGGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC
ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTCACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC
TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAATGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA
GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC WO 01/44467 .23 - PCTFROO/03496GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC WO 01/44467 .23 - PCTFROO / 03496
AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCCGC AGCCGCCTGA CGCACCTATC CCTGTCTCGA CGGGCATCCG AATCCTCCCT GTCGTCTGAA TCCTCCGAAT CTTCTGATGC AGGTCTCTGC ATGGATTCCC CCAGCCCTAT GGACCCCCAC ATGGCGGAGC AGACGTTTGA ACAGGCCATC CAGGCAGCCA GCCGGATCAT TCGAAACGAG CAGTTTGCCA TCAGACGCTT CCAGTCTATG CCGGTGAGGC TGCTGGGCCA CAGCCCCGTG CTTCGGAACA TCACCAACTC CCAGGCGCCC GACGGCCGGA GGAAGAGCGA GGCGGGCAGT GGAGCTGCCA GCAGCTCTGG GGAAGACAAG GAGAATGATG GATTTGTCTT CAAGATGCCA TGGAAGCCCA CACATCCCAG CTCCACCCAT GCTCTGGCAG AGTGGGCCAG CCGCAGGGAA GCCTTTGCCC AGAGACCCAG CTCGGCCCCC GACCTGATGT GTCTCAGTCC TGACCGGAAG ATGGAAGTGG AGGAGCTCAG CCCCCTGGCC CTAGGTCGCT TCTCTCTGAC CCCTGCAGAG GGGGATACTG AGGAAGATGA TGGATTTGTG GACATCCTAG AGAGTGACTT AAAGGATGAT GATGCAGTTC CCCCAGGCAT GGAGAGTCTC ATTAGTGCCC CACTGGTCAA GACCTTGGAA AAGGAAGAGG AAAAGGACCT CGTCATGTAC AGCAAGTGCC AGCGGCTCTT CCGCTCTCCG TCCATGCCCT GCAGCGTGAT CCGGCCCATC CTCAAGAGGC TGGAGCGGCC CCAGGACAGG GACACGCCCG TGCAGAATAA GCGGAGGCGG AGCGTGACCC CTCCTGAGGA GCAGCAGGAG GCTGAGGAAC CTAAAGCCCG CGTCCTCCGC TCAAAATCAC TGTGTCACGA TGAGATCGAG AACCTCCTGG ACAGTGACCA CCGAGAGCTG ATTGGAGATT ACTCTAAGGC CTTCCTCCTA CAGACAGTAG ACGGAAAGCA CCAAGACCTC AAGTACATCT CACCAGAAAC GATGGTGGCC CTATTGACGG GCAAGTTCAG CAACATCGTG GATAAGTTTG TGATTGTAGA CTGCAGATAC CCCTATGAAT ATGAAGGCGG GCACATCAAG ACTGCGGTGA ACTTGCCCCT GGAACGCGAC GCCGAGAGCT TCCTACTGAA GAGCCCCATC GCGCCCTGTA GCCTGGACAA GAGAGTCATC CTCATTTTCC ACTGTGAATT CTCATCTGAG CGTGGGCCCC GCATGTGCCG TTTCATCAGG GAACGAGACC GTGCTGTCAA CGACTACCCC AGCCTCTACT ACCCTGAGAT GTATATCCTG AAAGGCGGCT ACAAGGAGTT CTTCCCTCAG CACCCGAACT TCTGTGAACC CCAGGACTAC CGGCCCATGA ACCACGAGGC CTTCAAGGAT GAGCTAAAGA CCTTCCGCCT CAAGACTCGC AGCTGGGCTG GGGAGCGGAG CCGGCGGGAG CTCTGTAGCC GGCTGCAGGA CCAGTGAGGG GCCTGCGCCA GTCCTGCTAC CTCCCTTGCC TTTCGAGGCC TGAAGCCAGC TGCCCTATGG GCCTGCCGGG CTGAGGGCCT GCTGGAGGCC TCAGGTGCTG TCCATGGGAA AGATGGTGTG GTGTCCTGCC TGTCTGCCCC AGCCCAGATT CCCCTGTGTC ATCCCATCAT TTTCCATATC CTGGTGCCCC CCACCCCTGG AAGAGCCCAG TCTGTTGAGT TAGTTAAGTT GGGTTAATAC CAGCTTAAAG GCAGTATTTT GTGTCCTCCA GGAGCTTCTT GTTTCCTTGT TAGGGTTAAC CCTTCATCTT CCTGTGTCCT GAAACGCTCC TTTGTGTGTG TGTCAGCTGA GGATCC SEQ. ID n° 10 (PMAL-HsCdc25B2) :AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCCGC AGCCGCCTGA CGCACCTATC CCTGTCTCGA CGGGCATCCG AATCCTCCCT GTCGTCTGAA TCCTCCGAAT CTTCTGATGC AGGTCTCTGC ATGGATTCCC CCAGCCCTAT GGACCCCCAC ATGGCGGAGC AGACGTTTGA ACAGGCCATC CAGGCAGCCA GCCGGATCAT TCGAAACGAG CAGTTTGCCA TCAGACGCTT CCAGTCTATG CCGGTGAGGC TGCTGGGCCA CAGCCCCGTG CTTCGGAACA TCACCAACTC CCAGGCGCCC GACGGCCGGA GGAAGAGCGA GGCGGGCAGT GGAGCTGCCA GCAGCTCTGG GGAAGACAAG GAGAATGATG GATTTGTCTT CAAGATGCCA TGGAAGCCCA CACATCCCAG CTCCACCCAT GCTCTGGCAG AGTGGGCCAG CCGCAGGGAA GCCTTTGCCC AGAGACCCAG CTCGGCCCCC GACCTGATGT GTCTCAGTCC TGACCGGAAG ATGGAAGTGG AGGAGCTCAG CCCCCTGGCC CTAGGTCGCT TCTCTCTGAC CCCTGCAGAG GGGGATACTG AGGAAGATGA TGGATTTGTG GACATCCTAG AGAGTGACTT AAAGGATGAT GATGCAGTTC CCCCAGGCAT GGAGAGTCTC ATTAGTGCCC CACTGGTCAA GACCTTGGAA AAGGAAGAGG AAAAGGACCT CGTCATGTAC AGCAAGTGCC AGCGGCTCTT CCGCTCTCCG TCCATGCCCT GCAGCGTGAT CCGGCCCATC CTCAAGAGGC TGGAGCGGCC CCAGGACAGG GACACGCCCG TGCAGAATAA GCGGAGGCGG AGCGTGACCC CTCCTGAGGA GCAGCAGGAG GCTGAGGAAC CTAAAGCCCG CGTCCTCCGC TCAAAATCAC TGTGTCACGA TGAGATCGAG AACCTCCTGG ACAGTGACCA CCGAGAGCTG ATTGGAGATT ACTCTAAGGC CTTCCTCCTA CAGACAGTAG ACGGAAAGCA CCAAGACCTC AAGTACATCT CACCAGAAAC GATGGTGGCC CTATTGACGG GCAAGTTCAG CAACATCGTG GATAAGTTTG TGATTGTAGA CTGCAGATAC CCCTATGAAT ATGAAGGCGG GCACATCAAG ACTGCGGTGA ACTTGCCCCT GGAACGCGAC GCCGAGAGCT TCCTACTGAA GAGCCCCATC GCGCCCTGTA GCCTGGACAA GAGAGTCATC CTCATTTTCC ACTGTGAATT CTCATCTGAG CGTGGGCCCC GCATGTGCCG TTTCATCAGG GAACGAGACC GTGCTGTCAA CGACTACCCC AGCCTCTACT ACCCTGAGAT GTATATCCTG AAAGGCGGCT ACAAGGAGTT CTTCCCTCAG CACCCGAACT TCTGTGAACC CCAGGACTAC CGGCCCATGA ACCACGAGGC CTTCAAGGAT GAGCTAAAGA CCTTCCGCCT CAAGACTCGC AGCTGGGCTG GGGAGCGGAG CCGGCGGGAG CTCTGTAGCC GGCTGCAGGA CCAGTGAGGG GCCTGCGCCA GTCCTGCTAC CTCCCTTGCC TTTCGAGGCC TGAAGCCAGC TGCCCTATGG GCCTGCCGGG CTGAGGGCC T GCTGGAGGCC TCAGGTGCTG TCCATGGGAA AGATGGTGTG GTGTCCTGCC TGTCTGCCCC AGCCCAGATT CCCCTGTGTC ATCCCATCAT TTTCCATATC CTGGTGCCCC CCACCCCTGG AAGAGCCCAG TCTGTTGAGT TAGTTAAGTT GGGTTAATAC CAGCTTAAAG GCAGTATTTT GTGTCCTCCA GGAGCTTCTT GTTTCCTTGT TAGGGTTAAC CCTTCATCTT CCTGTGTCCT GAAACGCTCC TTTGTGTGTG TGTCAGCTGA GGATCC SEQ. ID No. 10 ( P MAL-HsCdc25B2):
ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCATATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT
CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATCATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTACACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC
AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAAAACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA
GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTGGATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG
AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCGAAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG
CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAACTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA
GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATTGACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT
AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAAAAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA
GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAAGGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA
GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTTGTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT
GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTCGGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC
CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTGCTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG
GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCCGGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC
ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTCACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC
TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAATGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA
GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAACGCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC
AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCG CAGGCT. CGGCTCTCAG.TCCAGCAGGC..GTGTGCGGTG..GCGCCCAGCG .TCCGGGCCAC CTCCCGGGCC .TC.CTGCTGGG .ATCTCATGGC...CTÇCTGGGCT ^ TCCTCGCCGG.TCACCACCCT...CACCCAGACC..ATGCA∞ ACC CAAAGA.GTCAGGTAGG...GACCÇ.TGÇTC..TTCCGÇAGÇÇ GÇAGÇCGÇÇT GACGCAÇÇTA TÇÇÇTGTÇT.C.GACGGGCATC. CG-^.TCCTÇ^..CTGTÇGTÇTG AAT.CCTCCGA ATCTT.CTGAT GÇAGGTCTCT GCATGGATTC ÇCÇÇAGCÇCT ATGG.ACCCCC..AÇATGGÇGGA. GCAGACGTTT G A.CAGGCÇA TÇCAGGCAGC CAGCCGGATC ATTÇG.AAACG..AGCAGTTTGC..ÇATCAGAÇGC TTCÇ.AGTCTA..TGCÇGGATGG..ATTTGTÇTTÇ. AAGATGÇC T GGAAGCCCAC AÇATCCCAGÇ TÇ.CACÇCATG.CTC.TGGCAGA..GTGGGÇCAGC. CGCAGGGAAG CÇT.TTGCCCA GAGACCCAGÇ TCGGCCÇÇCG..AÇCTGATGTG .T.ÇTCAGTCCT..GACCGGAAGA TGGAAGTGGA. GGAGCTCAGC CCCCTGGCCC TAGGTCGÇTT CT.TCTGACC CCTGCAGAGG..GGGATA.CTGA..G^AAGATGAT GGA.TTTGTGG AÇATCCTAG GAGTGACTTA AAGGATGATG .A.TGCAGTTCC..CCCAGGÇATG GAGAGTCTÇA TTAGTGCCCC ACTGGTCAAG ACÇT.TGGAAA.. .GGAAGAGGA. AAAGGACCTC GTCATGTAÇA GÇAAGTGCCA GCGGCTCTTC CGÇTCTCCGT. ÇCATGCCCTG .CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCC CAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAG CAGCAGGAGG CTGAGGAACC TAAAGCCCGCAACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCG CAGGCT . CGGCTCTCAG . TCCAGCAGGC .. GTGTGCGGTG .. GCGCCCAGCG . TCCGGGCCAC CTCCCGGGCC . TC . CTGCTGGG . ATCTCATGGC ... CTÇCTGGGCT ^ TCCTCGCCGG . TCACCACCCT ... CACCCAGACC .. ATGCA∞ ACC CAAAGA . GTCAGGTAGG ... GACCÇ . TGÇTC .. TTCCGÇAGÇÇ GÇAGÇCGÇÇT GACGCAÇÇTA TÇÇÇTGTÇT . C. GACGGGCATC . CG- ^ . TCCTÇ ^ .. CTGTÇGTÇTG AAT . CCTCCGA ATCTT . CTGAT GÇAGGTCTCT GCATGGATTC ÇCÇÇAGCÇCT ATGG . ACCCCC. . AÇATGGÇGGA . GCAGACGTTT GA . CAGGCÇA TÇCAGGCAGC CAGCCGGATC ATTÇG . AAACG. . AGCAGTTTGC .. ÇATCAGAÇGC TTCÇ . AGTCTA .. TGCÇGGATGG .. ATTTGTÇTTÇ . AAGATGÇC T GGAAGCCCAC AÇATCCCAGÇ TÇ . CACÇCATG . CTC . TGGCAGA .. GTGGGÇCAGC . CGCAGGGAAG CÇT . TTGCCCA GAGACCCAGÇ TCGGCCÇÇCG .. AÇCTGATGTG . T. ÇTCAGTCCT .. GACCGGAAGA TGGAAGTGGA . GGAGCTCAGC CCCCTGGCCC TAGGTCGÇTT CT . TCTGACC CCTGCAGAGG .. GGGATA . CTGA .. G ^ AAGATGAT GGA . TTTGTGG AÇATCCTAG GAGTGACTTA AAGGATGATG . A. TGCAGTTCC .. CCCAGGÇATG GAGAGTCTÇA TTAGTGCCCC ACTGGTCAAG ACÇT . TGGAAA ... GGAAGAGGA . AAAGGACCTC GTCATGTAÇA GÇAAGTGCCA GCGGCTCTTC CGÇTCTCCGT . ÇCATGCCCTG . CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCC CAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAG CAGCAGGAGG CTGAGGAACC TAAAGCCCGC
GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCACGTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC
CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCACCGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC
CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGCCAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC
AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGGAACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG
CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAGCACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG
AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTCAGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC
TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAACTCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC
GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTCGACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC
TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCCTTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC
TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGCTTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC
ÇGGÇGGGAGC.TCTGTAGÇCG.GCT CCTGCGCCAG TCCTGCTACCÇGGÇGGGAGC . TCTGTAGÇCG . GCT CCTGCGCCAG TCCTGCTACC
TCCCTTGCCT TTCGAGGCCT GAAGCCAGCT GCCCTATGGG CCTGCCGGGC TGAGGGCCTGTCCCTTGCCT TTCGAGGCCT GAAGCCAGCT GCCCTATGGG CCTGCCGGGC TGAGGGCCTG
CTGGAGGCCT CAGGTGCTGT CCATGGGAAA GATGGTGTGG TGTCCTGCCT GTCTGCCCCA GCCCAGATTC CCCTGTGTCA TCCCATCATT TTCCATATCC TGGTGCCCCC CACCCCTGGACTGGAGGCCT CAGGTGCTGT CCATGGGAAA GATGGTGTGG TGTCCTGCCT GTCTGCCCCA GCCCAGATTC CCCTGTGTCA TCCCATCATT TTCCATATCC TGGTGCCCCC CACCCCTGGA
AGAGCCCAGT CTGTTGAGTT AGTTAAGTTG GGTTAATACC AGCTTAAAGG CAGTATTTTGAGAGCCCAGT CTGTTGAGTT AGTTAAGTTG GGTTAATACC AGCTTAAAGG CAGTATTTTG
TGTCCTCCAG GAGCTTCTTG TTTCCTTGTT AGGGTTAACC CTTCATCTTC CTGTGTCCTG AAACGCTCCT TTGTGTGTGT GTCAGCTGAG GATCCTGTCCTCCAG GAGCTTCTTG TTTCCTTGTT AGGGTTAACC CTTCATCTTC CTGTGTCCTG AAACGCTCCT TTGTGTGTGT GTCAGCTGAG GATCC
• SEQ. ID n° 11 (pMAL-HsCdc25B3) :• SEQ. ID # 11 (pMAL-HsCdc25B3):
ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGTATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT
CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCATCTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT
CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATCATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTACACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC
AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAAAACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA
GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTGGATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG
AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAAAAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA
GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAACGCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC
AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCCAACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC
GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCTGCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT
TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCGAATCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCGAA
ACCCCAAAGA GTCAGGTAGG GACCCTGCTC TTCCGCAGCC GCAGCCGCCT GACGCACCTAACCCCAAAGA GTCAGGTAGG GACCCTGCTC TTCCGCAGCC GCAGCCGCCT GACGCACCTA
TCCCTGTCTC GACGGGCATC CGAATCCTCC CTGTCGTCTG AATCCTCCGA ATCTTCTGATTCCCTGTCTC GACGGGCATC CGAATCCTCC CTGTCGTCTG AATCCTCCGA ATCTTCTGAT
GCAGGTCTCT GCATGGATTC CCCCAGCCCT ATGGACCCCC ACATGGCGGA GCAGACGTTT GAACAGGCCA TCCAGGCAGC CAGCCGGATC ATTCGAAACG AGCAGTTTGC CATCAGACGCGCAGGTCTCT GCATGGATTC CCCCAGCCCT ATGGACCCCC ACATGGCGGA GCAGACGTTT GAACAGGCCA TCCAGGCAGC CAGCCGGATC ATTCGAAACG AGCAGTTTGC CATCAGACGC
TTCCAGTCTA TGCCGGTGAG GCTGCTGGGC CACAGCCCCG TGCTTCGGAA CATCACCAACTTCCAGTCTA TGCCGGTGAG GCTGCTGGGC CACAGCCCCG TGCTTCGGAA CATCACCAAC
TCCCAGGCGC CCGACGGCCG GAGGAAGAGC GAGGCGGGCA GTGGAGCTGC CAGCAGCTCTTCCCAGGCGC CCGACGGCCG GAGGAAGAGC GAGGCGGGCA GTGGAGCTGC CAGCAGCTCT
GGGGAAGACA AGGAGAATGA TGGATTTGTC TTCAAGATGC CATGGAAGCC CACACATCCCGGGGAAGACA AGGAGAATGA TGGATTTGTC TTCAAGATGC CATGGAAGCC CACACATCCC
AGCTCCACCC ATGCTCTGGC AGAGTGGGCC AGCCGCAGGG AAGCCTTTGC CCAGAGACCC AGCTCGGCCC CCGACCTGAT GTGTCTCAGT CCTGACCGGA AGATGGAAGT GGAGGAGCTCAGCTCCACCC ATGCTCTGGC AGAGTGGGCC AGCCGCAGGG AAGCCTTTGC CCAGAGACCC AGCTCGGCCC CCGACCTGAT GTGTCTCAGT CCTGACCGGA AGATGGAAGT GGAGGAGCTC
AGCCCCCTGG CCCTAGGTCG CTTCTCTCTG ACCCCTGCAG AGGGGGATAC TGAGGAAGATAGCCCCCTGG CCCTAGGTCG CTTCTCTCTG ACCCCTGCAG AGGGGGATAC TGAGGAAGAT
GATGGATTTG TGGACATCCT AGAGAGTGAC TTAAAGGATG ATGATGCAGT TCCCCCAGGCGATGGATTTG TGGACATCCT AGAGAGTGAC TTAAAGGATG ATGATGCAGT TCCCCCAGGC
ATGGAGAGTC TCATTAGTGC CCCACTGGTC AAGACCTTGG AAAAGGAAGA GGAAAAGGACATGGAGAGTC TCATTAGTGC CCCACTGGTC AAGACCTTGG AAAAGGAAGA GGAAAAGGAC
CTCGTCATGT ACAGCAAGTG CCAGCGGCTC TTCCGCTCTC CGTCCATGCC CTGCAGCGTG ATCCGGCCCA TCCTCAAGAG GCTGGAGCGG CCCCAGGACA GGGACACGCC CGTGCAGAATCTCGTCATGT ACAGCAAGTG CCAGCGGCTC TTCCGCTCTC CGTCCATGCC CTGCAGCGTG ATCCGGCCCA TCCTCAAGAG GCTGGAGCGG CCCCAGGACA GGGACACGCC CGTGCAGAAT
AAGCGGAGGC GGAGCGTGAC CCCTCCTGAG GAGCAGCAGG AGGCTGAGGA ACCTAAAGCCAAGCGGAGGC GGAGCGTGAC CCCTCCTGAG GAGCAGCAGG AGGCTGAGGA ACCTAAAGCC
CGCGTCCTCC GCTCAAAATC ACTGTGTCAC GATGAGATCG AGAACCTCCT GGACAGTGACCGCGTCCTCC GCTCAAAATC ACTGTGTCAC GATGAGATCG AGAACCTCCT GGACAGTGAC
CACCGAGAGC TGATTGGAGA TTACTCTAAG GCCTTCCTCC TACAGACAGT AGACGGAAAGCACCGAGAGC TGATTGGAGA TTACTCTAAG GCCTTCCTCC TACAGACAGT AGACGGAAAG
CACCAAGACC TCAAGTACAT CTCACCAGAA ACGATGGTGG CCCTATTGAC GGGCAAGTTC AGCAACATCG TGGATAAGTT TGTGATTGTA GACTGCAGAT ACCCCTATGA ATATGAAGGCCACCAAGACC TCAAGTACAT CTCACCAGAA ACGATGGTGG CCCTATTGAC GGGCAAGTTC AGCAACATCG TGGATAAGTT TGTGATTGTA GACTGCAGAT ACCCCTATGA ATATGAAGGC
