WO1998030680A2 - Special catalytic domains of cdc25c phosphatase - Google Patents

Special catalytic domains of cdc25c phosphatase Download PDF

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Publication number
WO1998030680A2
WO1998030680A2 PCT/US1998/000018 US9800018W WO9830680A2 WO 1998030680 A2 WO1998030680 A2 WO 1998030680A2 US 9800018 W US9800018 W US 9800018W WO 9830680 A2 WO9830680 A2 WO 9830680A2
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Prior art keywords
leu
cdc25c
glu
gin
seq
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PCT/US1998/000018
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French (fr)
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WO1998030680A3 (en
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Martin R. Deibel, Jr.
Anne F. Vosters
Anthony W. Yem
Keith D. Watenpaugh
Musiri N. Janakiraman
Luis A. Parodi
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Pharmacia & Upjohn Company
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Priority to AU60149/98A priority Critical patent/AU6014998A/en
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Publication of WO1998030680A3 publication Critical patent/WO1998030680A3/en

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    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to the field of protein phosphatases, specifically cdc25C like enzymes.
  • VHR Human dual specificity phosphatase VHR activates maturation promotion factor and triggers meiotic maturation in Xenopus oocytes. J. Biol. Chem. 9 June 1995; 270(23): 14229-34.
  • Speculation that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.
  • cdc25 + encodes a protein phosphatase that dephosphorylates p34 cdc2 . Mol. Biol. Cell. 3: 73-84 (1992). Discloses an expressed full length form of cdc25C. J.B.A. Millar, CH. McGowan, G. Lenaers, R. Jones, and P. Russell, EMBO J, vol. 10, pp. 4301-4309 (1991) "p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast.”
  • M-phase specific histone kinase or more simply as the M-phase kinase.
  • This kinase consists of at least three subunits; the catalytic subunit (cdc2), a regulatory subunit (cyclin B) and a low molecular weight subunit (pl3-Sucl).
  • cdc25 is the cdc2 phosphatase, Kumagai, A. and W. G. Dunphy, Cell 64:903-914 (1991) and Strausfeld, U. et al., Nature 351:242-245 (1991).
  • cdc25 acts as a cdc2 phosphatase which dephosphorylates tyrosine and possibly threonine residues on p34 cdc thus regulating MPF activation, Dunphy, W. G. and A. Kumagai, Cell 67:189-196 (1991) and Gautier, J. et al., Cell 67:197-211 (1991). Because cdc25 phosphatases are responsible for the dephosphorylation and activation of cyclin-dependent protein kinases, they help control cell cycle progression.
  • A, B, and C are responsible for the dephosphorylation and activation of cyclin- dependent protein kinases; thus, there is interest in understanding how cdc25 phosphatases are regulated.
  • genetic studies in fission yeast have established that the cdc25 gene is essential for the initiation of mitosis, P. Nurse, P. Thuriaux, and K. Nasmyth. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146: 167-178 (1976), and the cdc25 gene product has been shown to serve as a rate-determining activator of the cdc2 protein kinase. See, P. Russell and P. Nurse. Cdc25 + functions as an inducer in the mitotic control of fission yeast. Cell 45: 145-153 (1986).
  • This region of the protein is capable in some cases of exhibiting enzymatic activity or biological complementation in non-bacterial cell systems. See, A. Nagata, M. Igarashi, S. Jinno, K. Suto, and H. Okayama. An additional homolog of the fission yeast cdc25 + gene occurs in humans and is highly expressed in some cancer cells. New Biol. 3: 959-968 (1991). T. Horiguchi, K. Nishi, S. Hakoda, S. Tanida, A. Nagata, and H. Okayama. Dnacin Al and Dnacin Bl are antitumor antibiotics that inhibit cdc25B phosphatase activity.
  • Cdc25 + encodes a protein phosphatase that dephosphorylates p34 . Mol. Biol. Cell. 3: 73-84 (1992). J. Gautier, M.J. Solomon, R.N. Booker, J.F. Bazan, and M.W. Kirschner. Cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell 67: 197-211 (1991). A. Kumagai and W.G. Dunphy.
  • the cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system.
  • GST glutathione S-transferase
  • This invention comprises, the fusions comprising the fusions shown below
  • the fusion may be comprised of DNA or amino acids, where the different parts of the fusions are shown as different lines in the box, where a) the GST portion, is labeled GST, with a straight line in the box, b) the protease cleavage site is shown as a dotted line in the box, labeled "P,” c) the restriction site is shown as a wavey line in the box, labeled "R,” and d) the cdc25C like portion is shown as a heavy line in the box, labelled "cdc25C" where the numbers above the box indicate DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either DNA or amino acids, where the boxes, lines and numbers are not drawn, as shown, to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25C region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length
  • fusion protease cleavage site is created to be responsive to Factor Xa.
  • fusion restriction site when expressed, yields the amino acid sequence GIQ.
  • the fusion consisting of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues. The process of making any of the fusion proteins or peptides described above.
  • DNA that codes for the fusion protein are described and particular DNA constructs are described such as where the DNA coding for the various fusion proteins.
  • the fusion consists of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues.
  • the process of making any of the DNA constructs is also described.
  • Essential nucleic acid intermediates are described.
  • the process of making any of the DNA fusions is also described. In addition to the fusions above, the fusion below is disclosed.
  • the fusion may be comprised of DNA or amino acids, where the different parts of the fusions are shown as different lines in the box, where a) the GST portion, is labeled GST, with a straight line in the box, b) the protease cleavage site is shown as a dotted line in the box, labeled "P,” c) a stop codon appears in the DNA sequence, but not the amino acid sequence, d) the restriction site is shown as a wavey line in the box, labeled "R,” and e) the cdc25C like portion is shown as a heavy line in the box, labelled "cdc25C" where the numbers above the box indicate DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either DNA or amino acids, where the boxes, lines and numbers are not drawn, as shown, to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25C region has
  • fusion protease cleavage site is created to be responsive to Factor Xa.
  • fusion restriction site when expressed, yields the amino acid sequence GIQ.
  • the fusion consisting of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues. The process of making any of the fusion proteins or peptides described above.
  • DNA that codes for the fusion protein are described and particular DNA constructs are described such as where the DNA coding for the various fusion proteins.
  • the fusion consists of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues.
  • Essential nucleic acid intermediates are described.
  • the process of making any of the DNA fusions is also described.
  • the sequences from the CHARTS and SEQUENCE LISTINGS are disclosed in addition to the process of making the various fusions, constructs or molecules.
  • Figure LA shows the plasmid pGEX-5X-3 used in the cloning of recombinant human cdc25O This figure shows the position of the factor Xa cleavage site sequence, various restriction sites, and location of the cDNA inserts for cdc25C
  • Figure IB is an expanded portion of the plasmid shown in Figure 1A.
  • Figure IB Figure IB contains the expanded portion of the segment shown in Figure 1A.
  • Figure 2 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part A, DNA residues 982 through 1632, showing the product of the PCR reaction (lane P), as well as DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position “a” shows the migration of the linearized plasmid pGEX-5X-3, and position "b” shows the migration of the cdc25C DNA insert/PCR product. Lane A shows a 1 kb DNA molecular weight marker ladder.
  • Figure 3 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing the product of the PCR reaction (lane 1) with template DNA and primers, while lane 2 shows the results of a control PCR reaction lacking template DNA (lane 2). Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B.
  • Figure 4 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position “a” shows the migration of the linearized plasmid pGEX-5X-3, and position "b” shows the migration of the cdc25C DNA insert. Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B.
  • BCIP is 5-bromo-4-chloro-3-indolyl phosphate.
  • Bio 101® Many of the kits used in this invention such as “the GeneClean® kit” and “a RPM® plasmid isolation kit” (RPM-rapid pure minipreps) are obtained from Bio 101®., a company in LaJolla, California.
  • Competent E. coli cells JM109 are a strain of commonly available cells.
  • IPTG is isopropyl-J-D-thiogalactopyranoside from Boehringer Mannheim, Indianapolis, Indiana.
  • LB media is a solution containing tryptone, yeast extract, sodium chloride and water. It is commerically available from Gibco-BRL.®, Gaithersburg, Maryland.
  • M9 medium containes dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, and ammonium chloride 1 g per L of deionized water.
  • M9YE medium is the same medium as M9 plus yeast extract, 5 g per L of deionized water.
  • One hundred ml vols. of M9YE contained in 500 ml wide mouth fermentation flasks were sterilized by autoclaving for 30 min. The sterilization pH was adjusted to 7.3 with KOH.
  • One L of sterile basal M9YE was prepared through the aseptic addition of filtered sterilized 1M MgSO4, 2ml; 20% glucose, 20 ml and lM CaC12, 0.1 ml.
  • MORPH® is a site-specific plasmid DNA mutagenesis kit obtained from 5 PRIME->3 PRIME, Inc.®, Boulder, Colorado.
  • “native extraction/buffer systems” are common extraction buffer systems such as, lysozyme (1 mg/ml) and fresh dithiothreitol (DTT) (20 mM) in TEN buffer (50 mM Tris HCl, 0.5 mM EDTA, 300 mM NaCl, 0.2% NP-40, pH 8.0)
  • NBT nitroblue tetrazolium
  • PAGE polyacrylamide gel electrophoresis
  • PNPP hydrolase activity associated with cdc25B are conducted using the reagents described by Horiguchi et al. (Biochemical Pharmacology, Vol. 48 pp. 2139-2141, (1994)). This is what is meant by “enzymatically active in a defined way with the colorimetric substrate, p-nitrophenol phosphate (PNPP)." "PVDF 1 is polyvinylidene difluoride.
  • SDS sodium dodecyl sulfate
  • TA cloning kit containing the pCRII plasmid, and INVIF' cells is obtained from InVitroGen®, San Diego, California
  • Temperatures are in degrees celcius unless noted otherwise and may be indicated with a number, a number supercase o, a number uppercase c, a number supercase o uppercase c or other obvious combinations or methods, e.g. 37, 37°, 37 C, 37° C, etc.
  • the present invention relates to a method of regulating (inhibiting or enhancing) cell division and to agents or compositions useful for regulating the cell cycle.
  • the present invention has the same uses as previously disclosed human cdc25C in addition to having other uses not possible with previously disclosed human cdc25C because of its physical characteristics.
  • Described herein is a novel recombinant fusion construct that produces a macromolecule that is soluble and that performs similar biochemical functions as full length cdc25C constructs, but is more active, more soluble, and does not require refolding.
  • the compounds described herein are useful for crystallography and for drug development screening tools. These compounds should also allow improved structure-based design for the development of novel phosphatase antagonists, the latter being expected to result in an anti-neoplastic drug.
  • the macro molecules described herein will make superior drug screening tools over previously disclosed cdc25C proteins because of their enhanced activity.
  • the compounds described herein are superior over known compounds, proteins and peptides for studies of cdc25C enzyme kinetics and mechanistic studies because these novel compounds are monomeric in structure and because the uniquely designed sequences do not display anomalies present in inhibitor kinetics seen with known proteins (e.g. GST-cdc25 fusions).
  • the compounds described herein can be created without a subsequent refolding step, thus providing simple consistent procedures for making highly active compounds.
  • the compounds described herein would make superior subjects of crystallization studies because of their solubility properties.
  • the compounds disclosed herein should be particularly useful for transfection studies in mammalian cells designed to test in vivo mechanism of action and proof of concept studies.
  • the GST-cdc25C full length enzyme apparently cannot be purified to homogeneity using prior art descriptions of purification of GST fusion proteins.
  • the compounds and procedures disclosed herein do allow the creation of highly purified and homogeneous active protein, as defined by several criteria.
  • VHR vaccinia Hl-related phosphatase
  • cdc25C proteins both full length as well as truncated minimal domains, were usually created as a GST fusion with cdc25C These fusion proteins were expressed in E. coli as partially soluble and low activity proteins. No attempts were made to remove the GST fusion partner, see M.S. Lee, S. Ogg, M. Xu, L.L. Parker, D.J. Donoghue, J.L. Mailer, and H. Piwnica- Worms. cdc25 + encodes a protein phosphatase that dephosphorylates p34 c . Mol. Biol. Cell. 3: 73-84 (1992). Note that prominent in these domains is a highly conserved HCXXXXXR signature sequence.
  • the products of the ligations were used for transformation of competent E. coli cells (JM109).
  • the constructs expressed in E. coli were designed as fusion proteins of GST with cdc25C, with an intervening factor Xa cleavage site.
  • the special GST-cdc25C constructs are then optimized. Methods.
  • AFV- 3 GAT CCA GTT AAA GAA GAC AGT CT
  • AFV- 2 GCC TCG AGT CAT GGG CTC ATG TCC TT
  • a reaction (final volume of 100 ul) containing 20 pmol AFV-2 , 20 pmol AFV-3 , 12 ng cdc25C template in 200 ⁇ M dNTPs, IX PCR Buffer [(Perkin-Elmer GeneAmp), 1.5 mM MgCl 2 , 50 mM KC1, 10 mM Tris-HCl pH 8.3, 0.001% (w/v) gelatin], and sterile water was used in the PCR reaction to generate the desired insert.
  • IX PCR Buffer (Perkin-Elmer GeneAmp), 1.5 mM MgCl 2 , 50 mM KC1, 10 mM Tris-HCl pH 8.3, 0.001% (w/v) gelatin
  • the product of the ligation was transformed into INV ⁇ F 1 cells (according to manufacturer's directions), and after overnight incubation of the cells at 37°C on L broth agar plates with ampicillin and Xgal, the resulting DNA of selected colonies was isolated using a Bio 101 DNA isolation kit, digested with Bam HI and Xho I, and purified by agarose gel electrophoresis.
  • IX ligase buffer 50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene glycol 8000, (Gibco BRL)], 1 ul dATP (G
  • PGEX-5X-3/cdc25C (982-1632) insert.
  • E. Coli Strain JM109 5 ul of the ligation reaction was added to 100 ul of Promega competent cells (E. coli). This suspension was incubated on ice for 1 hour, incubated at 42°C for 90 seconds, and cooled on ice for 1 minute. 250 ul of SOC medium was added, followed by incubation at 37°C with shaking for 1 hour. The mixture was used for application of 10, 100 and 200 ul aliquots onto (1.5%) agar plates containing LB medium and ampicillin (100 ⁇ g ml). After streaking the agar plates with the transformation mixture, the plates were incubated overnight at 37°C Colonies were abundant on PGEX-5X-3/cdc25C insert plates and sparse on control plates.
  • Fluorescence-based sequencing of DNA templates were done using the ABI PRISM Ready Dye-Deoxy Terminator FS-kits with Taq FS polymerase (Perkin-Elmer/Applied Biosystems Division, PE/ABI, Foster City, CA) and the ABI373A fluorescence-based sequencer (stretch upgrade). The temperature cycles for cycle sequencing were controlled using an automated PCR thermal cycler (Perkin Elmer 9600). Fluorescence-based sequencing was done using about 0.5 ⁇ g of DNA and the following cycle sequencing reaction conditions: initial denaturation at 98°C for 1 min, followed by 35 cycles of 98°C for 15 sec, annealing at 50°C for 10 sec and extension at 60 °C for 4 min.
  • Extension products are purified using 1 ml spin columns (Centri-sep, from Princeton Separations, Inc., Adelphia, NJ). Each reaction product is loaded by pipette onto the column, which is then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B table top centrifuge) at 750 x g for 2 min at room temperature. Column- purified samples are dried under vacuum for about 40 min and then dissolved in 5 ul of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml blue dextran). The samples are then heated to 90°C for three min and loaded into the gel sample wells for sequence analysis by the ABI373A sequencer.
  • a DNA loading solution 83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml blue dextran
  • Sequence analysis was done by importing ABI373A files into the Sequencher program, obtained from Gene Codes (Ann Arbor, MI), and generally sequence readings of 700 to 800 bp were obtained. DNA sequencing errors were minimized by obtaining sequence information from both DNA strands.
  • E. coli was inoculated into 100 ml vols. of M9 medium containing yeast extract at 0.5% (M9YE) contained in 500 ml large mouth fermentation flasks. The medium contained 100 mg of ampicillin/L. The inoculated flasks were incubated for 16 hr at 37C while shaking at 200 rpm. M9YE was prepared as described below. Production fermentation: Production flasks (as above) containing M9YE with filter sterilized ampicillin (as above) were inoculated with the mature seed fermentation at a 3% rate.
  • M9YE medium contained dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, ammonium chloride 1 g and yeast extract, 5 g per L of deionized water.
  • M9YE contained in 500 ml wide mouth fermentation flasks were sterilzied by autoclaving for 30 min. The presterlization pH was adjusted to 7.3 with KOH. One L of sterile basal M9YE was completed through the aseptic addition of filtered sterilized 1M MgSO4, 2ml; 20% glucose, 20 ml and 1M CaC12, 0.1 ml.