GGGCACATCA AGACTGCGGT GAACTTGCCC CTGGAACGCG ACGCCGAGAG CTTCCTACTGGGGCACATCA AGACTGCGGT GAACTTGCCC CTGGAACGCG ACGCCGAGAG CTTCCTACTG
AAGAGCCCCA TCGCGCCCTG TAGCCTGGAC AAGAGAGTCA TCCTCATTTT CCACTGTGAAAAGAGCCCCA TCGCGCCCTG TAGCCTGGAC AAGAGAGTCA TCCTCATTTT CCACTGTGAA
TTCTCATCTG AGCGTGGGCC CCGCATGTGC CGTTTCATCA GGGAACGAGA CCGTGCTGTCTTCTCATCTG AGCGTGGGCC CCGCATGTGC CGTTTCATCA GGGAACGAGA CCGTGCTGTC
AACGACTACC CCAGCCTCTA CTACCCTGAG ATGTATATCC TGAAAGGCGG CTACAAGGAG TTCTTCCCTC AGCACCCGAA CTTCTGTGAA CCCCAGGACT ACCGGCCCAT GAACCACGAGAACGACTACC CCAGCCTCTA CTACCCTGAG ATGTATATCC TGAAAGGCGG CTACAAGGAG TTCTTCCCTC AGCACCCGAA CTTCTGTGAA CCCCAGGACT ACCGGCCCAT GAACCACGAG
GCCTTCAAGG ATGAGCTAAA GACCTTCCGC CTCAAGACTC GCAGCTGGGC TGGGGAGCGGGCCTTCAAGG ATGAGCTAAA GACCTTCCGC CTCAAGACTC GCAGCTGGGC TGGGGAGCGG
AGCCGGCGGG AGCTCTGTAG CCGGCTGCAG GACCAGTGAG GGGCCTGCGC CAGTCCTGCTAGCCGGCGGG AGCTCTGTAG CCGGCTGCAG GACCAGTGAG GGGCCTGCGC CAGTCCTGCT
ACCTCCCTTG CCTTTCGAGG CCTGAAGCCA GCTGCCCTAT GGGCCTGCCG GGCTGAGGGCACCTCCCTTG CCTTTCGAGG CCTGAAGCCA GCTGCCCTAT GGGCCTGCCG GGCTGAGGGC
CTGCTGGAGG CCTCAGGTGC TGTCCATGGG AAAGATGGTG TGGTGTCCTG CCTGTCTGCC CCAGCCCAGA TTCCCCTGTG TCATCCCATC ATTTTCCATA TCCTGGTGCC CCCCACCCCTCTGCTGGAGG CCTCAGGTGC TGTCCATGGG AAAGATGGTG TGGTGTCCTG CCTGTCTGCC CCAGCCCAGA TTCCCCTGTG TCATCCCATC ATTTTCCATA TCCTGGTGCC CCCCACCCCT
GGAAGAGCCC AGTCTGTTGA GTTAGTTAAG TTGGGTTAAT ACCAGCTTAA AGGCAGTATTGGAAGAGCCC AGTCTGTTGA GTTAGTTAAG TTGGGTTAAT ACCAGCTTAA AGGCAGTATT
TTGTGTCCTC CAGGAGCTTC TTGTTTCCTT GTTAGGGTTA ACCCTTCATC TTCCTGTGTCTTGTGTCCTC CAGGAGCTTC TTGTTTCCTT GTTAGGGTTA ACCCTTCATC TTCCTGTGTC
CTGAAACGCT CCTTTGTGTG TGTGTCAGCT GAGGATCC Ces clones sont conservés à - 80 °C sous forme de culture saturée additionnée de glycérol (concentration finale 25%). Ces souches sont ensuite utilisées pour toutes les étapes de production ultérieures.CTGAAACGCT CCTTTGTGTG TGTGTCAGCT GAGGATCC These clones are stored at -80 ° C. in the form of a saturated culture added with glycerol (final concentration 25%). These strains are then used for all subsequent production stages.
2 - Production et purification des protéines recombinantes MBP-Cdc25Bl, MBP-Cdc25B2 et MBP-Cdc25B3 :2 - Production and purification of the recombinant proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3:
Les protéines recombinantes MBP-Cdc25Bl, B2 et B3 sont produites exactement comme cela est décrit pour MBP-Cdc25C.The recombinant proteins MBP-Cdc25Bl, B2 and B3 are produced exactly as described for MBP-Cdc25C.
Les séquences SEQ. ID n° 12, SEQ. ID n° 13 et SEQ. ID n° 14 sont obtenues respectivement pour les protéines de fusion MBP-Cdc25Bl, MBP-Cdc25B2 et MBP-Cdc25B3 :SEQ sequences. ID No. 12, SEQ. ID # 13 and SEQ. ID No. 14 are obtained respectively for the fusion proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3:
• SEQ. ID n° 12 (MBP-Cdc25Bl) :• SEQ. ID # 12 (MBP-Cdc25Bl):
ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGTATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT
CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCATCTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT
CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATCATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTACACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC
AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAAAACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA
GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCCGCGATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCCGC
AGCCGCCTGA CGCACCTATC CCTGTCTCGA CGGGCATCCG AATCCTCCCT GTCGTCTGAAAGCCGCCTGA CGCACCTATC CCTGTCTCGA CGGGCATCCG AATCCTCCCT GTCGTCTGAA
TCCTCCGAAT CTTCTGATGC AGGTCTCTGC ATGGATTCCC CCAGCCCTAT GGACCCCCACTCCTCCGAAT CTTCTGATGC AGGTCTCTGC ATGGATTCCC CCAGCCCTAT GGACCCCCAC
ATGGCGGAGC AGACGTTTGA ACAGGCCATC CAGGCAGCCA GCCGGATCAT TCGAAACGAG CAGTTTGCCA TCAGACGCTT CCAGTCTATG CCGGTGAGGC TGCTGGGCCA CAGCCCCGTGATGGCGGAGC AGACGTTTGA ACAGGCCATC CAGGCAGCCA GCCGGATCAT TCGAAACGAG CAGTTTGCCA TCAGACGCTT CCAGTCTATG CCGGTGAGGC TGCTGGGCCA CAGCCCCGTG
CTTCGGAACA TCACCAACTC CCAGGCGCCC GACGGCCGGA GGAAGAGCGA GGCGGGCAGTCTTCGGAACA TCACCAACTC CCAGGCGCCC GACGGCCGGA GGAAGAGCGA GGCGGGCAGT
GGAGCTGCCA GCAGCTCTGG GGAAGACAAG GAGAATGATG GATTTGTCTT CAAGATGCCAGGAGCTGCCA GCAGCTCTGG GGAAGACAAG GAGAATGATG GATTTGTCTT CAAGATGCCA
TGGAAGCCCA CACATCCCAG CTCCACCCAT GCTCTGGCAG AGTGGGCCAG CCGCAGGGAATGGAAGCCCA CACATCCCAG CTCCACCCAT GCTCTGGCAG AGTGGGCCAG CCGCAGGGAA
GCCTTTGCCC AGAGACCCAG CTCGGCCCCC GACCTGATGT GTCTCAGTCC TGACCGGAAG ATGGAAGTGG AGGAGCTCAG CCCCCTGGCC CTAGGTCGCT TCTCTCTGAC CCCTGCAGAGGCCTTTGCCC AGAGACCCAG CTCGGCCCCC GACCTGATGT GTCTCAGTCC TGACCGGAAG ATGGAAGTGG AGGAGCTCAG CCCCCTGGCC CTAGGTCGCT TCTCTCTGAC CCCTGCAGAG
GGGGATACTG AGGAAGATGA TGGATTTGTG GACATCCTAG AGAGTGACTT AAAGGATGATGGGGATACTG AGGAAGATGA TGGATTTGTG GACATCCTAG AGAGTGACTT AAAGGATGAT
GATGCAGTTC CCCCAGGCAT GGAGAGTCTC ATTAGTGCCC CACTGGTCAA GACCTTGGAAGATGCAGTTC CCCCAGGCAT GGAGAGTCTC ATTAGTGCCC CACTGGTCAA GACCTTGGAA
AAGGAAGAGG AAAAGGACCT CGTCATGTAC AGCAAGTGCC AGCGGCTCTT CCGCTCTCCGAAGGAAGAGG AAAAGGACCT CGTCATGTAC AGCAAGTGCC AGCGGCTCTT CCGCTCTCCG
TCCATGCCCT GCAGCGTGAT CCGGCCCATC CTCAAGAGGC TGGAGCGGCC CCAGGACAGG GACACGCCCG TGCAGAATAA GCGGAGGCGG AGCGTGACCC CTCCTGAGGA GCAGCAGGAGTCCATGCCCT GCAGCGTGAT CCGGCCCATC CTCAAGAGGC TGGAGCGGCC CCAGGACAGG GACACGCCCG TGCAGAATAA GCGGAGGCGG AGCGTGACCC CTCCTGAGGA GCAGCAGGAG
GCTGAGGAAC CTAAAGCCCG CGTCCTCCGC TCAAAATCAC TGTGTCACGA TGAGATCGAGGCTGAGGAAC CTAAAGCCCG CGTCCTCCGC TCAAAATCAC TGTGTCACGA TGAGATCGAG
AACCTCCTGG ACAGTGACCA CCGAGAGCTG ATTGGAGATT ACTCTAAGGC CTTCCTCCTAAACCTCCTGG ACAGTGACCA CCGAGAGCTG ATTGGAGATT ACTCTAAGGC CTTCCTCCTA
CAGACAGTAG ACGGAAAGCA CCAAGACCTC AAGTACATCT CACCAGAAAC GATGGTGGCCCAGACAGTAG ACGGAAAGCA CCAAGACCTC AAGTACATCT CACCAGAAAC GATGGTGGCC
CTATTGACGG GCAAGTTCAG CAACATCGTG GATAAGTTTG TGATTGTAGA CTGCAGATAC CCCTATGAAT ATGAAGGCGG GCACATCAAG ACTGCGGTGA ACTTGCCCCT GGAACGCGAC GCCGAGAGCT TCCTACTGAA GAGCCCCATC GCGCCCTGTA GCCTGGACAA GAGAGTCATCCTATTGACGG GCAAGTTCAG CAACATCGTG GATAAGTTTG TGATTGTAGA CTGCAGATAC CCCTATGAAT ATGAAGGCGG GCACATCAAG ACTGCGGTGA ACTTGCCCCT GGAACGCGAC GCCGAGAGCT TCCTACTGAA GAGCCCCATC GCGCCCTGTA GCC
CTCATTTTCC ACTGTGAATT CTCATCTGAG CGTGGGCCCC GCATGTGCCG TTTCATCAGGCTCATTTTCC ACTGTGAATT CTCATCTGAG CGTGGGCCCC GCATGTGCCG TTTCATCAGG
GAACGAGACC GTGCTGTCAA CGACTACCCC AGCCTCTACT ACCCTGAGAT GTATATCCTG AAAGGCGGCT ACAAGGAGTT CTTCCCTCAG CACCCGAACT TCTGTGAACC CCAGGACTAC CGGCCCATGA ACCACGAGGC CTTCAAGGAT GAGCTAAAGA CCTTCCGCCT CAAGACTCGCGAACGAGACC GTGCTGTCAA CGACTACCCC AGCCTCTACT ACCCTGAGAT GTATATCCTG AAAGGCGGCT ACAAGGAGTT CTTCCCTCAG CACCCGAACT TCTGTGAACC CCAGGACTAC CGGCCCATGA ACCACGAGGC CTTCAAGGAT GAGCTAAAGA CCTTCCGCCT CA
AGCTGGGCTG GGGAGCGGAG CCGGCGGGAG CTCTGTAGCC GGCTGCAGGA CCAGTGAAGCTGGGCTG GGGAGCGGAG CCGGCGGGAG CTCTGTAGCC GGCTGCAGGA CCAGTGA
SEQ ID n° 13 (MBP-Cdc25B2)SEQ ID No. 13 (MBP-Cdc25B2)
ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA WO 01/44467 . 29 - PCT/FROO/03496ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA WO 01/44467. 29 - PCT / FROO / 03496
GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATTGACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT
AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAAAAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA
GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAAGGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA
GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTTGTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT
GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTCGGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC
CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTGCTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG
GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCCGGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC
ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTCACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC
TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAATGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA
GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAACGCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC
AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCGAA ACCCCAAAGA GTCAGGTAGG GACCCTGCTC TTCCGCAGCC GCAGCCGCCT GACGCACCTA TCCCTGTCTC GACGGGCATC CGAATCCTCC CTGTCGTCTG AATCCTCCGA ATCTTCTGAT GCAGGTCTCT GCATGGATTC CCCCAGCCCT ATGGACCCCC ACATGGCGGA GCAGACGTTT GAACAGGCCA TCCAGGCAGC CAGCCGGATC ATTCGAAACG AGCAGTTTGC CATCAGACGC TTCCAGTCTA TGCCGGATGG ATTTGTCTTC AAGATGCCAT GGAAGCCCAC ACATCCCAGC TCCACCCATG CTCTGGCAGA GTGGGCCAGC CGCAGGGAAG CCTTTGCCCA GAGACCCAGC TCGGCCCCCG ACCTGATGTG TCTCAGTCCT GACCGGAAGA TGGAAGTGGA GGAGCTCAGC CCCCTGGCCC TAGGTCGCTT CTCTCTGACC CCTGCAGAGG GGGATACTGA GGAAGATGAT GGATTTGTGG ACATCCTAGA GAGTGACTTA AAGGATGATG ATGCAGTTCC CCCAGGCATG GAGAGTCTCA TTAGTGCCCC ACTGGTCAAG ACCTTGGAAA AGGAAGAGGA AAAGGACCTC GTCATGTACA GCAAGTGCCA GCGGCTCTTC CGCTCTCCGT CCATGCCCTG CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCC CAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAG CAGCAGGAGG CTGAGGAACC TAAAGCCCGC GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCΛGC AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC CGGCGGGAGC TCTGTAGCCG GCTGCAGGAC CAGTGA • SEQ ID n° 14 (MBP-Cdc25B3)AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCGAA ACCCCAAAGA GTCAGGTAGG GACCCTGCTC TTCCGCAGCC GCAGCCGCCT GACGCACCTA TCCCTGTCTC GACGGGCATC CGAATCCTCC CTGTCGTCTG AATCCTCCGA ATCTTCTGAT GCAGGTCTCT GCATGGATTC CCCCAGCCCT ATGGACCCCC ACATGGCGGA GCAGACGTTT GAACAGGCCA TCCAGGCAGC CAGCCGGATC ATTCGAAACG AGCAGTTTGC CATCAGACGC TTCCAGTCTA TGCCGGATGG ATTTGTCTTC AAGATGCCAT GGAAGCCCAC ACATCCCAGC TCCACCCATG CTCTGGCAGA GTGGGCCAGC CGCAGGGAAG CCTTTGCCCA GAGACCCAGC TCGGCCCCCG ACCTGATGTG TCTCAGTCCT GACCGGAAGA TGGAAGTGGA GGAGCTCAGC CCCCTGGCCC TAGGTCGCTT CTCTCTGACC CCTGCAGAGG GGGATACTGA GGAAGATGAT GGATTTGTGG ACATCCTAGA GAGTGACTTA AAGGATGATG ATGCAGTTCC CCCAGGCATG GAGAGTCTCA TTAGTGCCCC ACTGGTCAAG ACCTTGGAAA AGGAAGAGGA AAAGGACCTC GTCATGTACA GCAAGTGCCA GCGGCTCTTC CGCTCTCCGT CCATGCCCTG CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCC CAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAG CAGCAGGAGG CTGAGGAACC TAAAGCCCGC GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCΛGC AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC CGGCGGGAGC TCTGTAGCCG GCTGCAGGAC CAGTGA • SEQ ID n ° 14 (MBP-Cdc25B3)
ATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGTATGAAAACTG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT
CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCATCTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT
CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATTCCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT
ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATCATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC
ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTACACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC
AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAAAACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA
GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTGGATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG
AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCGAAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG
CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAACTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA
GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATTGACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT
AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAAAAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA
GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAAGGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA
GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTTGTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT
GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTCGGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC
CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTGCTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG
GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCCGGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC
ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTCACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC
TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAATGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA
GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAACGCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC
AACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCGAA ACCCCAAAGA GTCAGGTAGG GACCCTGCTC TTCCGCAGCC GCAGCCGCCT GACGCACCTA TCCCTGTCTC GACGGGCATC CGAATCCTCC CTGTCGTCTG AATCCTCCGA ATCTTCTGAT GCAGGTCTCT GCATGGATTC CCCCAGCCCT ATGGACCCCC ACATGGCGGA GCAGACGTTT GAACAGGCCA TCCAGGCAGC CAGCCGGATC ATTCGAAACG AGCAGTTTGC CATCAGACGC TTCCAGTCTA TGCCGGTGAG GCTGCTGGGC CACAGCCCCG TGCTTCGGAA CATCACCAAC TCCCAGGCGC CCGACGGCCG GAGGAAGAGC GAGGCGGGCA GTGGAGCTGC CAGCAGCTCT GGGGAAGACA AGGAGAATGA TGGATTTGTC TTCAAGATGC CATGGAAGCC CACACATCCC AGCTCCACCC ATGCTCTGGC AGAGTGGGCC AGCCGCAGGG AAGCCTTTGC CCAGAGACCC AGCTCGGCCC CCGACCTGAT GTGTCTCAGT CCTGACCGGA AGATGGAAGT GGAGGAGCTC AGCCCCCTGG CCCTAGGTCG CTTCTCTCTG ACCCCTGCAG AGGGGGATAC TGAGGAAGAT GATGGATTTG TGGACATCCT AGAGAGTGAC TTAAAGGATG ATGATGCAGT TCCCCCAGGC ATGGAGAGTC TCATTAGTGC CCCACTGGTC AAGACCTTGG AAAAGGAAGA GGAAAAGGAC CTCGTCATGT ACAGCAAGTG CCAGCGGCTC TTCCGCTCTC CGTCCATGCC CTGCAGCGTG ATCCGGCCCA TCCTCAAGAG GCTGGAGCGG CCCCAGGACA GGGACACGCC CGTGCAGAAT AAGCGGAGGC GGAGCGTGAC CCCTCCTGAG GAGCAGCAGG AGGCTGAGGA ACCTAAAGCC CGCGTCCTCC GCTCAAAATC ACTGTGTCAC GATGAGATCG AGAACCTCCT GGACAGTGAC CACCGAGAGC TGATTGGAGA TTACTCTAAG GCCTTCCTCC TACAGACAGT AGACGGAAAG CACCAAGACC TCAAGTACAT CTCACCAGAA ACGATGGTGG CCCTATTGAC GGGCAAGTTC AGCAACATCG TGGATAAGTT TGTGATTGTA GACTGCAGAT ACCCCTATGA ATATGAAGGC GGGCACATCA AGACTGCGGT GAACTTGCCC CTGGAACGCG ACGCCGAGAG CTTCCTACTG AAGAGCCCCA TCGCGCCCTG TAGCCTGGAC AAGAGAGTCA TCCTCATTTT CCACTGTGAA TTCTCATCTG AGCGTGGGCC CCGCATGTGC CGTTTCATCA GGGAACGAGA CCGTGCTGTC AACGACTACC CCAGCCTCTA CTACCCTGAG ATGTATATCC TGAAAGGCGG CTACAAGGAG TTCTTCCCTC AGCACCCGAA CTTCTGTGAA CCCCAGGACT ACCGGCCCAT GAACCACGAG GCCTTCAAGG ATGAGCTAAA GACCTTCCGC CTCAAGACTC GCAGCTGGGC TGGGGAGCGG AGCCGGCGGG AGCTCTGTAG CCGGCTGCAG GACCAGTGAAACCTCGGGA TCGAGGGAAG GATTTCAGAA TTCCATATGG AGGTGCCCCA GCCGGAGCCC GCGCCAGGCT CGGCTCTCAG TCCAGCAGGC GTGTGCGGTG GCGCCCAGCG TCCGGGCCAC CTCCCGGGCC TCCTGCTGGG ATCTCATGGC CTCCTGGGGT CCCCGGTGCG GGCGGCCGCT TCCTCGCCGG TCACCACCCT CACCCAGACC ATGCACGACC