  • the slurry was poured into a column and washed extensively with the lysis buffer described above (w/o lysozyme). Finally the protein charged column matrix was washed with factor Xa digestion buffer (50mM Tris HCl, lOOmM NaCl, and ImM CaCl 2 pH 8.), followed by the addition of 125 ug of Factor Xa (Boehringer) with mixing in 20ml of digestion buffer. After 12-14hrs incubation at 8°C the Xa released material is collected and concentrated by Amicon ultrafiltration (YM-10 filter). The concentrate is diluted 3x with deionized water to lower the salt concentration for the ion exchange chromatography step.
  • factor Xa digestion buffer 50mM Tris HCl, lOOmM NaCl, and ImM CaCl 2 pH 8.
  • Electrophoresis- SDS gel electrophoresis were performed according to Laemmli. Western blots were completed using a semi-dry electroblotter onto PVDF membranes (Millipore) with a constant current set at 125 mA/gel (7x9 cm). Blots were visualized by staining with Coomassie blue R250 (.2% W/V) in 50% Ethanol, 5% Acetic acid followed by a destaining step using 50% ethanol solution.
  • Enzvme assay and Kinetic Analyses- Assays of PNPP hydrolase activity associated with cdc25C are conducted using the reagents described previously. These reagents include (as final concentrations in 125 ul): 25 mM Hepes, pH 8.0, 10 mM DTT, 0.1 mg/ml bovine serum albumin, and variable concentrations of pNPP. For assays where a single concentration of substrate is used at saturation, we customarily use a final concentration of 20 mM pNPP. Assay solution is prepared in a final volume of 100 ul, including the addition of freshly prepared dithiothreitol.
  • the experiment was designed so that the resulting protein should have been a glutathione S-transferase (GST) tag linked to the cdc25C protein encompassing amino acid residues 258-473 of the full length protein.
  • GST glutathione S-transferase
  • a factor Xa cleavage site allowed for cleavage of the GST tag from the truncated cdc25C protein.
  • Three additional amino acid residues (Gly-Ile-Gln) were engineered into the N-terminus preceding the cdc25C sequence, but after the factor Xa sequence, IEGR.
  • the fusion protein was purified away from E. coli proteins by affinity chromatography on glutathione Sepharose.
  • the cdc25C protein was proteolytically cleaved from the GST polypeptide, while the latter was still attached to the affinity matrix, using factor Xa protease.
  • the solubilized, truncated cdc25C protein was chromatographed on QAE Sepharose to provide a product consisting of only cdc25C protein.
  • the final product was a protein initiating at Leu258 and terminating at Arg446, with the Gly-Ile-Gln sequence preceding Leu258.
  • Validation of the sequence was completed by N-terminal sequencing which showed that the sequence initiated with GIQ..., and by mass spectroscopy which showed a mass ion consistent with a protein terminating with Arg446. Therefore, this truncated domain is produced by factor Xa cleavage at an unexpected site within the cdc25C protein sequence.
  • the protein which begins as expected (Gly-Ile-Gln-Leu258%), but terminates at an unexpected position (Arg446).
  • PNPP p-nitrophenylphosphate
  • This sequence is a human cdc25C minimal domain free of GST which is soluble and active.
  • the recombinant fusion protein produced in E. coli, is designed to have a glutathione S-transferase (GST) tag linked to the cdc25C protein encompassing amino acid residues 258-473 of the full length protein.
  • GST glutathione S-transferase
  • a factor Xa cleavage site (IEGR) is situated immediately after the GST polypeptide, and prior to the beginning of the cdc25C sequence. Three additional amino acid residues are positioned immediately after the factor Xa cleavage site as a result of the restriction site nucleotide coding sequence.
  • the factor Xa cleavage site allows for cleavage of the GST tag from the truncated cdc25C protein using factor Xa protease.
  • the progress can be followed by SDS polyacrylamide gel electrophoresis and Western blotting, leading to the observation of a 20-24 kD cdc25C product.
  • Small amounts of the two bacterial chaperonins (DnaK and GroEL) from E. coli were detected.
  • the products were resolved by anionic exchange chromatography.
  • the location of the cdc25C fractions was made by SDS PAGE as well as by PNPP hydrolytic activity profiles.
  • Mass spectrometry A mass ion representing the major product of the cdc25C protein preparation has been identified at a mass corresponding directly to the sequence of GIQ-cdc25C (258-446). This indicates the factor Xa not only processes the correct engineered factor Xa cleavage site, but also cleaves C-terminal to Arg446.
  • Figure 1A shows the plasmid pGEX-5X-3 used in the cloning of recombinant human cdc25C. This figure shows the position of the factor Xa cleavage site sequence, various restriction sites, and location of the cDNA inserts for cdc25C.
  • Figure 1A is an expanded portion of the plasmid shown in Figure IB.
  • Figure IB Figure IB shows the plasmid construction of plasmid pGEX-5X-3.
  • Figure IB contains the expanded portion of the segment shown in Figure 1A.
  • Figure 2 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part A, DNA residues 982 through 1632, showing the product of the PCR reaction (lane P), as well as DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel.
  • Position "a” shows the migration of the linearized plasmid pGEX-5X-3
  • position "b” shows the migration of the cdc25C DNA insert/PCR product.
  • Lane A shows a 1 kb DNA molecular weight marker ladder.
  • the colorimetric substrate, p-Nitrophenyl phosphate (PNPP) is from Sigma Diagnostics®. All other reagents used are of the purest grade available. Original Substrate.
  • the cDNA encoding the entire sequence of human cdc25C was prepared for use as a template for the polymerase chain reaction (PCR) by first linearizing with Hind III. Subsequently, the cDNA was isolated by gel electrophoresis in 0.8% agarose, followed by purification using a Geneclean kit® (Bio 101®). PCR Cloning.
  • CAVHR-4 (GCC TCG AGT CAC ATA GGG CAG TAG CTC) (SEQ. ID. 14) (the italicized letters indicate the positions of engineered restriction sites).
  • a reaction final volume of 100 ul) containing 20 pmol CAVHR-3, 20 pmol CAVHR-4 , 12 ng cdc25C template, 200 TM dNTPs, IX PCR Buffer ((Perkin-Elmer GeneAmp), 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.001% (w/v) gelatin), and sterile water was used in the PCR reaction to generate the desired insert.
  • Ligation of the TA cloning product into PGEX-5X-3- PGEX-5X-3 was prepared for ligation by digestion with the restriction enzymes BamHI and EcoRI at 37°C in 50 mM Tris-HCL (pH 8.0), 10 mM MgCl 2 and 50 mM NaCl (React 2 buffer, Gibco BRL®).
  • the resultant linearized plasmid was subjected to electrophoresis on a 0.8% agarose gel in IX TAE (Tris-acetate-EDTA buffer).
  • the gel was soaked in IX TAE with 0.5 Tg/ml ethidium bromide (EtBr) so that the DNA could be visualized under long-wave UV.
  • EtBr ethidium bromide
  • DNA ligase (Gibco BRL), IX ligase buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene glycol 8000, (Gibco BRL)), 1 mM dATP (Gibco BRL), and sterile H 2 0 was prepared to give a total volume of 10 ul.
  • IX ligase buffer 50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene glycol 8000, (Gibco BRL)), 1 mM dATP (Gibco BRL), and sterile H 2 0 was prepared to give a total volume of 10 ul.
  • E. coli was inoculated into 100 ml vols. of M9 medium containing yeast extract at 0.5% (M9YE) contained in 500 ml large mouth fermentation flasks. The medium contained 100 mg of ampicillin/L. The inoculated flasks were incubated for 16 hr at 37C while shaking at 200 rpm. M9YE was prepared as described below. Production fermentation: Production flasks (as above) containing M9YE with filter sterilized ampicillin (as above) were inoculated with the mature seed fermentation at a 3% rate. This fermentation was continued for 2.25 hr at 37C while shaking at 200 rpm until the turbidity at 660 nm reached ca. 1.0.
  • M9YE medium contained dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, ammonium chloride 1 g and yeast extract, 5 g per L of deionized water.
  • M9YE contained in 500 ml wide mouth fermentation flasks were sterilzied by autoclaving for 30 min.
  • the presterlization pH was adjusted to 7.3 with KOH.
  • One L of sterile basal M9YE was completed through the aseptic addition of filtered sterilized 1M MgS04, 2ml; 20% glucose, 20 ml and 1M CaC12, 0.1 ml.
  • the slurry was poured into a column and washed extensively with the lysis buffer described above (w/o lysozyme). Finally, the protein charged column matrix was washed with factor Xa digestion buffer (50 mM Tris HCl, 100 mM NaCl, and 1 mM CaCl 2 pH 8.), followed by the addition of 125 ug of Factor Xa with mixing in 20- 30 ml of digestion buffer. After 12-14 hrs incubation at 8°C the Xa released material is collected and concentrated by Amicon® ultrafiltration (YM-10 filter). The concentrate is diluted 3x with deionized water to lower the salt concentration for the ion exchange chromatography step.
  • factor Xa digestion buffer 50 mM Tris HCl, 100 mM NaCl, and 1 mM CaCl 2 pH 8.
  • Blots were visualized by either staining with Coomassie blue R250 (.2% W/V) in 50% Ethanol, 5% Acetic acid followed by a destaining step using 50% ethanol solution, or alternatively, were processed for immunostaining.
  • Assays of PNPP hydrolase activity associated with cdc25C are conducted using the reagents described previously. These reagents include (as final concentrations in 125 ul): 25 mM Hepes, pH 8.0, 10 mM DTT, 0.1 mg/ml bovine serum albumin, and variable concentrations of pNPP. For assays where a single concentration of substrate is used at saturation, we customarily use a final concentration of 20 mM pNPP. Assay solution is prepared in a final volume of 100 ul, including the addition of freshly prepared dithiothreitol.
  • a 96-well plate assay is also used for the purpose of rapidly handling multiple samples.
  • K ⁇ and V ma ⁇ specific activity
  • multiple pNPP concentrations are used at a constant enzyme concentration. Rates at each concentration of substrate are determined and the 1 ⁇ and V ma ⁇ calculated from line fitting to a Michaelis Menten equation.
  • the velocity of the reaction is defined as follows: One unit of activity is defined as nmoles of pNPP hydrolyzed per minute per milligram of enzyme protein at 25°C.
  • Characteristics of the purified truncated recombinant human cdc25C gg_ 35 The purified protein exhibits a single peak when analyzed by C4 reverse phase HPLC.
  • the amino terminus of the purified cdc25C protein was determined to be G-I-Q -D 266 - etc.
  • a mass ion representing the major product of the cdc25C protein preparation has been identified at about 20,112 daltons. This mass corresponds directly to the sequence of GIQ-cdc25C (266-435). Both the fusion protein and the minimal domain of cdc25C (266-435) were shown to be enzymatically active as a phosphatase against p-nitrophenylphosphate (PNPP).
  • PNPP p-nitrophenylphosphate
  • Figure 1A Figure LA shows the plasmid pGEX-5X-3 used in the cloning of recombinant human cdc25C. This figure shows the position of the factor Xa cleavage site sequence, various restriction sites, and location of the cDNA inserts for cdc25C.
  • Figure 1A is an expanded portion of the plasmid shown in Figure IB.
  • Figure IB Figure IB shows the plasmid construction of plasmid pGEX-5X-3.
  • Figure IB contains the expanded portion of the segment shown in Figure 1A.
  • Figure 3 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing the product of the PCR reaction (lane 1) with template DNA and primers, while lane 2 shows the results of a control PCR reaction lacking template DNA (lane 2).
  • Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B.
  • Figure 4 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position “a” shows the migration of the linearized plasmid pGEX-5X-3, and position "b” shows the migration of the cdc25C DNA insert. Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B. Part C (Advanced Forms of the Macromolecule)
  • cdc25C An improved form of cdc25C (258-473) is also disclosed.
  • Arg-Cys-Arg there are two arginines, one of which is readily attacked by factor Xa during the cleavage step.
  • these arginines were replaced by alanines, and the single cysteine was replaced with serine using mutagenesis strategies. These substitutions are designed to prevent the factor Xa processing of this site which occurs during the normal processing of the GST fusion protein.
  • wild type protein is digested by factor Xa it usually generates cdc25C (258-446), which contains a large amino acid truncation at the C-terminus.
  • ADVANCED FORM SEQUENCE COMPARISON Experimental design.
  • the top line represents the mutated cDNA sequence, while the second line represents the wild type sequence. These sequences are followed by the corresponding antisense sequences.
  • SEQ. ID. NO. 15 ...AAGACTGAGTTGCTGAGGTGTCGAAGCCAGAGCAAAGTGCAGGAAGGGGAGCGGCAGCTG..
  • GST-IEGR-GIQ-cdc25C (258-473, A446S447A448) was created using the clone pGEX-5X-3 with the insert 982-1632 as a template.
  • the quickchange (Stratagene) system was used with two mutagenic oligonucleotide primers designed to allow change of Arg446-Cys-447-Arg448 into Ala446-Ser447-Ala448.
  • MRD597-1 5' ACT GAG TTG CTG GCG TCT GCA AGC CAG AGC AAA (SEQ ID 23 corresponding to SEQ. ID.
  • the steps utilized in the mutagenesis are as follows: The original cDNA construct which codes for GST-IEGR-GIQ-cdc25C (Leu258-Pro473) was mutagenized using the mutagenic oligonucleotide primers described above in the Stratagene Quickchange system and transformed into XLl-Blue. Colonies were selected by first cutting out and purifying the BamHi XhoI insert and then by cutting with Taq I. The desired mutations eliminated the Taq I site from the original clone. Therefore, clones were selected based on the absence of the Taq I cutting site. Three clones maintaining the BamHI/XhoI insert but lacking the Taq I site were transformed into the expression line JM109.
  • the cDNA was sequenced and shown to be as predicted.
  • the protein was successfully expressed as a fusion protein in E. coli in soluble form, and was cleaved away from the GST moiety by factor Xa digestion.
  • the process of expressing the protein encoded by the construct was similar to that described for the wildtype protein.
  • the purification of the cdc25C protein was conducted in a manner similar to that described for the wild type enzyme. Frozen E. coli cell paste was thawed and washed in deionized water, and then the washed pellet was resuspended in TEN buffer containing lysozyme, and the solution was incubated on ice for 10 minutes.
  • Supernatant was obtained by centrifugation at 20K RPM using an SS-34 rotor.
  • the fusion protein was purified away from E. coli proteins by affinity chromatography on glutathione Sepharose affinity columns. After collection of the non-bound pool and additional washes, the resin (containing bound GST-cdc25C) was incubated with equilibration buffer containing factor Xa. After a period of time, the released protein is collected and concentrated by Amicon ultrafiltration. Next, the products were resolved from the contaminants and the factor Xa protease by anionic exchange chromatography (Q fast flow) using a linear gradient of NaCl.
  • the released mutated cdc25C protein was found to contain residues 258-473, exhibiting no truncation at the C-terminus.
  • This improved version of the protein was analyzed for solubility and kinetic constants. Surprisingly, we found that the protein had greater solubility, making it an ideal version for the crystallography effort.
  • the cdc25C counterpart did not have a reduced Km for pNPP. In fact, the Km was nearly identical to that observed for cdc25C (258-446).
  • wild type cdc25C intermediate SEQ. ID. NO. 25 & 26
  • PART A - INTERMEDIATE The sequence below is from the construct from Part A, intermediate, before Xa cleavage, is shown here.
  • the single arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A.
  • the construct begins with a GST fusion, it has an Xa restriction site (IEGR) and immediately following the restriction site is a GIQ sequence, immediately following this is the cdc25C segment.
  • IEGR Xa restriction site
  • GIQ GIQ sequence, immediately following this is the cdc25C segment.
  • the sequence below, beginning with GIQ is also listed as Sequence ID. no. 2.
  • the underlined amino acids were also mutated to create an improved protein . The improved mutation is shown in the next chart .
  • PART C ADVANCED MUTANT INTERMEDIATE The sequence below is from the construct from Part C, intermediate, before Xa cleavage, is shown here.
  • the single arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A.
  • the construct begins with a GST fusion, it has an Xa restriction site (IEGR) and immediately following the restriction site is a GIQ sequence, immediately following this is the cdc25C segment.
  • IEGR Xa restriction site
  • GIQ Xa restriction site
  • the sequence below, beginning with GIQ is also listed as Sequence ID. no. 33.
  • the underlined amino acids were mutated to create an improved protein . The improved mutation is shown in the next chart.
  • the sequence of the construct from Part A, final construct, after Xa cleavage, is shown here.
  • the arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A.
  • the construct begins with a GIQ sequence. The sequence below is also listed as Sequence ID. no. 3.
  • PART B - INTERMEDIATE The sequence from the construct from Part B, intermediate, is shown here.
  • the arrows indicate corresponding residues in full length cdc25C sequence.
  • the construct begins with a GST fusion, it has a Xa restriction site, (IEGR), immediately followed by a GIQ sequence, immediately following this is the cdc25C segment.
  • IEGR Xa restriction site
  • GIQ GIQ sequence
  • PART B - FINAL CONSTRUCT The sequence from the construct from Part B, final construct, is shown here.
  • the arrows indicate corresponding residues in full length cdc25C sequence.
  • the construct begins with a GIQ sequence .
  • the sequence below is also listed as Sequence ID. no. 5.
  • NUCLEOTIDE CHARTS correspond to the AMINO ACID CHARTS above, and provide the nucleotide sequences that code for the amino acids in those charts .
  • the numbers shown here correspond to full length cDNA and do not include GST, restrictions sites or residues that code for GIQ .
  • the arrows/positions indicate the first or last included nucleotide. Stop codons may be indicated by a star " ⁇ " where naturally occurring, as in Nucleotide Chart 1 and 2, they may be omitted when added as part of the creation of the construct, as in Nucleotide Chart 3, where a stop codon would be added to the end of the sequence, this is explained from the textual description of the construct.
  • the nucleic acids corresponding to the amino acids in AMINO CHART 1 are marked here in the same manner as in AMINO CHART 1.
  • the first arrow below, T (L, A), is at position 982; the second arrow, t(B), is at position 1006; the third arrow, f(B), is at position 1515; the fourth arrow, ! (A), is at position 1548; and the last arrow, If (L) , is at position 1632.
  • the Part B construct has a stop codon added to become position 1516-1518 of that construct.
  • the sequence below is also listed as Sequence ID. no. 6.
  • Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 2 the Part A construct.
  • the nucleic acids corresponding to the amino acids in AMINO ACID CHART 2 are marked here in the same manner as in AMINO ACID CHART 2.
  • the GST and IEGR segments are shown, the GGGATCCAG residues codes for GIQ .
  • the first arrow below, t (L, A) is at position 982; the second arrow, t (A), is at position 1548; and the third arrow, t (L) , is at position 1632.
  • a natural stop codon is from position
  • the nucleic acids corresponding to the amino acids in AMINO ACID CHART 2m are marked here in the same manner as in AMINO ACID CHART 2m.
  • the GST and IEGR segments are shown, the GGGATCCAG residues codes for GIQ .
  • the first arrow below, t (L, A) is at position 982; there is no corresponding arrow as the second arrow at 1548 in Nucleotide Chart 2 because this mutant does not cleave at that point; and the second arrow, If (L) , is at position 1632.
  • a natural stop codon is from position
  • the sequence below is also listed as Sequence ID . no . 8.
  • the mutated DNA sequences are underlined. The sequence below is also listed as Sequence ID. no.36.
  • the nucleic acids corresponding to the amino acids in AMINO ACID CHART 4 are marked here in the same manner as in AMINO ACID CHART 4.
  • the GST and IEGR segments are shown, the GGGATCCAG residues code for GIQ .
  • the first arrow below, T(B), is at position 1006; the second arrow, 1(B), is at position 1515.
  • a stop codon is introduced after the residue 1515, see text.
  • the sequence below, beginning with GGGATCCAG-, is also listed as Sequence ID. no. 9. (GST) - (IEGR) - GGGAT
  • the nucleic acids corresponding to the amino acids in AMINO ACID CHART 5 are marked here in the same manner as in AMINO ACID CHART 5.
  • the GGGATCCAG residues code for GIQ The first arrow below, T(B), is at position 1006; the second arrow, t(B), is at position 1515. A stop codon is introduced after the residue 1515, see text.
  • the sequence below, beginning with GGGATCCAG-, is also listed as Sequence ID. no. 10.
  • ADDRESSEE Pharmacia & Upjohn Company
  • STREET 301 Henrietta Street
  • Lys Lys Pro lie Val Pro Leu Asp Thr Gin Lys Arg lie lie lie Val 100 105 110
  • Lys Lys Pro lie Val Pro Leu Asp Thr Gin Lys Arg lie lie lie Val
  • TCTGCAAACC AACAGGCTAC CAACTTGTAT CCAGGCCTGG GAATGGATTA GGTTTCAGCA 1800
  • CTTTCTTTGT ATTTTCCTTC TTTGTTTCCC CCTCTTTCTT TTTTAAAAAT GGAAAAATAA 2040
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • ATGCATCATC AGGACCACAA GACTGAGTTG CTGAGGTGTC GAAGCCAGAG CAAAGTGCAG 600
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE YES
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:19:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • TTCCACTGTG AATTCTCCTC AGAGAGGGGC CCCCGAATGT GCCGCTGTCT GCGTGAAGAG 420 GACAGGTCTC TGAACCAGTA TCCTGCATTG TACTACCCAG AGCTATATAT CCTTAAAGGC 480
  • ATGCATCATC AGGACCACAA GACTGAGTTG CTGGCGTCTG CAAGCCAGAG CAAAGTGCAG 600
  • MOLECULE TYPE cDNA (ill) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
  • TTCCACTGTG AATTCTCCTC AGAGAGGGGC CCCCGAATGT GCCGCTGTCT GCGTGAAGAG 420 GACAGGTCTC TGAACCAGTA TCCTGCATTG TACTACCCAG AGCTATATAT CCTTAAAGGC 480
  • ATGCATCATC AGGACCACAA GACTGAGTTG CTGGCGTCTG CAAGCCAGAG CAAAGTGCAG 600

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Abstract

This invention discloses novel forms of catalytic macro molecules that are related to cdc25C, a cell cycle specific phosphatase. These special domains of cdc25C, and the method of making them, are especially useful.

Description

SPECIAL CATALYTIC DOMAINS OF CDC25C PHOSPHATASE
Field of the Invention This invention relates to the field of protein phosphatases, specifically cdc25C like enzymes.
Information Disclosure P. Aroca, D.P. Bottaro, T. Ishibashi, S.A. Aaronson, and E. Santos. Human dual specificity phosphatase VHR activates maturation promotion factor and triggers meiotic maturation in Xenopus oocytes. J. Biol. Chem. 9 June 1995; 270(23): 14229-34. Speculation that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.
David H. Beach and Konstantin Galaktionov, U.S. Patent 5,441,880, issued Aug. 15, 1995. "Human cdc25 genes, encoded products and uses thereof."
David H. Beach and Konstantin Galaktionov, United States Patent 5,441,880, issued Aug. 15, 1995, entitled "Human cdc25 genes, encoded products and uses thereof. J.M. Denu, Y.G. Zhou, and J.E. Dixon. The catalytic role of aspartic acid- 92 in a human dual-specific protein-tyrosine phosphatase. Biochemistry 34: 3396- 3403 (1995).
J.M. Denu and J.E. Dixon. A catalytic mechanism for the dual-specific phosphatases. Proc. Natl. Acad. Sci.U-S-A. 1995 Jun 20; 92(13): 5910-4. J.M. Denu, G. Zhou, L. Wu, R. Zhao, J. Yuvaniyama, M.A. Saper, and J.E.
Dixon. The purification and characterization of a human dual-specific protein tyrosine phosphatase. J. Biol. Chem.. 1995 Feb 24; 270(8): 3796-803. An expression and purification method was developed to obtain the recombinant human dual-specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. Conditions were developed to obtain crystals which were suitable for x-ray structure determination.
J.W. Eckstein, P. Beer-Romero, and I. Berdo. Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases. Protein Sceince 5: 5-12 (1996). I. Hoffman, P.R. Clarke, M.J. Marcote, E. Karsenti, and G. Draetta, EMBO J, vol.12 pp. 53-63 (1993). "Phosphorylation and activation of human cdc25C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis."
I. Hoffman, G. Draetta, and E. Karsenti, EMBO J, vol. 13, pp. 4302-4310 (1994). "Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the Gl/S transition. "
Takashi Horiguchi, et al., Biochemical Pharmacology, vol. 48 pp. 2139-2141, (1994). "Dnacin Al and Dnacin Bl are antitumor antibiotics that inhibit cdc25b phosphatase activity." Report of a GST-cdc25B (residues 355-566) using PNPP as substrate at 37°C. T. Ishibashi; D.P. Bottaro; P. Michieli; CA. Kelley; S.A. Aaronson. J. Biol.
Chem. 1994 Nov 25; 269(47): 29897-902. "A novel dual specificity phosphatase induced by serum stimulation and heat shock," discloses an amino acid sequence of human VHR phosphatase.
T. Ishibashi, D.P. Bottaro, A. Chan, T. Miki, and S.A. Aaronson. Expression cloning of a human dual-specificity phosphatase. Proc. Natl. Acad. Sci. U-S-A. 1992 Dec 15; 89(24): 12170-4.
A. Kamb, P.A. Futreal, J. Rosenthal, C. Cochran, K.D. Harshman, Q. Liu, R.S. Phelps, S.V. Tavtigian, T. Tran, C. Hussey, et-al. Localization of the VHR phosphatase gene and its analysis as a candidate for BRCA1. Genomics. 1994 Sep 1; 23(1): 163-7.
A. Kumagai and W.G. Dunphy, Cell, vol. 64 pp. 903-914, (1991). "The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system." A. Kumagai and W.G. Dunphy, Cell, vol. 70 pp. 139-151 (1992). "Regulation of the cdc25 protein during the cell cycle in Xenopus extracts." Reporting an engineered recombinant C-terminal domain of the Drosophila cdc25 protein,) at 37 C using PNPP as substrate.
S.P. Kwak, D.J. Hakes, K.J. Martell, and J.E. Dixon. Isolation and characterization of a human dual specificity protein-tyrosine phosphatase gene. J. Biol.Chem. 1994 Feb 4; 269(5): 3596-604. U.K. Laemmli, Nature, vol. 227 pp.680-685 (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
M.S. Lee, S. Ogg, M. Xu, L.L. Parker, D.J. Donoghue, J.L. Mailer, and H. Piwnica-Worms. cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2. Mol. Biol. Cell. 3: 73-84 (1992). Discloses an expressed full length form of cdc25C. J.B.A. Millar, CH. McGowan, G. Lenaers, R. Jones, and P. Russell, EMBO J, vol. 10, pp. 4301-4309 (1991) "p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast."
U. Strausfeld, A. Fernandez, J-P. Capony, F. Girard, N. Lautredou, J. Derancourt, J-C. Labbe, and N.J.C. Lamb, J. Biol. Chem., vol. 269 pp. 5989-6000 (1994). "Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells."
KM. Walton and J.E. Dixon. Protein tyrosine phosphatases. Ann. Rev. Biochem. 62: 101-120 (1993). Xu Xu and S.P. Burke, Journal of Biological Chemistry, vol. 271, no.9, pp
5118-5124 (1996) "Roles of Active Site Residues and the NH2-terminal Domain in the Catalysis and Substrate Binding of Human Cdc25."
J. Yuvaniyama, J.M. Denu, J.E. Dixon, and M.A. Saper. Crystal Structure of the Dual Specificity Protein Phosphatase VHR. Science 272: 1328-1331 Z.Y. Zhang, Y. Wang, L. Wu, E.B. Fauman, J.A. Stuckey, H.L. Schubert, M.A.
Saper, and J.E. Dixon. The Cys(X)5Arg catalytic motif in phosphoester hydrolysis. Biochemistry. 1994 Dec 27; 33(51): 15266-70.
G. Zhou, J.M. Denu, L. Wu, and J.E. Dixon. The catalytic role of Cysl24 in the dual specificity phosphatase VHR. J. Biol. Chem. 1994 Nov 11; 269(45): 28084-90. Results demonstrate that the dual specificity phosphatases and the tyrosine-specific PTPases employ similar catalytic mechanisms.
Background of the Invention In eukaryotic cells, mitosis is initiated following the activation of a protein kinase known as MPF, the M-phase specific histone kinase or more simply as the M-phase kinase. This kinase consists of at least three subunits; the catalytic subunit (cdc2), a regulatory subunit (cyclin B) and a low molecular weight subunit (pl3-Sucl).
There is much interest in the regulation of the phosphatase which dephosphorylates cdc2 because of its role in the activation of MPF. Genetic studies in fission yeast have established that the cdc25 gene function is essential for the initiation of mitosis, Nurse, P. et al., Mol. Gen. Genet. 146:167-178 (1976). The cdc25 gene product serves as a rate-determining activator of the cdc2 protein kinase, Russell, P. and P. Nurse, Cell 45:145-153 (1986); Ducommun, B. et al, Biochem. Biophys. Res. Common. 167:301-309 (1990); Moreno, S. et al., Nature 344:549-552 (1990)). Mutant cdc2-F15, whose product cannot be phosphorylated on tyrosine, bypasses the requirement for cdc25 protein function, Gould, K. and P. Nurse, Nature 342:39-45 (1989)). Additional work has suggested that cdc25 is the cdc2 phosphatase, Kumagai, A. and W. G. Dunphy, Cell 64:903-914 (1991) and Strausfeld, U. et al., Nature 351:242-245 (1991). Apparently cdc25 acts as a cdc2 phosphatase which dephosphorylates tyrosine and possibly threonine residues on p34cdc thus regulating MPF activation, Dunphy, W. G. and A. Kumagai, Cell 67:189-196 (1991) and Gautier, J. et al., Cell 67:197-211 (1991). Because cdc25 phosphatases are responsible for the dephosphorylation and activation of cyclin-dependent protein kinases, they help control cell cycle progression.
During the cell cycle, at least three forms of a phosphatase, known as cdc25
A, B, and C, are responsible for the dephosphorylation and activation of cyclin- dependent protein kinases; thus, there is interest in understanding how cdc25 phosphatases are regulated. As mentioned above, genetic studies in fission yeast have established that the cdc25 gene is essential for the initiation of mitosis, P. Nurse, P. Thuriaux, and K. Nasmyth. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146: 167-178 (1976), and the cdc25 gene product has been shown to serve as a rate-determining activator of the cdc2 protein kinase. See, P. Russell and P. Nurse. Cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45: 145-153 (1986).
B. Ducommun, G. Draetta, P. Young, and D. Beach. Fission yeast cdc25 is a cell- cycle regulated protein. Biochem. Biophys. Res. Commun. 167: 301-309 (1990). S. Moreno, P. Nurse, and P. Russell. Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic infjUcer in fission yeast. Nature 344: 549-552 (1990)). A full length form of cdc25C has been expressed in E. coli, see, M.S. Lee, S.
Ogg, M. Xu, L.L. Parker, D.J. Donoghue, J.L. Mailer, and H. Pi wnica- Worms. "Cdc25+ encodes a protein phosphatase that dephosphorylates p34 ." Mol. Biol. Cell. 3: 73-84 (1992). And reports have clearly shown that cdc25A, cdc25B, and cdc25C share sequence similarity at the C-terminus. K. Galaktionov and D. Beach. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell 67: 1181-1194. This region of the protein is capable in some cases of exhibiting enzymatic activity or biological complementation in non-bacterial cell systems. See, A. Nagata, M. Igarashi, S. Jinno, K. Suto, and H. Okayama. An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells. New Biol. 3: 959-968 (1991). T. Horiguchi, K. Nishi, S. Hakoda, S. Tanida, A. Nagata, and H. Okayama. Dnacin Al and Dnacin Bl are antitumor antibiotics that inhibit cdc25B phosphatase activity. Takashi Horiguchi, et al., Biochemical Pharmacology, vol. 48 pp. 2139-2141, (1994). Cdc25+ encodes a protein phosphatase that dephosphorylates p34 . Mol. Biol. Cell. 3: 73-84 (1992). J. Gautier, M.J. Solomon, R.N. Booker, J.F. Bazan, and M.W. Kirschner. Cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell 67: 197-211 (1991). A. Kumagai and W.G. Dunphy. The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system. Cell 64: 903-914 (1991). But it has not been proven that a soluble active minimal domain of cdc25C in E. coli can be designed, created and expressed, without being a fusion partner with glutathione S-transferase (GST). Furthermore, successful expression of a soluble and active minimal domain lacking a fusion tag, such as glutathione S transferase, has not been reported for any human cdc25 protein.
It has been known for sometime that cdc25A, cdc25B, and cdc25C share significant sequence similarity at the C-terminus. KGalaktionov and D. Beach. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell 67: 1181-1194. In fact, these proteins can be aligned with a variety of tyrosine and dual specificity phosphatases within a putative catalytic domain of around 170 amino acids. J.M. Denu, Y.G. Zhou, and J.E. Dixon. The catalytic role of aspartic acid-92 in a human dual-specific protein- tyrosine phosphatase. Biochemistry 34: 3396-3403 (1995). KM. Walton and J.E. Dixon. Protein tyrosine phosphatases. Ann. Rev. Biochem. 62: 101-120 (1993).