TCGCCGGGCT CGGCAGCGAA ACCCCAAAGA GTCAGGTAGG GACCCTGCTC TTCCGCAGCC GCAGCCGCCT GACGCACCTA TCCCTGTCTC GACGGGCATC CGAATCCTCC CTGTCGTCTG AATCCTCCGA ATCTTCTGAT GCAGGTCTCT GCATGGATTC CCCCAGCCCT ATGGACCCCC ACATGGCGGA GCAGACGTTT GAACAGGCCA TCCAGGCAGC CAGCCGGATC ATTCGAAACG AGCAGTTTGC CATCAGACGC TTCCAGTCTA TGCCGGTGAG GCTGCTGGGC CACAGCCCCG TGCTTCGGAA CATCACCAAC TCCCAGGCGC CCGACGGCCG GAGGAAGAGC GAGGCGGGCA GTGGAGCTGC CAGCAGCTCT GGGGAAGACA AGGAGAATGA TGGATTTGTC TTCAAGATGC CATGGAAGCC CACACATCCC AGCTCCACCC ATGCTCTGGC AGAGTGGGCC AGCCGCAGGG AAGCCTTTGC CCAGAGACCC AGCTCGGCCC CCGACCTGAT GTGTCTCAGT CCTGACCGGA AGATGGAAGT GGAGGAGCTC AGCCCCCTGG CCCTAGGTCG CTTCTCTCTG ACCCCTGCAG AGGGGGATAC TGAGGAAGAT GATGGATTTG TGGACATCCT AGAGAGTGAC TTAAAGGATG ATGATGCAGT TCCCCCAGGC ATGGAGAGTC TCATTAGTGC CCCACTGGTC AAGACCTTGG AAAAGGAAGA GGAAAAGGAC CTCGTCATGT ACAGCAAGTG CCAGCGGCTC TTCCGCTCTC CGTCCATGCC CTGCAGCGTG ATCCGGCCCA TCCTCAAGAG GCTGGAGCGG CCCCAGGACA GGGACACGCC CGTGCAGAAT AAGCGGAGGC GGAGCGTGAC CCCTCCTGAG GAGCAGCAGG AGGCTGAGGA ACCTAAAGCC CGCGTCCTCC GCTCAAAATC ACTGTGTCAC GATGAGATCG AGAACCTCCT GGACAGTGAC CACCGAGAGC TGATTGGAGA TTACTCTAAG GCCTTCCTCC TACAGACAGT AGACGGAAAG CACCAAGACC TCAAGTACAT CTCACCAGAA ACGATGGTGG CCCTATTGAC GGGCAAGTTC AGCAACATCG TGGATAAGTT TGTGATTGTA GACTGCAGAT ACCCCTATGA ATATGAAGGC GGGCACATCA AGACTGCGGT GAACTTGCCC CTGGAACGCG ACGCCGAGAG CTTCCTACTG AAGAGCCCCA TCGCGCCCTG TAGCCTGGAC AAGAGAGTCA TCCTCATTTT CCACTGTGAA TTCTCATCTG AGCGTGGGCC CCGCATGTGC CGTTTCATCA GGGAACGAGA CCGTGCTGTC AACGACTACC CCAGCCTCTA CTACCCTGAG ATGTATATCC TGAAAGGCGG CTACAAGGAG TTCTTCCCTC AGCACCCGAA CTTCTGTGAA CCCCAGGACT ACCGGCCCAT GAACCACGAG GCCTTCAAGG ATGAGCTAAA GACCTTCCGC CTCAAGACTC GCAGCTGGGC TGGGGAGCGG AGCCGGCGGG AGCTCTGTAG CCGGCTGCAG GACCAGTGA
3 - Détermination de l'activité des protéines de fusion MBP-Cdc25Bl, MBP-Cdc25B2 et MBP-Cdc25B3 :3 - Determination of the activity of the fusion proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3:
Les activités des protéines de fusion MBP-Cdc25Bl, MBP-Cdc25B2 et MBP-Cdc25B3 sont mesurées dans les mêmes conditions que celles décπtes pour la protéine de fusion MBP-Cdc25C. Les résultats obtenus sont reportés en figure 3The activities of the fusion proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3 are measured under the same conditions as those determined for the fusion protein MBP-Cdc25C. The results obtained are reported in Figure 3
LEGENDE DES FIGURESLEGEND OF FIGURES
La figure 1 (FIG 1) représente la chromatographie d'analyse relative à l'induction d'expression de la protéine de fusion MBP-Cdc25C Les lignes 1 et 2 de la figure 1 correspondent respectivement à l'extrait total de JM109/pMAL-Cdc25C avec ou sans ajout d'IPTG. La ligne 3 correspond à l'extrait soluble Les lignes 4 et 5 correspondent respectivement aux fractions non retenue et retenue sur amylose-agarose Enfin, la ligne 6 de la figure 1 correspond a la fraction d'elution n° 12 qui ne contient pratiquement que la protéine de fusionFIG. 1 (FIG 1) represents the analysis chromatography relating to the expression induction of the fusion protein MBP-Cdc25C The lines 1 and 2 of FIG. 1 correspond respectively to the total extract of JM109 / pMAL- Cdc25C with or without addition of IPTG. Line 3 corresponds to the soluble extract. Lines 4 and 5 correspond respectively to the fractions not retained and retained on amylose-agarose. Finally, line 6 of FIG. 1 corresponds to the elution fraction No. 12 which contains practically only fusion protein
La figure 2 (FIG. 2) représente les résultats de la mesure de l'activité de la protéine recombinante MBP-Cdc25C (un "+" signifiant que de la ménadione a été ajoutée à l'échantillon, un "-" que l'échantillon n'a pas été traité par de la ménadione) La figure 3 (FIG. 3) représente les résultats de la mesure de l'activité des protéines recombinantes MBP-Cdc25Bl, MBP-Cdc25B2 et MBP-Cdc25B3. La réaction est réalisée avec 300 ng d'enzyme par essai. La MBP à la même concentration est utilisée dans le contrôle. Les valeurs de fluorescence mesurées permettent le calcul des pentes : 0,0025 Δfluo/sec pour le contrôle, 0,0361 Δfluo/sec pour Cdc25Bl, 0,0350 Δfluo/sec pour Cdc25B2 et 0,0372 Δfluo/sec pour Cdc25B3. FIG. 2 (FIG. 2) represents the results of the measurement of the activity of the recombinant protein MBP-Cdc25C (a "+" signifying that menadione was added to the sample, a "-" that the sample was not treated with menadione) FIG. 3 (FIG. 3) represents the results of the measurement of the activity of the recombinant proteins MBP-Cdc25Bl, MBP-Cdc25B2 and MBP-Cdc25B3. The reaction is carried out with 300 ng of enzyme per test. MBP at the same concentration is used in the control. The measured fluorescence values allow the slopes to be calculated: 0.0025 Δfluo / sec for the control, 0.0361 Δfluo / sec for Cdc25Bl, 0.0350 Δfluo / sec for Cdc25B2 and 0.0372 Δfluo / sec for Cdc25B3.