Various studies have also provided clues as to the correct size of the dual specificity phosphatase catalytic domain. Nagata et al. demonstrated that the minimal catalytic domain for cdc25 required for complementation of cdc25 deficient yeast was represented by cdc25B residues 405-566. A. Nagata, M. Igarashi, S. Jinno, K Suto, and H. Okayama. An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells. New Biol. 3: 959-968 (1991). Other examples of cdc25 cloning and expression of C-terminal residues which resulted in a positive biological response have also been published. See A. Nagata, et al., New Biol. 3: 959-968 (1991). T. Horiguchi, et al. Biochemical Pharmacology 48: 2139-2141, 1994. M.S. Lee, et al. Mol. Biol. Cell. 3: 73-84 (1992). J. Gautier, et al. Cell 67: 197-211 (1991), and A. Kumagai and W.G. Dunphy. Cell 64: 903-914 (1991). Interestingly, in all examples of bacterial expression of cdc25 C-terminal domains, a determination of activity of cdc25 has been based entirely on the activity of GST fusion proteins, and not on the independent cdc25 protein alone. Only in one example has there been a suggestion of improved solubility of a GST-cdc25 minimal domain. For that example, Lee et al. had reported that of several deletion mutants of cdc25C, only one was soluble (GST-cdc25C 258-473). M.S. Lee, S. Ogg, M. Xu, L.L. Parker, D.J. Donoghue, J.L. Mailer, and H. Piwnica- Worms. cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2. Mol. Biol. Cell. 3: 73-84 (1992). However, despite the lack of an explanation for improved solubility, that parameter did not correlate with activity, since all of the enzymatically active constructs had similar Vmaχ's.
Yet another construct of cdc25B was reported by Horiguchi et al. as the expression with GST as a fusion protein (GST-cdc25B (355-566)). T. Horiguchi, K Nishi, S. Hakoda, S. Tanida, A. Nagata, and H. Okayama. Dnacin Al and Dnacin Bl are antitumor antibiotics that inhibit cdc25B phosphatase activity. Biochemical Pharmacology 48: 2139-2141, 1994. Unfortunately, little information was presented regarding the activity or solubility of the protein, and there was no mention as to whether an attempt was made to remove the GST moiety.
Still, the belief that a minimal domains of cdc25 should be more active than the full length protein persists and it is based on the belief that there is stricter regulation by the N-terminus of the protein. Regulation studies of cdc25 have been reported in the literature. For example, Kumagai and Dunphy showed that Xenopus extracted cdc25C protein had low activity during interphase, but a rapid rise in activity at the onset of mitosis, coincident with protein phosphorylation. A. Kumagai and W.G. Dunphy. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. Cell 70: 139-151 (1992). Other reports have shown that both cdc25C and cdc25A appear to require prior phosphorylation by a cyclin dependent kinase for activation. Strausfeld et al. showed that cdc25C expressed in bacteria could be phosphorylated in vitro by cdc2/cyclin B to give a protein with 2-3 fold higher phosphatase activity. In addition they identified the sites of Cdc2: cyclin B phosphorylation of human cdc25C by isolation of phosphopeptides. U. Strausfeld, A. Fernandez, J-P. Capony, F. Girard, N. Lautredou, J-C Derancourt Labbe, and N.J.C. Lamb. Activation of p34cdc protein kinase by microinjection of human cdc25C into mammalian cells. J. Biol. Chem. 269: 5989-6000 (1994). Several of these putative phosphorylation sites are also found in human cdc25B. Almost all of the phosphorylation sites in cdc25C are at the N-terminus. Since regulation of cdc25C activity by phosphorylation is likely important for phosphatase activity of full length cdc25C, it is important to consider the implications of removing these sites away from the active catalytic domain. Such a modification is likely to produce a constitutive enzyme activity, which would be highly desired for use in screening assays.
Here we report stable recombinant forms of cdc25O These isoforms have better solubility characteristics and improved activity when compared to previously reported forms of the proteins. The stable recombinant forms of the protein reported here are also capable of easy manipulation for crystallography studies in order to better understand and characterize these types of phosphatases by structural analyses and models. This invention provides macro molecules having these and other desirable characteristics.
Summary of the Invention This invention comprises, the fusions comprising the fusions shown below
1548
Figure imgf000009_0001
446 where, the fusion may be comprised of DNA or amino acids, where the different parts of the fusions are shown as different lines in the box, where a) the GST portion, is labeled GST, with a straight line in the box, b) the protease cleavage site is shown as a dotted line in the box, labeled "P," c) the restriction site is shown as a wavey line in the box, labeled "R," and d) the cdc25C like portion is shown as a heavy line in the box, labelled "cdc25C" where the numbers above the box indicate DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either DNA or amino acids, where the boxes, lines and numbers are not drawn, as shown, to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25C region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length cdc25C, where the numbers near the dotted lines show one construct and the numbers at the ends of the cdc25C box with the heavy line shows a different construct, or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues, where the circle indicates that mutations can be made.
Numerous subsets of the fusions are disclosed, such as where the fusion protease cleavage site is created to be responsive to Factor Xa. Where the fusion restriction site, when expressed, yields the amino acid sequence GIQ. The fusion consisting of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues. The process of making any of the fusion proteins or peptides described above.
DNA that codes for the fusion protein are described and particular DNA constructs are described such as where the DNA coding for the various fusion proteins. The fusion consists of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues. The process of making any of the DNA constructs is also described. Essential nucleic acid intermediates are described. The process of making any of the DNA fusions is also described. In addition to the fusions above, the fusion below is disclosed.
(DNA)
GST p R 1006 cdc25C 15ι5
Figure imgf000010_0001
GST p R 266 cdc25C 435
(peptide) where, the fusion may be comprised of DNA or amino acids, where the different parts of the fusions are shown as different lines in the box, where a) the GST portion, is labeled GST, with a straight line in the box, b) the protease cleavage site is shown as a dotted line in the box, labeled "P," c) a stop codon appears in the DNA sequence, but not the amino acid sequence, d) the restriction site is shown as a wavey line in the box, labeled "R," and e) the cdc25C like portion is shown as a heavy line in the box, labelled "cdc25C" where the numbers above the box indicate DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either DNA or amino acids, where the boxes, lines and numbers are not drawn, as shown, to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25C region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length cdc25C, where the numbers near the dotted lines show one construct and the numbers at the ends of the cdc25C box with the heavy line shows a different construct, or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
Numerous subsets of the fusions are disclosed, such as where the fusion protease cleavage site is created to be responsive to Factor Xa. Where the fusion restriction site, when expressed, yields the amino acid sequence GIQ. The fusion consisting of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues. The process of making any of the fusion proteins or peptides described above.
DNA that codes for the fusion protein are described and particular DNA constructs are described such as where the DNA coding for the various fusion proteins. The fusion consists of various sequences described by the Charts and Sequence Listings or similar sequences obtained by deleting, adding or replacing one to several amino acid residues. The process of making any of the DNA constructs described above. Essential nucleic acid intermediates are described. The process of making any of the DNA fusions is also described. The sequences from the CHARTS and SEQUENCE LISTINGS are disclosed in addition to the process of making the various fusions, constructs or molecules.
Brief Description of the Drawings Figure LA. Figure LA shows the plasmid pGEX-5X-3 used in the cloning of recombinant human cdc25O This figure shows the position of the factor Xa cleavage site sequence, various restriction sites, and location of the cDNA inserts for cdc25C Figure IB is an expanded portion of the plasmid shown in Figure 1A.
Figure IB. Figure IB contains the expanded portion of the segment shown in Figure 1A. Figure 2. Figure 2 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part A, DNA residues 982 through 1632, showing the product of the PCR reaction (lane P), as well as DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position "a" shows the migration of the linearized plasmid pGEX-5X-3, and position "b" shows the migration of the cdc25C DNA insert/PCR product. Lane A shows a 1 kb DNA molecular weight marker ladder.
Figure 3. Figure 3 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing the product of the PCR reaction (lane 1) with template DNA and primers, while lane 2 shows the results of a control PCR reaction lacking template DNA (lane 2). Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B.
Figure 4. Figure 4 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position "a" shows the migration of the linearized plasmid pGEX-5X-3, and position "b" shows the migration of the cdc25C DNA insert. Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B.
Additional More Detailed Description of the Invention Definitions. Definitions are included throughout this document in addition to the specific definitions and sources of materials noted below.
"BCIP" is 5-bromo-4-chloro-3-indolyl phosphate.
"Bio 101®." Many of the kits used in this invention such as "the GeneClean® kit" and "a RPM® plasmid isolation kit" (RPM-rapid pure minipreps) are obtained from Bio 101®., a company in LaJolla, California.
"competent E. coli cells (JM109)" are a strain of commonly available cells. "IPTG" is isopropyl-J-D-thiogalactopyranoside from Boehringer Mannheim, Indianapolis, Indiana.
"LB media" is a solution containing tryptone, yeast extract, sodium chloride and water. It is commerically available from Gibco-BRL.®, Gaithersburg, Maryland. "M9 medium" containes dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, and ammonium chloride 1 g per L of deionized water. M9YE medium is the same medium as M9 plus yeast extract, 5 g per L of deionized water. One hundred ml vols. of M9YE contained in 500 ml wide mouth fermentation flasks were sterilized by autoclaving for 30 min. The sterilization pH was adjusted to 7.3 with KOH. One L of sterile basal M9YE was prepared through the aseptic addition of filtered sterilized 1M MgSO4, 2ml; 20% glucose, 20 ml and lM CaC12, 0.1 ml.
"MORPH®" is a site-specific plasmid DNA mutagenesis kit obtained from 5 PRIME->3 PRIME, Inc.®, Boulder, Colorado.
"native extraction/buffer systems" are common extraction buffer systems such as, lysozyme (1 mg/ml) and fresh dithiothreitol (DTT) (20 mM) in TEN buffer (50 mM Tris HCl, 0.5 mM EDTA, 300 mM NaCl, 0.2% NP-40, pH 8.0)
"NBT" is nitroblue tetrazolium. "PAGE" is polyacrylamide gel electrophoresis.
Assays of PNPP hydrolase activity associated with cdc25B are conducted using the reagents described by Horiguchi et al. (Biochemical Pharmacology, Vol. 48 pp. 2139-2141, (1994)). This is what is meant by "enzymatically active in a defined way with the colorimetric substrate, p-nitrophenol phosphate (PNPP)." "PVDF1 is polyvinylidene difluoride.
"SDS" is sodium dodecyl sulfate.
"TA cloning kit," containing the pCRII plasmid, and INVIF' cells is obtained from InVitroGen®, San Diego, California
Temperatures are in degrees celcius unless noted otherwise and may be indicated with a number, a number supercase o, a number uppercase c, a number supercase o uppercase c or other obvious combinations or methods, e.g. 37, 37°, 37 C, 37° C, etc.
The Inventions and Their Utilities.
The present invention relates to a method of regulating (inhibiting or enhancing) cell division and to agents or compositions useful for regulating the cell cycle. The present invention has the same uses as previously disclosed human cdc25C in addition to having other uses not possible with previously disclosed human cdc25C because of its physical characteristics. Described herein is a novel recombinant fusion construct that produces a macromolecule that is soluble and that performs similar biochemical functions as full length cdc25C constructs, but is more active, more soluble, and does not require refolding. The compounds described herein are useful for crystallography and for drug development screening tools. These compounds should also allow improved structure-based design for the development of novel phosphatase antagonists, the latter being expected to result in an anti-neoplastic drug.
The macro molecules described herein will make superior drug screening tools over previously disclosed cdc25C proteins because of their enhanced activity. The compounds described herein are superior over known compounds, proteins and peptides for studies of cdc25C enzyme kinetics and mechanistic studies because these novel compounds are monomeric in structure and because the uniquely designed sequences do not display anomalies present in inhibitor kinetics seen with known proteins (e.g. GST-cdc25 fusions). Furthermore, the compounds described herein can be created without a subsequent refolding step, thus providing simple consistent procedures for making highly active compounds. The compounds described herein would make superior subjects of crystallization studies because of their solubility properties. These compounds would make superior templates for studies of structure activity relationships because their structure is more suitable for structure based design strategies than known cdc25C compounds. The compounds disclosed herein should be particularly useful for transfection studies in mammalian cells designed to test in vivo mechanism of action and proof of concept studies. The GST-cdc25C full length enzyme apparently cannot be purified to homogeneity using prior art descriptions of purification of GST fusion proteins. The compounds and procedures disclosed herein do allow the creation of highly purified and homogeneous active protein, as defined by several criteria.
The design of a model of cdc25C protein sequence is probable because the 3D structure of a similar phosphatase called VHR (vaccinia Hl-related phosphatase) was recently crystallized. See, J. Yuvaniyama, J.M. Denu, J.E. Dixon, and M.A. Saper; "Crystal Structure of the Dual Specificity Protein Phosphatase VHR." Science 272: 1328-1331. VHR shows limited sequence identity to cdc25 proteins, and has been used frequently as the model for studies devoted to understanding the mechanism of dual specificity phosphatases such as cdc25. It is also useful as a structural model for cdc25 proteins, since it has a relatively low molecular weight mass, of about 20,500. See, J.M. Denu, G. Zhou, L. Wu, R. Zhao, J. Yuvaniyama, M.A. Saper, and J.E. Dixon. "The purification and characterization of a human dual-specific protein tyrosine phosphatase." J. Biol. Chem. 1995 Feb 24; 270(8): 3796-803.
The previously described cdc25C proteins, both full length as well as truncated minimal domains, were usually created as a GST fusion with cdc25C These fusion proteins were expressed in E. coli as partially soluble and low activity proteins. No attempts were made to remove the GST fusion partner, see M.S. Lee, S. Ogg, M. Xu, L.L. Parker, D.J. Donoghue, J.L. Mailer, and H. Piwnica- Worms. cdc25+ encodes a protein phosphatase that dephosphorylates p34 c . Mol. Biol. Cell. 3: 73-84 (1992). Note that prominent in these domains is a highly conserved HCXXXXXR signature sequence.
General Discussion of the Methods. Procedures and Constructs. The methods, procedures and constructs of this invention will be described in several parts. This invention is comprised of at least two distinctive constructs of cdc25C These constructs differ from one another in their compositions, their properties and the manner of creating them, even though many of the procedures used are common or related, and these procedures can be used in various combinations and obvious variations to produce substantially similar variants. One type of construct, is described in Part A, another type construct, in Part B, and a third type construct in Part C. The procedures used below describe how the constructs were initially created, however; because we provide the sequences of the intermediate and final products, it is expected that one skilled in the art may use alternative procedures obvious to one skilled in the art to create these same or substantially similar constructs.
Part A ■ Preparation of special cdc25C (258-446) or cdc25C258"446. Experimental Approach.
This special soluble and active truncated form of cdc25C is suitable for enzymatic studies and for screening. The sequence of the DNA used in these experiments is defined by Genbank accession number M34065.gb_pr. To begin, linearize the pBSK-1 vector, which contains the full length cdc25C insert, and use PCR primers (containing Bam HI (5' sense) and Xho I (3' antisense) restriction sites) which allow for the amplification of the cDNA for cdc25O Several coding regions were chosen for the cloning experiments. Following PCR reactions and subcloning into TA vectors, the resulting purified inserts can be ligated into pGEX-5X-3 in separate experiments. The products of the ligations were used for transformation of competent E. coli cells (JM109). The constructs expressed in E. coli were designed as fusion proteins of GST with cdc25C, with an intervening factor Xa cleavage site. The special GST-cdc25C constructs are then optimized. Methods.
PCR Cloning. AFV- 3 (GCG GAT CCA GTT AAA GAA GAC AGT CT ) (SEQ. ID. 11) and AFV- 2 (GCC TCG AGT CAT GGG CTC ATG TCC TT ) (SEQ. ID. 12) were designed as primers for the PCR reaction to amplify M34065.gb_pr between 982-1632. A reaction (final volume of 100 ul) containing 20 pmol AFV-2 , 20 pmol AFV-3 , 12 ng cdc25C template in 200 μM dNTPs, IX PCR Buffer [(Perkin-Elmer GeneAmp), 1.5 mM MgCl2, 50 mM KC1, 10 mM Tris-HCl pH 8.3, 0.001% (w/v) gelatin], and sterile water was used in the PCR reaction to generate the desired insert. 2.5 units Amplitaq DNA Polymerase were used for this PCR reaction, and cycling conditions were as follows: 8 minutes at 95°, (1 minute at 95°, 90 seconds at 50°, 2 minutes at 72°, for a total of 25 cycles); finally, cycling was completed with incubation for 5 minutes at 72°, followed by a 4°C hold temperature. The PCR reaction product was immediately (without prior purification) ligated into the TA Cloning vector pCRII (Invitrogen TA Cloning Kit) according to the manufacturer's directions. The product of the ligation was transformed into INVαF1 cells (according to manufacturer's directions), and after overnight incubation of the cells at 37°C on L broth agar plates with ampicillin and Xgal, the resulting DNA of selected colonies was isolated using a Bio 101 DNA isolation kit, digested with Bam HI and Xho I, and purified by agarose gel electrophoresis.
Ligation of Bam HI I Xho I TA cloning product into PGEX-5X-3- PGEX-5X-3 (Pharmacia Biotech) was prepared for ligation by simultaneous digestion with the restriction enzyme BamHI and EcoRI at 37°C in 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2 and 100 mM NaCl (React 3 buffer, Gibco BRL). The resultant double- digested plasmid was subjected to electrophoresis on a 0.8% agarose gel in IX TAE (Tris-acetate-EDTA buffer, containing 0.5 μg/ml ethidium bromide (EtBr)). The product was excised from the gel and was purified using Geneclean III from Bio 101 as recommended by the manufacturer. The following ligation conditions were utilized:
A. PGEX-5X-3 alone/representing a no insert control. A mixture of -250 ng PGEX-5X-3, was digested with Bam HI/EcoRI, together with 1 ul of T4 DNA ligase (Gibco BRL), IX ligase buffer [50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene glycol 8000, (Gibco BRL)], 1 ul dATP (Gibco BRL), and sterile H20 was prepared to give a total volume of 10 ul. B. PGEX-5X-3/cdc25C (982-1632) insert. A mixture of =250 ng PGEX-5X-3, was Bam HI/EcoRI digested, =100 ng cdc25C minimal domain from the TA cloning experiment (Bam HllXho I digested), 1 ul of T4 DNA ligase (Gibco BRL), IX ligase buffer (Gibco BRL), 1 ul dATP (Gibco BRL), and sterile H20 was prepared to give a total volume of 10 ul. Both ligation reactions were allowed to proceed overnight at 15°C.
Transformation of E. Coli Strain JM109. 5 ul of the ligation reaction was added to 100 ul of Promega competent cells (E. coli). This suspension was incubated on ice for 1 hour, incubated at 42°C for 90 seconds, and cooled on ice for 1 minute. 250 ul of SOC medium was added, followed by incubation at 37°C with shaking for 1 hour. The mixture was used for application of 10, 100 and 200 ul aliquots onto (1.5%) agar plates containing LB medium and ampicillin (100 μg ml). After streaking the agar plates with the transformation mixture, the plates were incubated overnight at 37°C Colonies were abundant on PGEX-5X-3/cdc25C insert plates and sparse on control plates.
Miniprep Analysis. DNA was purified from cultures using 1.5 ml of culture in a Bio 101 RPM kit. The DNA was eluted in 50 ul sterile H20, and digested with Bam HI and Xho I simultaneously in React 2 buffer at 37°C for 1 hour. Samples were run on a 0.8% agarose gel containing IX TAE buffer and 0.5 μg/ml EtBr. DNA Sequencing. The sequence of the engineered insert of pGEX-5X/cdc25C was determined in the DNA Sequencing Core Laboratory. Fluorescence-based sequencing of DNA templates were done using the ABI PRISM Ready Dye-Deoxy Terminator FS-kits with Taq FS polymerase (Perkin-Elmer/Applied Biosystems Division, PE/ABI, Foster City, CA) and the ABI373A fluorescence-based sequencer (stretch upgrade). The temperature cycles for cycle sequencing were controlled using an automated PCR thermal cycler (Perkin Elmer 9600). Fluorescence-based sequencing was done using about 0.5 μg of DNA and the following cycle sequencing reaction conditions: initial denaturation at 98°C for 1 min, followed by 35 cycles of 98°C for 15 sec, annealing at 50°C for 10 sec and extension at 60 °C for 4 min. Extension products are purified using 1 ml spin columns (Centri-sep, from Princeton Separations, Inc., Adelphia, NJ). Each reaction product is loaded by pipette onto the column, which is then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B table top centrifuge) at 750 x g for 2 min at room temperature. Column- purified samples are dried under vacuum for about 40 min and then dissolved in 5 ul of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml blue dextran). The samples are then heated to 90°C for three min and loaded into the gel sample wells for sequence analysis by the ABI373A sequencer. Sequence analysis was done by importing ABI373A files into the Sequencher program, obtained from Gene Codes (Ann Arbor, MI), and generally sequence readings of 700 to 800 bp were obtained. DNA sequencing errors were minimized by obtaining sequence information from both DNA strands.
Expression of the human recombinant truncated cdc25C protein (GST-cdc25C (258-473 )) in E. coli. Seed fermentation: E. coli was inoculated into 100 ml vols. of M9 medium containing yeast extract at 0.5% (M9YE) contained in 500 ml large mouth fermentation flasks. The medium contained 100 mg of ampicillin/L. The inoculated flasks were incubated for 16 hr at 37C while shaking at 200 rpm. M9YE was prepared as described below. Production fermentation: Production flasks (as above) containing M9YE with filter sterilized ampicillin (as above) were inoculated with the mature seed fermentation at a 3% rate. This fermentation was continued for 2.25 hr at 37C while shaking at 200 rpm until the turbidity at 660 nm reached ca. 1.0. At this time, filter sterilized IPTG was added to a final concentration of 0.2 mM, and the temperature was shifted to 30C The production fermentation was continued at 200 rpm for an additional 3.5 hr until the turbidity reached ca. 3.0 when harvest was done by centrifugation. Eighty flasks were fermented to achieve an 8L production batch. M9YE medium contained dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, ammonium chloride 1 g and yeast extract, 5 g per L of deionized water. One hundred ml vols. of M9YE contained in 500 ml wide mouth fermentation flasks were sterilzied by autoclaving for 30 min. The presterlization pH was adjusted to 7.3 with KOH. One L of sterile basal M9YE was completed through the aseptic addition of filtered sterilized 1M MgSO4, 2ml; 20% glucose, 20 ml and 1M CaC12, 0.1 ml.
Affinity purification of GST-cdc25C bv a Glutathione Sepharose chromatography column and factor Xa processing to the final product. Four liters of frozen E. coli cell paste were thawed and washed in deionized water, and the washed pellet was resuspended in .5mM EDTA, 20mmM DTT, .3M NaCl, 0.2% NP- 40 and 50mM Tris.Hcl pH 8.0. Egg lysozyme (Sigma) was added at lmg/ml, and the solution was incubated on ice for 10'. Supernatant was obtained by centrifugation at 20K RPM using an SS-34 rotor for 35' at 5°C The fusion protein was purified away from E. coli proteins by affinity chromatography on the glutathione Sepharose affinity column. After collection of the non-bound pool, the resin, which contains bound GST-GIQ-cdc25C (258-473) was incubated with equilibration buffer containing factor Xa (50 mM Tris HCl, 100 mM NaCl, 1 mM CaC12, pH 8.0). Specifically, the collected lysate was batch mixed with 15ml of packed glutathione resin. The slurry was poured into a column and washed extensively with the lysis buffer described above (w/o lysozyme). Finally the protein charged column matrix was washed with factor Xa digestion buffer (50mM Tris HCl, lOOmM NaCl, and ImM CaCl2 pH 8.), followed by the addition of 125 ug of Factor Xa (Boehringer) with mixing in 20ml of digestion buffer. After 12-14hrs incubation at 8°C the Xa released material is collected and concentrated by Amicon ultrafiltration (YM-10 filter). The concentrate is diluted 3x with deionized water to lower the salt concentration for the ion exchange chromatography step.
Q Fast Flow column (ion exchange) chromato raphy. Upon the desired completion of Xa digestion of cdc25C, the products are resolved from the contaminants by anionic exchange chromatography. Proteins were resolved from one another by a linear gradient of 100-190 mM NaCl in a total volume of 36 ml over a period of 36 min.
Analytical size exclusion chromatography (SEC)- An aliquot of the protein sample was analyzed on a Pharmacia Smart system equipped with a Superose-12 column (PC3.2/30cm) at a flow rate of 50ul/min in tris buffer saline. No additional reducing reagent was present in the column buffer. Protein elution was monitored at 280nm and fractions were collected at 1 min/fraction.
Analytical C4 Reverse phase HPLC- An aliquot of the purified truncated cdc25C protein was analyzed by C4 reverse phase HPLC. Using a starting solvent of 0.1% trifluoroacetic acid (TFA), the cdc25C protein was finally eluted in a linear gradient of acetonitrile from 24% to 48% in 0.1% TFA (58 min gradient).
Electrophoresis- SDS gel electrophoresis were performed according to Laemmli. Western blots were completed using a semi-dry electroblotter onto PVDF membranes (Millipore) with a constant current set at 125 mA/gel (7x9 cm). Blots were visualized by staining with Coomassie blue R250 (.2% W/V) in 50% Ethanol, 5% Acetic acid followed by a destaining step using 50% ethanol solution.
Enzvme assay and Kinetic Analyses- Assays of PNPP hydrolase activity associated with cdc25C are conducted using the reagents described previously. These reagents include (as final concentrations in 125 ul): 25 mM Hepes, pH 8.0, 10 mM DTT, 0.1 mg/ml bovine serum albumin, and variable concentrations of pNPP. For assays where a single concentration of substrate is used at saturation, we customarily use a final concentration of 20 mM pNPP. Assay solution is prepared in a final volume of 100 ul, including the addition of freshly prepared dithiothreitol. At the initiation of the assay, 25 ul of enzyme is added with mixing, and a continuous recording of absorbances at 405 nm is completed over a short time period. For the determination of 1^ and Vmaχ (specific activity), multiple pNPP concentrations are used at a constant enzyme concentration. Rates at each concentration of substrate are determined and the 1^ and Vmaχ calculated from line fitting to a Michaelis Menten equation. The velocity of the reaction is defined as follows: One unit of activity is defined as nmoles of pNPP hydrolyzed per minute per milligram of enzyme protein at 25°C
Sequence analysis and mass spectroscopy- Amino terminal sequencing of purified cdc25C proteins was performed on an ABI 476A protein sequencer. Database searches were conducted using the Genetics Computer Group software package (GCG). The amino acid sequences of several homologous proteins were examined by the program FASTA.
Preparation of a Rabbit anti-cdc25C Antibody- Antibodies directed against recombinant human truncated cdc25C were produced in a rabbit. Immunization of the rabbit and production of sera was contracted with HRP, Inc. Response is measured in our lab by Western blotting. Experimental Results
To clone the desired sequence of cdc25C, defined by Genbank accession number M34065.gb_pr, we designed PCR primers containing Bam HI and Xho I restriction sites which would amplify cdc25C DNA residues 982 through 1632. This sequence of the cDNA has also been utilized by Lee et al. , Mol. Cell. Biol. 3: 73-84. 1992, for the cloning and expression of GST-cdc25O The unpurified products of the PCR reaction were ligated into a TA cloning vector (pCRII), and the resulting amplified product was used to transform INVαF' cells. Appropriate colonies were selected, DNA was isolated, and the insert was purified. The purified insert DNA was then ligated into pGEX-5X-3, and the products of the ligation were used for transformation of competent E. coli cells (JM109). Positive colonies were selected and used for induction of the gene and expression of GST-cdc25C protein.
The experiment was designed so that the resulting protein should have been a glutathione S-transferase (GST) tag linked to the cdc25C protein encompassing amino acid residues 258-473 of the full length protein. A factor Xa cleavage site allowed for cleavage of the GST tag from the truncated cdc25C protein. Three additional amino acid residues (Gly-Ile-Gln) were engineered into the N-terminus preceding the cdc25C sequence, but after the factor Xa sequence, IEGR. The fusion protein was purified away from E. coli proteins by affinity chromatography on glutathione Sepharose. The cdc25C protein was proteolytically cleaved from the GST polypeptide, while the latter was still attached to the affinity matrix, using factor Xa protease. The solubilized, truncated cdc25C protein was chromatographed on QAE Sepharose to provide a product consisting of only cdc25C protein.
The final product was a protein initiating at Leu258 and terminating at Arg446, with the Gly-Ile-Gln sequence preceding Leu258. Validation of the sequence was completed by N-terminal sequencing which showed that the sequence initiated with GIQ..., and by mass spectroscopy which showed a mass ion consistent with a protein terminating with Arg446. Therefore, this truncated domain is produced by factor Xa cleavage at an unexpected site within the cdc25C protein sequence. As a result, the protein which begins as expected, (Gly-Ile-Gln-Leu258...), but terminates at an unexpected position (Arg446).
The minimal domain of cdc25C produced was enzymatically active as a phosphatase against p-nitrophenylphosphate (PNPP), with a Km =18 mM and Vmax = 250 nmoles/min per mg of protein at 25°C.
This sequence is a human cdc25C minimal domain free of GST which is soluble and active.
A. Affinity purification of GST-GIQ-cdc25C (258-473) and subsequent processing and purification of truncated GIQ-cdc25C (258-446)
The recombinant fusion protein, produced in E. coli, is designed to have a glutathione S-transferase (GST) tag linked to the cdc25C protein encompassing amino acid residues 258-473 of the full length protein. A factor Xa cleavage site (IEGR) is situated immediately after the GST polypeptide, and prior to the beginning of the cdc25C sequence. Three additional amino acid residues are positioned immediately after the factor Xa cleavage site as a result of the restriction site nucleotide coding sequence. The factor Xa cleavage site allows for cleavage of the GST tag from the truncated cdc25C protein using factor Xa protease. From several liters of frozen E. coli cell paste, we were able to generate milligrams of purified truncated cdc25C. As described in detail in Materials and Methods, for the procedure we take advantage of the affinity of GST-cdc25C for glutathione Sepharose. After binding the fusion protein from crude lysates, E. coli proteins are easily washed away. The resin, which contains bound GST-GIQ-cdc25C (258-473), can then be treated with factor Xa to proteolytically cleave the cdc25C away from the bound GST moiety. The reaction progress of the factor Xa digestion can be followed by analyzing the released products by analysis of the PNPP hydrolytic activity of the enzyme. The progress can be followed by SDS polyacrylamide gel electrophoresis and Western blotting, leading to the observation of a 20-24 kD cdc25C product. Small amounts of the two bacterial chaperonins (DnaK and GroEL) from E. coli were detected. To remove these chaperonins as well as the factor Xa protease, the products were resolved by anionic exchange chromatography. The location of the cdc25C fractions was made by SDS PAGE as well as by PNPP hydrolytic activity profiles.
B. Characteristics of the purified truncated recombinant human cdc25C. C4 reverse phase HPLC. The purified protein exhibits a single peak when analyzed by C4 reverse phase HPLC using a gradient defined in Materials and Methods, above. N-terminal sequence analysis. The amino terminus of the purified cdc25C protein was determined to be Gly-Ile-Gln-cdc25C (258 — ). This data validates that the factor Xa processes on the carboxyl side of the IEGR sequence following the GST fusion protein sequence.
Mass spectrometry. A mass ion representing the major product of the cdc25C protein preparation has been identified at a mass corresponding directly to the sequence of GIQ-cdc25C (258-446). This indicates the factor Xa not only processes the correct engineered factor Xa cleavage site, but also cleaves C-terminal to Arg446.
Description of the Figures. Figure 1A. Figure 1A shows the plasmid pGEX-5X-3 used in the cloning of recombinant human cdc25C. This figure shows the position of the factor Xa cleavage site sequence, various restriction sites, and location of the cDNA inserts for cdc25C. Figure 1A is an expanded portion of the plasmid shown in Figure IB.
Figure IB. Figure IB shows the plasmid construction of plasmid pGEX-5X-3. Figure IB contains the expanded portion of the segment shown in Figure 1A. Figure 2. Figure 2 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part A, DNA residues 982 through 1632, showing the product of the PCR reaction (lane P), as well as DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position "a" shows the migration of the linearized plasmid pGEX-5X-3, and position "b" shows the migration of the cdc25C DNA insert/PCR product. Lane A shows a 1 kb DNA molecular weight marker ladder.
Part B (VHR-like domain of cdc25C) Experimental Approach. Software tools were used to obtain a tentative sequence alignment of cdc25C with other protein tyrosine phosphatases. We created a small active domain, very amenable for NMR studies, that has none of what are apparently floppy regions that may adversely effect crystallization studies. Materials. Glutathione Sepharose 4B and Q fast flow ion exchange matrix were obtained from Pharmacia Biotech®, restriction protease Factor Xa is purchased from Boehringer Mannheim®. A polyclonal antibody made against the C-terminus of cdc25C was purchased from Santa Cruz Biotech. Electrophoresis reagents are from Bio Rad®. The colorimetric substrate, p-Nitrophenyl phosphate (PNPP) is from Sigma Diagnostics®. All other reagents used are of the purest grade available. Original Substrate. The cDNA encoding the entire sequence of human cdc25C was prepared for use as a template for the polymerase chain reaction (PCR) by first linearizing with Hind III. Subsequently, the cDNA was isolated by gel electrophoresis in 0.8% agarose, followed by purification using a Geneclean kit® (Bio 101®). PCR Cloning. A defined region of cdc25C was desired, requiring the isolation of a section of the cdc25C cDNA (residues 1006 to 1515) which codes for amino acid residues 266-435. To amplify only this sequence, the following DNA primers, listed in a 5'->3' orientation were prepared (Genosys):
CAVHR-3 (GCG GAT CCA GGA CAT TAC TAT CAC TCA) (SEG. ID.13) and
CAVHR-4 (GCC TCG AGT CAC ATA GGG CAG TAG CTC) (SEQ. ID. 14) (the italicized letters indicate the positions of engineered restriction sites). A reaction (final volume of 100 ul) containing 20 pmol CAVHR-3, 20 pmol CAVHR-4 , 12 ng cdc25C template, 200 TM dNTPs, IX PCR Buffer ((Perkin-Elmer GeneAmp), 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.001% (w/v) gelatin), and sterile water was used in the PCR reaction to generate the desired insert. 2.5 units Amplitaq Gold DNA Polymerase was used for this PCR reaction, and cycling conditions were as follows: 8 minutes at 95°, (1 minute at 95°, 90 seconds at 50°, 2 minutes at 72°, for a total of 25 cycles); finally, cycling is completed with incubation for 5 minutes at 72°, followed by a 4°C hold temperature. The PCR reaction product was immediately (without prior purification) ligated into the TA Cloning vector pCRII (Invitrogen TA Cloning Kit®) according to the manufacturer's directions. The product of the ligation was transformed into INVIF' cells. Isolated colonies were selected on a basis of ampicillin resistance, and after overnight incubation of selected colonies at 37°C, the resulting DNA was isolated using a Bio 101® DNA isolation kit, digested with Bam HI and Xho I, size selected by agarose gel electrophoresis, and purified using a GeneClean Spin Kit® (Bio 101®).