Claims

Revendications claims
1. Protéine caractérisée en ce qu'il s'agit d'une protéine de fusion entre la protéine liant le maltose (MBP) et une protéine choisie parmi les protéines Cdc25Bl, Cdc25B2, Cdc25B3 et Cdc25C.1. Protein characterized in that it is a fusion protein between the maltose-binding protein (MBP) and a protein chosen from the proteins Cdc25Bl, Cdc25B2, Cdc25B3 and Cdc25C.
2. Protéine selon la revendication 1, caractérisée en ce qu'elle est choisie parmi les protéines suivantes : - une protéine de fusion entre la phosphatase Cdc25Bl humaine et la MBP, laquelle est codée par la séquence SEQ. ID n° 12 ;2. Protein according to claim 1, characterized in that it is chosen from the following proteins: - a fusion protein between human phosphatase Cdc25Bl and MBP, which is coded by the sequence SEQ. ID No. 12;
- une protéine de fusion entre la phosphatase Cdc25B2 humaine et la MBP, laquelle est codée par la séquence SEQ. ID n° 13 ;- a fusion protein between human Cdc25B2 phosphatase and MBP, which is coded by the sequence SEQ. ID No. 13;
- une protéine de fusion entre la phosphatase Cdc25B3 humaine et la MBP, laquelle est codée par la séquence SEQ. ID n° 14 ; et- a fusion protein between human Cdc25B3 phosphatase and MBP, which is coded by the sequence SEQ. ID # 14; and
- une protéine de fusion entre la phosphatase Cdc25C humaine et la MBP, laquelle est caractérisée en ce qu'elle est codée par la séquence SEQ. ID n° 1.- A fusion protein between human Cdc25C phosphatase and MBP, which is characterized in that it is coded by the sequence SEQ. ID # 1.
3. Protéine selon la revendication 2, caractérisée en ce qu'elle est codée par la séquence SEQ. ID n° l.3. Protein according to claim 2, characterized in that it is coded by the sequence SEQ. ID # 1.
4. ADN caractérisé en ce qu'il code pour une protéine selon l'une des revendications 1 à 3.4. DNA characterized in that it codes for a protein according to one of claims 1 to 3.
5. ADN complémentaire à l'ADN selon la revendication 4.5. DNA complementary to the DNA according to claim 4.
6. Souche bactérienne caractérisée en ce qu'il s'agit d'une souche bactérienne JM 109 transfectée par un plasmide choisi parmi le plasmide de séquence SEQ. ID n° 9, le plasmide de séquence SEQ. ID n° 10, le plasmide de séquence SEQ. ID n° 11 et le plasmide de séquence SEQ. ID n° 5.6. Bacterial strain characterized in that it is a bacterial strain JM 109 transfected with a plasmid chosen from the plasmid of sequence SEQ. ID No. 9, the plasmid with sequence SEQ. ID No. 10, the plasmid with sequence SEQ. ID No. 11 and the plasmid with sequence SEQ. ID # 5.
7. Procédé de préparation d'une protéine selon la revendication 1 , caractérisé en ce qu'il comporte les étapes successives suivantes :7. Method for preparing a protein according to claim 1, characterized in that it comprises the following successive steps:
- culture de la souche bactérienne JM 109 transfectée par le plasmide de séquence SEQ. ID n° 9, le plasmide de séquence SEQ. ID n° 10, le plasmide de séquence- Culture of the bacterial strain JM 109 transfected with the plasmid of sequence SEQ. ID No. 9, the plasmid with sequence SEQ. ID No. 10, the plasmid with sequence
SEQ. ID n° 11 ou le plasmide de séquence SEQ. ID n° 5, dans un milieu LB additionné d'ampicilline ; - induction de la synthèse de la protéine de fusion par ajout d'isopropylthiogalactoside ;SEQ. ID No. 11 or the plasmid with sequence SEQ. ID No. 5, in LB medium supplemented with ampicillin; - induction of the synthesis of the fusion protein by addition of isopropylthiogalactoside;
- lyse des bactéries ;- lysis of bacteria;
- purification de la protéine de fusion obtenue par chromatographie sur résine d'amylose-agarose et récupération des fractions contenant la protéine purifiée.- Purification of the fusion protein obtained by chromatography on amylose-agarose resin and recovery of the fractions containing the purified protein.
8. Application d'une protéine selon la revendication 1 à une méthode d'identification de modulateurs de la protéine Cdc25Bl, Cdc25B2, Cdc25B3 ou Cdc25C, caractérisée en ce que ladite méthode comporte les étapes successives suivantes :8. Application of a protein according to claim 1 to a method of identifying modulators of the protein Cdc25Bl, Cdc25B2, Cdc25B3 or Cdc25C, characterized in that said method comprises the following successive steps:
- ajout, à une solution de phosphate de 3-O-méthylfluorescéine, de la protéine de fusion telle qu'obtenue par un procédé de préparation selon la revendication 7 et d'un composé présumé être un modulateur de la protéine Cdc25Bl, Cdc25B2, Cdc25B3 ou Cdc25C ;- addition, to a 3-O-methylfluorescein phosphate solution, of the fusion protein as obtained by a preparation process according to claim 7 and of a compound presumed to be a modulator of the protein Cdc25Bl, Cdc25B2, Cdc25B3 or Cdc25C;
- détermination de la quantité de 3-O-méthylfluorescéine produite rapportée à la quantité initiale de phosphate de 3-O-méthylfluorescéine.- Determination of the amount of 3-O-methylfluorescein produced compared to the initial amount of 3-O-methylfluorescein phosphate.
9. Application selon la revendication 8, caractérisée en ce que la détermination de la quantité de 3-O-méthylfluorescéine produite rapportée à la quantité initiale de phosphate de 3-O-méthylfluorescéine est effectuée par mesure de l'absorbance liée à la 3-O-méthylfluorescéine à la longueur d'onde de 477 nm.9. Application according to claim 8, characterized in that the determination of the amount of 3-O-methylfluorescein produced relative to the initial amount of 3-O-methylfluorescein phosphate is carried out by measuring the absorbance linked to 3- O-methylfluorescein at the wavelength of 477 nm.
10. Application selon la revendication 8, caractérisée en ce que la détermination de la quantité de 3-O-méthylfluorescéine produite rapportée à la quantité initiale de phosphate de 3-O-méthylfluorescéine est effectuée par fluorimétrie en utilisant une excitation à la longueur d'onde de 475 nm et une lecture à la longueur d'onde de 510 nm. 10. Application according to claim 8, characterized in that the determination of the amount of 3-O-methylfluorescein produced relative to the initial amount of 3-O-methylfluorescein phosphate is carried out by fluorimetry using excitation to the length of 475nm wave and a 510nm wavelength reading.
EP00993418A 1999-12-14 2000-12-13 Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators Withdrawn EP1242599A2 (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
FR9915722 1999-12-14
FR9915722A FR2815347A1 (en) 1999-12-14 1999-12-14 New fusion protein of maltose-binding protein and Cdc phosphatase, useful for identifying phosphatase modulators for regulating the cell cycle
FR0006883 2000-05-30
FR0006883A FR2812292A1 (en) 2000-05-30 2000-05-30 New fusion protein of maltose-binding protein and Cdc phosphatase, useful for identifying phosphatase modulators for regulating the cell cycle
FR0012008 2000-09-21
FR0012008 2000-09-21
PCT/FR2000/003496 WO2001044467A2 (en) 1999-12-14 2000-12-13 Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators

Publications (1)

Publication Number Publication Date
EP1242599A2 true EP1242599A2 (en) 2002-09-25

Family

ID=27248667

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00993418A Withdrawn EP1242599A2 (en) 1999-12-14 2000-12-13 Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators

Country Status (5)

Country Link
EP (1) EP1242599A2 (en)
JP (1) JP2003516756A (en)
AU (1) AU2855901A (en)
CA (1) CA2394152A1 (en)
WO (1) WO2001044467A2 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0286239B1 (en) * 1987-03-10 1996-03-13 New England Biolabs, Inc. Production and purification of a protein fused to a binding protein
AU2921697A (en) * 1996-05-02 1997-11-19 Pharmacia & Upjohn Company Catalytic macro molecules having cdc25b like activity
WO1998030680A2 (en) * 1997-01-13 1998-07-16 Pharmacia & Upjohn Company Special catalytic domains of cdc25c phosphatase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0144467A3 *

Also Published As

Publication number Publication date
WO2001044467A2 (en) 2001-06-21
WO2001044467A3 (en) 2001-12-27
CA2394152A1 (en) 2001-06-21
JP2003516756A (en) 2003-05-20
AU2855901A (en) 2001-06-25

Similar Documents

Publication Publication Date Title
EP2013337B1 (en) A group of esterases for the enantioselective production of fine and speciality chemicals
CN102421892A (en) Diguanylate cyclase, method for producing same and use thereof for producing cyclic di-GMP and analogues thereof
CN111041011B (en) Glyphosate oxidase mutant and cloning, expression and application thereof
CN107267474B (en) Dihydrothiooctanoic amide dehydrogenase mutant protein, and preparation method and application thereof
CN111187785B (en) Cloning expression and application of serpentis grass tryptophan decarboxylase gene OpTDC2
CN109706130A (en) Fructoamino-acid-oxidase mutant, detection kit and detection method
KR20050074313A (en) Cephalosporin c acylases
EP1242599A2 (en) Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators
CN108034646B (en) PvEH3 mutant with improved catalytic activity and improved enantiotropic normalization
US20040029204A1 (en) Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators
CN113604453B (en) Marine streptomycete phospholipase D mutant and application
Olchowy et al. Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase
Pan et al. The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport
TW200531978A (en) Removal of n-terminal methionine from proteins by engineered methionine aminopeptidase
FR2812292A1 (en) New fusion protein of maltose-binding protein and Cdc phosphatase, useful for identifying phosphatase modulators for regulating the cell cycle
CN110951711B (en) Esterase with activity of degrading chiral ester and coding gene and application thereof
KR101383546B1 (en) Esterases KTL4 Isolated from Deep Sea Sediment
JPH03502042A (en) Gene encoding glucuronide permease
Hsu et al. A Flexible peptide tether controls accessibility of a unique C-terminal RNA-binding domain in leucyl-tRNA synthetases
Tsai et al. The structure of human leucine carboxyl methyltransferase 1 that regulates protein phosphatase PP2A
Moe et al. Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod
KR20120129852A (en) Esterases KTL 9 Isolated from Deep Sea Sediment
KR20170027599A (en) Paraoxonase 1 mutants with enhanced hydrolytic proficiency for organophosphate paraoxone and and method for manufacturing the same
KR20220072793A (en) Method for synthesizing a biocatalyst of formaldehyde using a multi-enzyme-catalyzed fusion protein scaffold and its use
CN116590268A (en) Bile salt hydrolase with enhanced thermal stability, preparation method and application

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020715

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20031229

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040709