Ligation of the TA cloning product into PGEX-5X-3- PGEX-5X-3 (Pharmacia Biotech) was prepared for ligation by digestion with the restriction enzymes BamHI and EcoRI at 37°C in 50 mM Tris-HCL (pH 8.0), 10 mM MgCl2 and 50 mM NaCl (React 2 buffer, Gibco BRL®). The resultant linearized plasmid was subjected to electrophoresis on a 0.8% agarose gel in IX TAE (Tris-acetate-EDTA buffer). After electrophoresis, the gel was soaked in IX TAE with 0.5 Tg/ml ethidium bromide (EtBr) so that the DNA could be visualized under long-wave UV. The product was excised from the gel and was purified using Geneclean III® from Bio 101® as recommended by the manufacturer.
The following ligation conditions were utilized:
A. PGEX-5X-3 alone/ representing a no insert control. A mixture of ~ 100 ng PGEX-5X-3, which was linearized with Bam HI/EcoRI, together with 1 Unit of T4
DNA ligase (Gibco BRL), IX ligase buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene glycol 8000, (Gibco BRL)), 1 mM dATP (Gibco BRL), and sterile H20 was prepared to give a total volume of 10 ul.
B. PGEX-5X-3/cdc25C insert. A mixture of =100 ng PGEX-5X-3, which was Bam Hl/Xho I digested, =100 ng cdc25C minimal domain from the TA cloning experiment (Bam HUXho I digested), 1 Unit of T4 DNA ligase (Gibco BRL), IX ligase buffer (Gibco BRL), 1 mM dATP (Gibco BRL), and sterile H20 was prepared to give a total volume of 10 ul. Both ligation reactions were allowed to proceed overnight at 15°C. Transformation of E. Coli Strain JM109. 5 ul of the ligation reaction was added to 100 ul of JM109 (Promega®) competent cells (E. coli). This suspension was incubated on ice for 1 hour, incubated at 42°C for 90 seconds, and cooled on ice for 1 minute. 250 ul of SOC medium was added, followed by incubation at 37°C with shaking for 1 hour. The mixture was used for application of 10, 50 and 200 ul aliquots onto (1%) agar plates containing LB medium and ampicillin (100 Tg ml). After streaking the agar plates with the transformation mixture, the plates were incubated overnight at 37°C. Colonies were picked and grown overnight in 5 ml cultures of LB + 100 ug/ml ampicillin.
Miniprep Analysis. DNA was purified from cultures using the Bio 101 RPM kit, and then digested with Bam HI and Xho I simultaneously in React 2 buffer at 37°C for 1 hour. Samples were run on a 0.8% agarose gel containing IX TAE buffer and 0.5 μg/ml EtBr.
Seed fermentation. E. coli was inoculated into 100 ml vols. of M9 medium containing yeast extract at 0.5% (M9YE) contained in 500 ml large mouth fermentation flasks. The medium contained 100 mg of ampicillin/L. The inoculated flasks were incubated for 16 hr at 37C while shaking at 200 rpm. M9YE was prepared as described below. Production fermentation: Production flasks (as above) containing M9YE with filter sterilized ampicillin (as above) were inoculated with the mature seed fermentation at a 3% rate. This fermentation was continued for 2.25 hr at 37C while shaking at 200 rpm until the turbidity at 660 nm reached ca. 1.0. At this time, filter sterilized IPTG was added to a final concentration of 0.2 mM, and the temperature was shifted to 30C. The production fermentation was continued at 200 rpm for an additional 3.5 hr until the turbidity reached ca. 3.0 when harvest was done by centrifugation. Eighty flasks were fermented to achieve an 8L production batch. * M9YE medium contained dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, ammonium chloride 1 g and yeast extract, 5 g per L of deionized water. One hundred ml vols. of M9YE contained in 500 ml wide mouth fermentation flasks were sterilzied by autoclaving for 30 min. The presterlization pH was adjusted to 7.3 with KOH. One L of sterile basal M9YE was completed through the aseptic addition of filtered sterilized 1M MgS04, 2ml; 20% glucose, 20 ml and 1M CaC12, 0.1 ml.
Affinity purification of GST-cdc25C bv a Glutathione Sepharose chromatography column and factor Xa processing to the final product. Six liters of frozen E. coli cell paste were thawed and washed in deionized water, and the washed pellet was resuspended in .5mM EDTA, 20mmM DTT, .3M NaCl, 0.2% NP- 40 and 50mM Tris.Hcl pH 8.0. Egg lysozyme (Sigma®) was added at lmg/ml, and the solution was incubated on ice for 10'. Supernatant was obtained by centrifugation at 14k RPM using an SS-34 rotor for 35' at 5°C. The fusion protein is purified away from E. coli proteins by affinity chromatography on the glutathione Sepharose affinity column. After collection of the non-bound pool, the resin, which contains bound GST-GIQ-cdc25C (266-435) was incubated with equilibration buffer containing factor Xa (50 mM Tris HCl, 100 mM NaCl, 1 mM CaC12, pH 8.0). Specifically, the collected lysate was batch mixed with 15ml of packed glutathione resin. The slurry was poured into a column and washed extensively with the lysis buffer described above (w/o lysozyme). Finally, the protein charged column matrix was washed with factor Xa digestion buffer (50 mM Tris HCl, 100 mM NaCl, and 1 mM CaCl2 pH 8.), followed by the addition of 125 ug of Factor Xa with mixing in 20- 30 ml of digestion buffer. After 12-14 hrs incubation at 8°C the Xa released material is collected and concentrated by Amicon® ultrafiltration (YM-10 filter). The concentrate is diluted 3x with deionized water to lower the salt concentration for the ion exchange chromatography step.
Q Fast Flow column (ion exchange) chromatography. Upon the desired completion of Xa digestion of cdc25C, the products are resolved from the contaminants by anionic exchange chromatography. Proteins were resolved from one another by a linear gradient of 100-190 mM NaCl over a period of 36 min.
Analytical C4 Reverse phase HPLC. An aliquot of the purified truncated cdc25C protein was analyzed by C4 reverse phase HPLC. Using a starting solvent of 0.1% trifluoroacetic acid (TFA), the cdc25C protein was finally eluted in a linear gradient of acetonitrile from 24% to 48% in 0.1% TFA (58 min gradient). Electrophoresis and Western Blotting. SDS gel electrophoresis were performed according to Laemmli. Western blots were completed using a semi-dry electroblotter onto PVDF membranes (Millipore®) with a constant current set at 125 mA/gel (7x9 cm). Blots were visualized by either staining with Coomassie blue R250 (.2% W/V) in 50% Ethanol, 5% Acetic acid followed by a destaining step using 50% ethanol solution, or alternatively, were processed for immunostaining.
Enzvme assay and Kinetic Analyses. Assays of PNPP hydrolase activity associated with cdc25C are conducted using the reagents described previously. These reagents include (as final concentrations in 125 ul): 25 mM Hepes, pH 8.0, 10 mM DTT, 0.1 mg/ml bovine serum albumin, and variable concentrations of pNPP. For assays where a single concentration of substrate is used at saturation, we customarily use a final concentration of 20 mM pNPP. Assay solution is prepared in a final volume of 100 ul, including the addition of freshly prepared dithiothreitol. At the initiation of the assay, 25 ul of enzyme is added with mixing, and a continuous recording of absorbances at 405 nm is completed over a short time period. A 96-well plate assay is also used for the purpose of rapidly handling multiple samples. For the determination of K^ and Vmaχ (specific activity), multiple pNPP concentrations are used at a constant enzyme concentration. Rates at each concentration of substrate are determined and the 1^ and Vmaχ calculated from line fitting to a Michaelis Menten equation. The velocity of the reaction is defined as follows: One unit of activity is defined as nmoles of pNPP hydrolyzed per minute per milligram of enzyme protein at 25°C.
Sequence analysis and mass spectroscopy. Amino terminal sequencing of purified cdc25C proteins were performed on an ABI 476A protein sequencer. Database searches were conducted using the Genetics Computer Group software package (GCG). The amino acid sequences of several homologous proteins were examined by the program FASTA.
Preparation of a Rabbit anti-cdc25C Antibody. Antibodies directed against recombinant human truncated GIQ-cdc25C (266-435) were produced in a rabbit.
Characteristics of the purified truncated recombinant human cdc25C gg_ 35. The purified protein exhibits a single peak when analyzed by C4 reverse phase HPLC. The amino terminus of the purified cdc25C protein was determined to be G-I-Q -D266- etc. A mass ion representing the major product of the cdc25C protein preparation has been identified at about 20,112 daltons. This mass corresponds directly to the sequence of GIQ-cdc25C (266-435). Both the fusion protein and the minimal domain of cdc25C (266-435) were shown to be enzymatically active as a phosphatase against p-nitrophenylphosphate (PNPP). r^ and Vmaχ determinations were made using the assay conditions defined in Materials and Methods. Enzymatically, the catalytic domain represented by residues 266-435 is active as an enzyme with PNPP as substrate, giving a K^ of 20-30 mM and a Vmaχ of 250 nmoles/min/mg. For 6 liters of E. coli culture, an approximate average yield of cdc25C protein can be expected greater than 15 mg. Description of the Figures.
Figure 1A. Figure LA shows the plasmid pGEX-5X-3 used in the cloning of recombinant human cdc25C. This figure shows the position of the factor Xa cleavage site sequence, various restriction sites, and location of the cDNA inserts for cdc25C. Figure 1A is an expanded portion of the plasmid shown in Figure IB.
Figure IB. Figure IB shows the plasmid construction of plasmid pGEX-5X-3. Figure IB contains the expanded portion of the segment shown in Figure 1A. Figure 3. Figure 3 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing the product of the PCR reaction (lane 1) with template DNA and primers, while lane 2 shows the results of a control PCR reaction lacking template DNA (lane 2). Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B.
Figure 4. Figure 4 is an agarose gel electrophoresis profile of construct cdc25C, disclosed in Part B, DNA residues 1006 through 1515, showing DNA from plasmid mini preps obtained from transformed E. coli JM109 cultures (lanes 1-12). The latter DNA preps were first double digested with the restriction enzymes BamHI and Xhol prior to loading on the gel. Position "a" shows the migration of the linearized plasmid pGEX-5X-3, and position "b" shows the migration of the cdc25C DNA insert. Lanes A and B show the migration of standard DNA molecular weight ladders (1 kb and 100 bp respectively). The DNA markers represented by 1500 bp and 600 bp are found in lane B. Part C (Advanced Forms of the Macromolecule)
An improved form of cdc25C (258-473) is also disclosed. In the region spanning residues 446 through 448 (Arg-Cys-Arg) there are two arginines, one of which is readily attacked by factor Xa during the cleavage step. For an improvement, these arginines were replaced by alanines, and the single cysteine was replaced with serine using mutagenesis strategies. These substitutions are designed to prevent the factor Xa processing of this site which occurs during the normal processing of the GST fusion protein. When wild type protein is digested by factor Xa it usually generates cdc25C (258-446), which contains a large amino acid truncation at the C-terminus. The replacement with the new sequence described here allows for the generation of a cdc25C molecule with an intact C-terminus. A construct was designed based on the mutagenesis of cdc25C sequence initiating with Leu258 and terminating with Pro473. The rationale for this construct was to engineer in an improvement in stability and ease of isolation of the expressed protein. One principle construct of cdc25C was created which when expressed in E. coli would be expected to give the sequence changes shown below, both design and actual results are shown.
ADVANCED FORM SEQUENCE COMPARISON Experimental design. The top line represents the mutated cDNA sequence, while the second line represents the wild type sequence. These sequences are followed by the corresponding antisense sequences. ... ACTGAGTTGCTGGCGTCTGCAAGCCAGAGCAAA ... (SEQ. ID. NO. 15) ...AAGACTGAGTTGCTGAGGTGTCGAAGCCAGAGCAAAGTGCAGGAAGGGGAGCGGCAGCTG.. (16)
1531 + + + + + -*• 1590
...TTCTGACTCAACGACTCCACAGCTTCGGTCTCGTTTCACGTCCTTCCCCTCGCCGTCGAC.. (17) ... TGACTCAACGACCGCAGACGTTCGGTCTCGTTT ... (SEQ. ID. NO. 18)
Predicted protein sequences (shown below are terminal fragments). Wild type
421...PEYMELCEPQ SYCP HHQDH KTELLRCRSQ SKVQEGERQL REQIALLVKD MSP 473 (SEQ ID 19) Mutant
421...PEYMELCEPQ SYCPMHHQDH KTELLASASQ SKVQEGERQL REQIALLVKD MSP 473 (SEQ ID 20)
The actual protein sequences after factor Xa processing is shown below, (shown below are terminal fragments): Wild type
421...PEYMELCEPQ SYCPMHHQDH KTELLE 446 (SEQ ID 21)
Mutant
421...PEYMELCEPQ SYCPMHHQDH KTELLASASQ SKVQEGERQL REQIALLVKD MSP 473 (SEQ ID 22)
GST-IEGR-GIQ-cdc25C (258-473, A446S447A448) was created using the clone pGEX-5X-3 with the insert 982-1632 as a template. The quickchange (Stratagene) system was used with two mutagenic oligonucleotide primers designed to allow change of Arg446-Cys-447-Arg448 into Ala446-Ser447-Ala448. MRD597-1: 5' ACT GAG TTG CTG GCG TCT GCA AGC CAG AGC AAA (SEQ ID 23 corresponding to SEQ. ID. 15, above) and MRD597-2: 5' TTT GCT CTG GCT TGC AGA CGC CAG CAA CTC AGT (SEQ ID 24 corresponding to SEQ. ID. 18, above). The outcome of these changes are summarized below, with the changes made in the DNA sequence highlighted. Original 5 ' ACT GAG TTG CTG AGG TGT CGA AGC CAG AGC AAA
(SEQ ID 23)
Mutant : 5 ' ACT GAG TTG CTG GCG TC.T GCA AGC CAG AGC AAA
(SEQ ID 24)
The steps utilized in the mutagenesis are as follows: The original cDNA construct which codes for GST-IEGR-GIQ-cdc25C (Leu258-Pro473) was mutagenized using the mutagenic oligonucleotide primers described above in the Stratagene Quickchange system and transformed into XLl-Blue. Colonies were selected by first cutting out and purifying the BamHi XhoI insert and then by cutting with Taq I. The desired mutations eliminated the Taq I site from the original clone. Therefore, clones were selected based on the absence of the Taq I cutting site. Three clones maintaining the BamHI/XhoI insert but lacking the Taq I site were transformed into the expression line JM109. Correct clones were then selected by first cutting out and purifying the BamHI/XhoI insert, then checking for cutting with Bsa HI. Correct mutagenesis introduced a novel BsaHI site so clones were selected for the presence of the BsaHI cutting site. Cdc25C Mutant Cl-5 JM109 Clone #7 was chosen for further analysis. The cells were grown up and examined for expression of cdc25C protein after IPTG induction as before.
The cDNA was sequenced and shown to be as predicted. The protein was successfully expressed as a fusion protein in E. coli in soluble form, and was cleaved away from the GST moiety by factor Xa digestion. The process of expressing the protein encoded by the construct was similar to that described for the wildtype protein. The purification of the cdc25C protein was conducted in a manner similar to that described for the wild type enzyme. Frozen E. coli cell paste was thawed and washed in deionized water, and then the washed pellet was resuspended in TEN buffer containing lysozyme, and the solution was incubated on ice for 10 minutes. Supernatant was obtained by centrifugation at 20K RPM using an SS-34 rotor. The fusion protein was purified away from E. coli proteins by affinity chromatography on glutathione Sepharose affinity columns. After collection of the non-bound pool and additional washes, the resin (containing bound GST-cdc25C) was incubated with equilibration buffer containing factor Xa. After a period of time, the released protein is collected and concentrated by Amicon ultrafiltration. Next, the products were resolved from the contaminants and the factor Xa protease by anionic exchange chromatography (Q fast flow) using a linear gradient of NaCl. Fractions were assayed for phosphatase activity (hydrolysis of p-nitrophenylphosphate, PNPP), as well as by Western blotting using our own anti-cdc25C antibody. The purified protein was analyzed by mass spectrometry and shown to be as predicted.
Unlike the wild type protein, which truncated at residue 446, the released mutated cdc25C protein was found to contain residues 258-473, exhibiting no truncation at the C-terminus. This improved version of the protein was analyzed for solubility and kinetic constants. Surprisingly, we found that the protein had greater solubility, making it an ideal version for the crystallography effort. However, unlike the corresponding cdc25B protein (356-566), the cdc25C counterpart did not have a reduced Km for pNPP. In fact, the Km was nearly identical to that observed for cdc25C (258-446). wild type cdc25C intermediate (SEQ. ID. NO. 25 & 26)
GST-IEGR-GIQ-LKK TVSLCDITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ
301 DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ
351 EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELL££R.SQ
451 SKVQEGERQL REQIALLVKD MSP mutant cdc25C intermediate (SEQ. ID. NO. 27 & 28)
GST-IEGR-GIQ-LKK TVSLCDITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ
301 DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ 351 EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ
401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLASASQ
451 SKVQEGERQL REQIALLVKD MSP wild type cdc25C final product (SEQ. ID. NO. 29 & 30)
258 GIQLKK TVSLCDITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ 301 DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ 351 EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLR mutant c c25C final product (SEQ. ID. NO. 31 & 32)
258 GIQLKK TVSLCDITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ 301 DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ 351 EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLASASQ 451 SKVQEGERQL REQIALLVKD MSP
From the procedures disclosed above, one skilled in the art could practice this invention. The following CHARTS, are provided to better describe and illustrate but not limit the invention.
AMINO ACID CHARTS
AMINO ACID CHART 1 FULL LENGTH CDC25C
Full length cdc25C, amino acids, as reported in M.S. Lee, et. al., Mol. Biol. Cell. 3: 73-84 (1992). The single arrows mark the first amino acids of selected regions or special contructs, and the double arrows mark the last amino acids of these special constructs. The region corresponding to the construct described by Lee, et al., id., (258 to 473) is noted (L), so is the region corresponding to the construct described in Part A of this document (A) (258-446), as well as the region corresponding to part B, (B) (266-435). The sequence below is also listed as Sequence ID. no. 1.
1 MSTELFSSTR EEGSSGSGPS FRSNQRKMLN LLERDTSFT 40
41 VCPDVPRTPV GKFLGDSANL SILSGGTPKC CLDLSNLSSG 80
81 EITATQLTTS ADLDETGHLD SSGLQEVHLA GMNHDQHLMK 120
121 CSPAQLLCST PNGLDRGHRK RDAMCSSSAN KENDNGNLVD 160
161 SEMKYLGSPI TTVPKLDKNP NLGEDQAEEI SDELMEFSLK 200 201 DQEAKVSRSG LYRSPS PEN LNRPRLKQVE KFKDNTIPDK 240
241 VKKKYFSGQG KLRKGLCLKK TVSLCDITIT QMLEEDSNQG 280
T(L,A258)!(B266)
281 HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG 320
321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360
361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400 401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH 440 f(B435) 441 KTELLRCRSQ SKVQEGERQL REQIALLVKD MSP 473
Figure imgf000032_0001
AMINO ACID CHART 2
PART A - INTERMEDIATE The sequence below is from the construct from Part A, intermediate, before Xa cleavage, is shown here. The single arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A. The construct begins with a GST fusion, it has an Xa restriction site (IEGR) and immediately following the restriction site is a GIQ sequence, immediately following this is the cdc25C segment. The sequence below, beginning with GIQ, is also listed as Sequence ID. no. 2. The underlined amino acids were also mutated to create an improved protein . The improved mutation is shown in the next chart .
(GST) -(IEGR)- GIQ-LKK TVSLCDITIT QMLEEDSNQG 280
T (A258 ) 281 HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG 320
321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360
361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400
401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH 440
441 KTELLR££SQ SKVQEGERQL REQIALLVKD MSP 473
Figure imgf000033_0001
AMINO ACID CHART 2m
PART C ADVANCED MUTANT INTERMEDIATE The sequence below is from the construct from Part C, intermediate, before Xa cleavage, is shown here. The single arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A. The construct begins with a GST fusion, it has an Xa restriction site (IEGR) and immediately following the restriction site is a GIQ sequence, immediately following this is the cdc25C segment. The sequence below, beginning with GIQ, is also listed as Sequence ID. no. 33. The underlined amino acids were mutated to create an improved protein . The improved mutation is shown in the next chart.
(GST) - (IEGR) -GIQ-LKK TVSLCDITIT QMLEEDSNQG 280
T (A258) 281 HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG 320
321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360 361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400 401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH 440
441 KTELLASASQ SKVQEGERQL REQIALLVKD MSP 473
Figure imgf000034_0001
AMINO ACID CHART 3
PART A, FINAL CONSTRUCT
The sequence of the construct from Part A, final construct, after Xa cleavage, is shown here. The arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A. The construct begins with a GIQ sequence. The sequence below is also listed as Sequence ID. no. 3.
GIQ-LKK TVSLCDITIT QMLEEDSNQG 280 T(A258)
281 HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG 320
321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360 361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400
401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH 440
441 KTELLR 446
Figure imgf000035_0001
AMINO ACID CHART 3m
PART C, ADVANCED FORM, FINAL CONSTRUCT The sequence of the construct from Part C, final construct, after Xa cleavage, is shown here. The arrows indicate corresponding residues from the full length cdc25C sequence. Numbers are from the full length numbers, CHART A. The construct begins with a GIQ sequence. The sequence below is also listed as Sequence ID. no. 34. The underlined amino acids are changed from the wild type.
GIQ-LKK TVSLCDITIT QMLEEDSNQG 280 25 Q ) 281 HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG 320
321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360
361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400
401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH 440 441 KTELLASASQ SKVQEGERQL REQIALLVKD MSP 473
!(A473 )
AMINO ACID CHART 4
PART B - INTERMEDIATE The sequence from the construct from Part B, intermediate, is shown here. The arrows indicate corresponding residues in full length cdc25C sequence. The construct begins with a GST fusion, it has a Xa restriction site, (IEGR), immediately followed by a GIQ sequence, immediately following this is the cdc25C segment. The sequence below, beginning with GIQ, is also listed as Sequence ID. no. 4.
(GST) - (IEGR) -GIQ-DITIT QMLEEDSNQG 280
281 HLIGDFSKVC ALPTVSGKHQ 320
321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360 361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400
401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPM 435
Figure imgf000037_0002
AMINO ACID CHART 5
PART B - FINAL CONSTRUCT The sequence from the construct from Part B, final construct, is shown here. The arrows indicate corresponding residues in full length cdc25C sequence. The construct begins with a GIQ sequence . The sequence below is also listed as Sequence ID. no. 5.
GIQ-DITIT QMLEEDSNQG 280
T (B266) 281 HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG 320 321 LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK 360
361 PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ 400
401 YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPM 435 fl(B435)
NUCLEOTTDE CHARTS
The following NUCLEOTIDE CHARTS correspond to the AMINO ACID CHARTS above, and provide the nucleotide sequences that code for the amino acids in those charts . The numbers shown here correspond to full length cDNA and do not include GST, restrictions sites or residues that code for GIQ . The arrows/positions indicate the first or last included nucleotide. Stop codons may be indicated by a star "★" where naturally occurring, as in Nucleotide Chart 1 and 2, they may be omitted when added as part of the creation of the construct, as in Nucleotide Chart 3, where a stop codon would be added to the end of the sequence, this is explained from the textual description of the construct. These Charts are intended to be used in conjunction with the entire specification and drawings and are not intended to stand alone or to suggest limitations to the invention. Where the words "(GST)- (IEGR)- " appear, they are, of course, not nucleotide sequences per se, rather they refer to nucleotides that code for GST (Glutathion S-Transferase) and any suitable protease cleavage site, such as IEGR. IEGR or lie, Glu, Gly, Arg, can be coded for by the following sequences, ATC, GAA, GGT, CGT , see text and Figure 1. The GGGATCCAG residues code for GIQ .
NUCLEOTIDE CHART 1
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 1. The nucleic acids corresponding to the amino acids in AMINO CHART 1 are marked here in the same manner as in AMINO CHART 1. The first arrow below, T (L, A), is at position 982; the second arrow, t(B), is at position 1006; the third arrow, f(B), is at position 1515; the fourth arrow, ! (A), is at position 1548; and the last arrow, If (L) , is at position 1632. There is a natural stop codon at position 1629 to 1632, this is used in the Part A construct . The Part B construct has a stop codon added to become position 1516-1518 of that construct. The sequence below is also listed as Sequence ID. no. 6.
1 CAGGAAGACT CTGAGTCCGA CGTTGGCCTA CCCAGTCGGA 41 AGGCAGAGCT GCAATCTAGT TAACTACCTC CTTTCCCCTA
81 GATTTCCTTT CATTCTGCTC AAGTCTTCGC CTGTGTCCGA
121 TCCCTATCTA CTTTCTCTCC TCTTGTAGCA AGCCTCAGAC
161 TCCAGGCTTG AGCTAGGTTT TGTTTTTCTC CTGGTGAGAA
201 TTCGAAGACC ATGTCTACGG AACTCTTCTC ATCCACAAGA 241 GAGGAAGGAA GCTCTGGCTC AGGACCCAGT TTTAGGTCTA
281 ATCAAAGGAA AATGTTAAAC CTGCTCCTGG AGAGAGACAC 321 TTCCTTTACC GTCTGTCCAG ATGTCCCTAG AACTCCAGTG
361 GGCAAATTTC TTGGTGATTC TGCAAACCTA AGCATTTTGT
401 CTGGAGGAAC CCCAAAATGT TGCCTCGATC TTTCGAATCT 441 TAGCAGTGGG GAGATAACTG CCACTCAGCT TACCACTTCT
481 GCAGACCTTG ATGAAACTGG TCACCTGGAT TCTTCAGGAC 521 TTCAGGAAGT GCATTTAGCT GGGATGAATC ATGACCAGCA
561 CCTAATGAAA TGTAGCCCAG CACAGCTTCT TTGTAGCACT
601 CCGAATGGTT TGGACCGTGG CCATAGAAAG AGAGATGCAA
641 TGTGTAGTTC ATCTGCAAAT AAAGAAAATG ACAATGGAAA
681 CTTGGTGGAC AGTGAAATGA AATATTTGGG CAGTCCCATT 721 ACTACTGTTC CAAAATTGGA TAAAAATCCA AACCTAGGAG
761 AAGACCAGGC AGAAGAGATT TCAGATGAAT TAATGGAGTT
801 TTCCCTGAAA GATCAAGAAG CAAAGGTGAG CAGAAGTGGC
841 CTATATCGCT CCCCGTCGAT GCCAGAGAAC TTGAACAGGC
881 CAAGACTGAA GCAGGTGGAA AAATTCAAGG ACAACACAAT 921 ACCAGATAAA GTTAAAAAAA AGTATTTTTC TGGCCAAGGA
961 AAGCTCAGGA AGGGCTTATG TTTAAAGAAG ACAGTCTCTC
!(L, A982)
1001 TGTGTGACAT TACTATCACT CAGATGCTGG AGGAAGATTC T(B1006>
1041 TAACCAGGGG CACCTGATTG GTGATTTTTC CAAGGTATGT
1081 GCGCTGCCAA CCGTGTCAGG GAAACACCAA GATCTGAAGT 1121 ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA
1161 GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT
1201 CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG
1241 CCTTAAACTT ATATAGTCAG GAAGAACTGT TTAACTTCTT
1281 TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA 1321 ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG
1361 GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC 1401 TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT
1441 ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA
1481 TGGAACTGTG TGAACCACAG AGCTACTGCC CTATGCATCA f (B1515)
1521 TCAGGACCAC AAGACTGAGT TGCTGAGGTG TCGAAGCCAG
1561 AGCAAAGTGC AGGAAGGGGA
Figure imgf000041_0001
1601 TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT GATAACATTC
★ ★★★★★
!(L, A1632)
1641 CAGCCACTGG CTGCTAACAA GTCACCAAAA AGACACTGCA 1681 GAAACCCTGA GCAGAAAGAG GCCTTCTGGA TGGCCAAACC
1721 CAAGATTATT AAAAGATGTC TCTGCAAACC AACAGGCTAC
1761 CAACTTGTAT CCAGGCCTGG GAATGGATTA GGTTTCAGCA
1801 GAGCTGAAAG CTGGTGGCAG AGTCCTGGAG CTGGCTCTAT
1841 AAGGCAGCCT TGAGTTGCAT AGAGATTTGT ATTGGTTCAG 1881 GGAACTCTGG CATTCCTTTT CCCAACTCCT CATGTCTTCT
1921 CACAAGCCAG CCAACTCTTT CTCTCTGGGC TTCGGGCTAT
1961 GCAAGAGCGT TGTCTACCTT CTTTCTTTGT ATTTTCCTTC
2001 TTTGTTTCCC CCTCTTTCTT TTTTAAAAAT GGAAAAATAA
2041 ACACTACAGA ATGAG - 2055
NUCLEOTIDE CHART 2
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 2, the Part A construct. The nucleic acids corresponding to the amino acids in AMINO ACID CHART 2 are marked here in the same manner as in AMINO ACID CHART 2. The GST and IEGR segments are shown, the GGGATCCAG residues codes for GIQ . The first arrow below, t (L, A), is at position 982; the second arrow, t (A), is at position 1548; and the third arrow, t (L) , is at position 1632. A natural stop codon is from position
1630 to 1632. The 1548 position indicates the last included amino acid after cleavage of the GST and restriction site. The sequence below, beginning with GGGATCCAG-, is also listed as Sequence ID. no. 7.
982 (GST) - (IEGR) - GGGATCCAG-TTAAAGAAG
T(L, A982) 991 ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG
1031 AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
1071 CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA 1111 GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
1151 TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
1191 CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC
1231 ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
1271 TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC 1311 CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
1351 TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
1391 AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC
1431 AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
1471 CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC 1511 CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGAGGTG 1551 TCGAAGCCAG AGCAAAGTGC AGGAAGGGGA
Figure imgf000042_0001
1591 CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT ★
1631 GA ★★
11 <L' A1632) NUCLEOTIDE CHART 2m
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 2m, the Part C construct, intermediate. The nucleic acids corresponding to the amino acids in AMINO ACID CHART 2m are marked here in the same manner as in AMINO ACID CHART 2m. The GST and IEGR segments are shown, the GGGATCCAG residues codes for GIQ . The first arrow below, t (L, A), is at position 982; there is no corresponding arrow as the second arrow at 1548 in Nucleotide Chart 2 because this mutant does not cleave at that point; and the second arrow, If (L) , is at position 1632. A natural stop codon is from position
1630 to 1632. The sequence below, beginning with GGGATCCAG-, is also listed as Sequence ID. no.35.
982 (GST) - (IEGR) - GGGATCCAG-TTAAAGAAG
T(L, A982)
991 ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG
1031 AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
1071 CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA 1111 GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
1151 TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
1191 CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC
1231 ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
1271 TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC 1311 CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
1351 TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
1391 AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC
1431 AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
1471 CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC 1511 CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGGCGTC
1551 TG_£AAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG
1591 CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT ★
1631 GA ★★ f (L' A1632> NUCLEOTIDE CHART 3
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 3, the Part A construct. The GGGATCCAG residues code for GIQ . The first arrow below, T (L, A), is at position 982; the second arrow, I (A), is at position 1548. The sequence below is also listed as Sequence ID . no . 8.
982 GGGATCCAG-TTAAAGAAG
T(L, A982) 991 ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG 1031 AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
1071 CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA
1111 GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
1151 TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
1191 CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC 1231 ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
1271 TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC
1311 CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
1351 TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
1391 AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC 1431 AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
1471 CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC
1511 CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGAGG
Figure imgf000044_0001
NUCLEOTIDE CHART 3m
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 3m, the Part C construct. The GGGATCCAG residues code for GIQ . The first arrow below, T (L, A), is at position 982; the second arrow, t (A), is at position . The mutated DNA sequences are underlined. The sequence below is also listed as Sequence ID. no.36.
982 GGGATCCAG-TTAAAGAAG
T(L, A982) 991 ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG
1031 AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
1071 CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA 1111 GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
1151 TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
1191 CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC
1231 ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
1271 TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC 1311 CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
1351 TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
1391 AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC
1431 AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
1471 CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC 1511 CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGGCGTC
1551 TG.C.AAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG
1591 CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT ★
1631 GA ★★ f(L' A1632) NUCLEOTIDE CHART 4
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 4, the Part B construct, intermediate . The nucleic acids corresponding to the amino acids in AMINO ACID CHART 4 are marked here in the same manner as in AMINO ACID CHART 4. The GST and IEGR segments are shown, the GGGATCCAG residues code for GIQ . The first arrow below, T(B), is at position 1006; the second arrow, 1(B), is at position 1515. A stop codon is introduced after the residue 1515, see text. The sequence below, beginning with GGGATCCAG-, is also listed as Sequence ID. no. 9. (GST) - (IEGR) - GGGAT
1006 CCAG-GACAT TACTATCACT CAGATGCTGG AGGAAGATTC
T(B) 1041 TAACCAGGGG CACCTGATTG GTGATTTTTC CAAGGTATGT
1081 GCGCTGCCAA CCGTGTCAGG GAAACACCAA GATCTGAAGT
1121 ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA 1161 GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT
1201 CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG
1241 CCTTAAACTT ATATAGTCAG GAAGAACTGT TTAACTTCTT
1281 TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA
1321 ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG 1331 GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC
1401 TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT
1441 ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA
1481 TGGAACTGTG TGAACCACAG AGCTACTGCC CTATG f (B1548) NUCLEOTIDE CHART 5
Nucleic acids corresponding to the amino acids shown in AMINO ACID CHART 4, the Part B construct. The nucleic acids corresponding to the amino acids in AMINO ACID CHART 5 are marked here in the same manner as in AMINO ACID CHART 5. The GGGATCCAG residues code for GIQ . The first arrow below, T(B), is at position 1006; the second arrow, t(B), is at position 1515. A stop codon is introduced after the residue 1515, see text. The sequence below, beginning with GGGATCCAG-, is also listed as Sequence ID. no. 10.
GGGAT
1006 CCAG-GACAT TACTATCACT CAGATGCTGG AGGAAGATTC
T(B)
1041 TAACCAGGGG CACCTGATTG GTGATTTTTC CAAGGTATGT 1081 GCGCTGCCAA CCGTGTCAGG GAAACACCAA GATCTGAAGT
1121 ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA
1161 GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT
1201 CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG
1241 CCTTAAACTT ATATAGTCAG GAAGAACTGT TTAACTTCTT 1281 TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA
1321 ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG
1361 GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC
1401 TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT
1441 ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA 1481 TGGAACTGTG TGAACCACAG AGCTACTGCC CTATG
I(B1515)
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Pharmacia & Upjohn Company
(ii) TITLE OF INVENTION: Special Catalytic Domains of cdc25C Phosphatase
(iii) NUMBER OF SEQUENCES: 36
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Pharmacia & Upjohn Company (B) STREET: 301 Henrietta Street
(C) CITY: Kalamazoo
(D) STATE: MI
(E) COUNTRY: USA
(F) ZIP: 49001
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Wootton , Thomas A.
(B) REGISTRATION NUMBER: 35,004 (C) REFERENCE/DOCKET NUMBER: 6084.P CP
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 616-833-7914
(B) TELEFAX: 616-833-8897
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 473 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI- SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
Met Ser Thr Glu Leu Phe Ser Ser Thr Arg Glu Glu Gly Ser Ser Gly 1 5 10 15
Ser Gly Pro Ser Phe Arg Ser Asn Gin Arg Lys Met Leu Asn Leu Leu 20 25 30
Leu Glu Arg Asp Thr Ser Phe Thr Val Cys Pro Asp Val Pro Arg Thr 35 40 45
Pro Val Gly Lys Phe Leu Gly Asp Ser Ala Asn Leu Ser lie Leu Ser 50 55 60
Gly Gly Thr Pro Lys Cys Cys Leu Asp Leu Ser Asn Leu Ser Ser Gly 65 70 75 80 Glu lie Thr Ala Thr Gin Leu Thr Thr Ser Ala Asp Leu Asp Glu Thr
85 90 95
Gly His Leu Asp Ser Ser Gly Leu Gin Glu Val His Leu Ala Gly Met 100 105 110
Asn His Asp Gin His Leu Met Lys Cys Ser Pro Ala Gin Leu Leu Cys 115 120 125
Ser Thr Pro Asn Gly Leu Asp Arg Gly His Arg Lys Arg Asp Ala Met 130 135 140
Cys Ser Ser Ser Ala Asn Lys Glu Asn Asp Asn Gly Asn Leu Val Asp 145 150 155 160 Ser Glu Met Lys Tyr Leu Gly Ser Pro lie Thr Thr Val Pro Lys Leu
165 170 175
Asp Lys Asn Pro Asn Leu Gly Glu Asp Gin Ala Glu Glu lie Ser Asp
180 185 190
Glu Leu Met Glu Phe Ser Leu Lys Asp Gin Glu Ala Lys Val Ser Arg
195 200 205
Ser Gly Leu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro 210 215 220
Arg Leu Lys Gin Val Glu Lys Phe Lys Asp Asn Thr lie Pro Asp Lys
225 230 235 240 Val Lys Lys Lys Tyr Phe Ser Gly Gin Gly Lys Leu Arg Lys Gly Leu
245 250 255
Cys Leu Lys Lys Thr Val Ser Leu Cys Asp lie Thr lie Thr Gin Met
260 265 270
Leu Glu Glu Asp Ser Asn Gin Gly His Leu lie Gly Asp Phe Ser Lys
275 280 285
Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu Lys Tyr 290 295 300
Val Asn Pro Glu Thr val Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly 305 310 315 320 Leu lie Glu Lys Phe Tyr Val lie Asp Cys Arg Tyr Pro Tyr Glu Tyr
325 330 335
Leu Gly Gly His lie Gin Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu 340 345 350
Leu Phe Asn Phe Phe Leu Lys Lys Pro lie Val Pro Leu Asp Thr Gin 355 360 365 Lys Arg lie lie lie Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly 370 375 380
Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin 385 390 395 400
Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr lie Leu Lys Gly Gly Tyr
405 410 415
Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr
420 425 430
Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg Cys Arg 435 440 445
Ser Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin lie 450 455 460 Ala Leu Leu Val Lys Asp Met Ser Pro
465 470
(2) INFORMATION FOR SEQ ID NO 2 (1) SEQUENCE CHARACTERISTICS
(A) LENGTH. 219 amino acids
(B) TYPE, amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(11) MOLECULE TYPE, protein (in) HYPOTHETICAL: NO ( V) ANTI- SENSE: NO
(v) FRAGMENT TYPE- N- terminal
(XI) SEQUENCE DESCRIPTION: SEQ ID NO : 2.
Gly lie Gin Leu Lys Lys Thr Val Ser Leu Cys Asp lie Thr lie Thr 1 5 10 15
Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu lie Gly Asp Phe 20 25 30
Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu 35 40 45
Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe
50 55 60 Gin Gly Leu lie Glu Lys Phe Tyr Val lie Asp Cys Arg Tyr Pro Tyr
65 70 75 80
Glu Tyr Leu Gly Gly His lie Gin Gly Ala Leu Asn Leu Tyr Ser Gin
85 90 95
Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro lie Val Pro Leu Asp
100 105 110
Thr Gin Lys Arg lie lie lie Val Phe His Cys Glu Phe Ser Ser Glu 115 120 125
Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu 130 135 140
Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr lie Leu Lys Gly 145 150 155 160
Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin 165 170 175
Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg 180 185 190
Cys Arg Ser Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu 195 200 205 Gin lie Ala Leu Leu Val Lys Asp Met Ser Pro
210 215
(2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Gly lie Gin Leu Lys Lys Thr Val Ser Leu Cys Asp lie Thr lie Thr 1 5 10 15
Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu lie Gly Asp Phe 20 25 30
Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu 35 40 45
Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe 50 55 60 Gin Gly Leu lie Glu Lys Phe Tyr Val lie Asp Cys Arg Tyr Pro Tyr
65 70 75 80
Glu Tyr Leu Gly Gly His lie Gin Gly Ala Leu Asn Leu Tyr Ser Gin 85 90 95
Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro lie Val Pro Leu Asp
100 105 110
Thr Gin Lys Arg lie lie lie Val Phe His Cys Glu Phe Ser Ser Glu 115 120 125
Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu
130 135 140 Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr lie Leu Lys Gly
145 150 155 160 Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin
165 170 175
Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg
180 185 190
(2) INFORMATION FOR SEQ ID NO : 4 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 173 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Gly lie Gin Asp lie Thr lie Thr Gin Met Leu Glu Glu Asp Ser Asn 1 5 10 15
Gin Gly His Leu lie Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr 20 25 30
Val Ser Gly Lys His Gin Asp Leu Lys Tyr Val Asn Pro Glu Thr Val 35 40 45
Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly Leu lie Glu Lys Phe Tyr 50 55 60 Val lie Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His lie Gin
65 70 75 80
Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu Leu Phe Asn Phe Phe Leu 85 90 95
Lys Lys Pro lie Val Pro Leu Asp Thr Gin Lys Arg lie lie lie Val 100 105 110
Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys 115 120 125
Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin Tyr Pro Ala Leu Tyr Tyr
130 135 140 Pro Glu Leu Tyr lie Leu Lys Gly Gly Tyr Arg Asp Phe Phe Pro Glu
145 150 155 160
Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys Pro Met 165 170
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 173 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
Gly lie Gin Asp lie Thr lie Thr Gin Met Leu Glu Glu Asp Ser Asn 1 5 10 15
Gin Gly His Leu lie Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr 20 25 30 Val Ser Gly Lys His Gin Asp Leu Lys Tyr Val Asn Pro Glu Thr Val
35 40 45
Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly Leu lie Glu Lys Phe Tyr 50 55 60
Val lie Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His lie Gin 65 70 75 80
Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu Leu Phe Asn Phe Phe Leu 85 90 95
Lys Lys Pro lie Val Pro Leu Asp Thr Gin Lys Arg lie lie lie Val
100 105 110 Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys
115 120 125
Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin Tyr Pro Ala Leu Tyr Tyr
130 135 140
Pro Glu Leu Tyr lie Leu Lys Gly Gly Tyr Arg Asp Phe Phe Pro Glu
145 150 155 160
Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys Pro Met 165 170
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2055 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
( i) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : CAGGAAGACT CTGAGTCCGA CGTTGGCCTA CCCAGTCGGA AGGCAGAGCT GCAATCTAGT 60 TAACTACCTC CTTTCCCCTA GATTTCCTTT CATTCTGCTC AAGTCTTCGC CTGTGTCCGA 120 TCCCTATCTA CTTTCTCTCC TCTTGTAGCA AGCCTCAGAC TCCAGGCTTG AGCTAGGTTT 180
TGTTTTTCTC CTGGTGAGAA TTCGAAGACC ATGTCTACGG AACTCTTCTC ATCCACAAGA 240
GAGGAAGGAA GCTCTGGCTC AGGACCCAGT TTTAGGTCTA ATCAAAGGAA AATGTTAAAC 300
CTGCTCCTGG AGAGAGACAC TTCCTTTACC GTCTGTCCAG ATGTCCCTAG AACTCCAGTG 360 GGCAAATTTC TTGGTGATTC TGCAAACCTA AGCATTTTGT CTGGAGGAAC CCCAAAATGT 420
TGCCTCGATC TTTCGAATCT TAGCAGTGGG GAGATAACTG CCACTCAGCT TACCACTTCT 480
GCAGACCTTG ATGAAACTGG TCACCTGGAT TCTTCAGGAC TTCAGGAAGT GCATTTAGCT 540
GGGATGAATC ATGACCAGCA CCTAATGAAA TGTAGCCCAG CACAGCTTCT TTGTAGCACT 600
CCGAATGGTT TGGACCGTGG CCATAGAAAG AGAGATGCAA TGTGTAGTTC ATCTGCAAAT 660 AAAGAAAATG ACAATGGAAA CTTGGTGGAC AGTGAAATGA AATATTTGGG CAGTCCCATT 720
ACTACTGTTC CAAAATTGGA TAAAAATCCA AACCTAGGAG AAGACCAGGC AGAAGAGATT 780
TCAGAIGAAT TAATGGAGTT TTCCCTGAAA GATCAAGAAG CAAAGGTGAG CAGAAGTGGC 840
CTATATCGCT CCCCGTCGAT GCCAGAGAAC TTGAACAGGC CAAGACTGAA GCAGGTGGAA 900
AAATTCAAGG ACAACACAAT ACCAGATAAA GTTAAAAAAA AGTATTTTTC TGGCCAAGGA 960 AAGCTCAGGA AGGGCTTATG TTTAAAGAAG ACAGTCTCTC TGTGTGACAT TACTATCACT 1020
CAGATGCTGG AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC CAAGGTATGT 1080
GCGCTGCCAA CCGTGTCAGG GAAACACCAA GATCTGAAGT ATGTCAACCC AGAAACAGTG 1140
GCTGCCTTAC TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT 1200
CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG CCTTAAACTT ATATAGTCAG 1260 GAAGAACTGT TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA 1320
ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT 1380
CTGCGTGAAG AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT 1440
ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA TGGAACTGTG TGAACCACAG 1500
AGCTACTGCC CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGAGGTG TCGAAGCCAG 1560 AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC 1620
ATGAGCCCAT GATAACATTC CAGCCACTGG CTGCTAACAA GTCACCAAAA AGACACTGCA 1680
GAAACCCTGA GCAGAAAGAG GCCTTCTGGA TGGCCAAACC CAAGATTATT AAAAGATGTC 1740
TCTGCAAACC AACAGGCTAC CAACTTGTAT CCAGGCCTGG GAATGGATTA GGTTTCAGCA 1800
GAGCTGAAAG CTGGTGGCAG AGTCCTGGAG CTGGCTCTAT AAGGCAGCCT TGAGTTGCAT 1860 AGAGATTTGT ATTGGTTCAG GGAACTCTGG CATTCCTTTT CCCAACTCCT CATGTCTTCT 1920
CACAAGCCAG CCAACTCTTT CTCTCTGGGC TTCGGGCTAT GCAAGAGCGT TGTCTACCTT 1980
CTTTCTTTGT ATTTTCCTTC TTTGTTTCCC CCTCTTTCTT TTTTAAAAAT GGAAAAATAA 2040
ACACTACAGA ATGAG 2055 (2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 660 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
GGGATCCAGT TAAAGAAGAC AGTCTCTCTG TGTGACATTA CTATCACTCA GATGCTGGAG 60
GAAGATTCTA ACCAGGGGCA CCTGATTGGT GATTTTTCCA AGGTATGTGC GCTGCCAACC 120
GTGTCAGGGA AACACCAAGA TCTGAAGTAT GTCAACCCAG AAACAGTGGC TGCCTTACTG 180 TCGGGGAAGT TCCAGGGTCT GATTGAGAAG TTTTATGTCA TTGATTGTCG CTATCCATAT 240
GAGTATCTGG GAGGACACAT CCAGGGAGCC TTAAACTTAT ATAGTCAGGA AGAACTGTTT 300
AACTTCTTTC TGAAGAAGCC CATCGTCCCT TTGGACACCC AGAAGAGAAT AATCATCGTG 360
TTCCACTGTG AATTCTCCTC AGAGAGGGGC CCCCGAATGT GCCGCTGTCT GCGTGAAGAG 420
GACAGGTCTC TGAACCAGTA TCCTGCATTG TACTACCCAG AGCTATATAT CCTTAAAGGC 480 GGCTACAGAG ACTTCTTTCC AGAATATATG GAACTGTGTG AACCACAGAG CTACTGCCCT 540
ATGCATCATC AGGACCACAA GACTGAGTTG CTGAGGTGTC GAAGCCAGAG CAAAGTGCAG 600
GAAGGGGAGC GGCAGCTGCG GGAGCAGATT GCCCTTCTGG TGAAGGACAT GAGCCCATGA 660
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 576 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
( i) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : GGGATCCAGT TAAAGAAGAC AGTCTCTCTG TGTGACATTA CTATCACTCA GATGCTGGAG 60
GAAGATTCTA ACCAGGGGCA CCTGATTGGT GATTTTTCCA AGGTATGTGC GCTGCCAACC 120
GTGTCAGGGA AACACCAAGA TCTGAAGTAT GTCAACCCAG AAACAGTGGC TGCCTTACTG 180
TCGGGGAAGT TCCAGGGTCT GATTGAGAAG TTTTATGTCA TTGATTGTCG CTATCCATAT 240 GAGTATCTGG GAGGACACAT CCAGGGAGCC TTAAACTTAT ATAGTCAGGA AGAACTGTTT 300
AACTTCTTTC TGAAGAAGCC CATCGTCCCT TTGGACACCC AGAAGAGAAT AATCATCGTG 360
TTCCACTGTG AATTCTCCTC AGAGAGGGGC CCCCGAATGT GCCGCTGTCT GCGTGAAGAG 420
GACAGGTCTC TGAACCAGTA TCCTGCATTG TACTACCCAG AGCTATATAT CCTTAAAGGC 480
GGCTACAGAG ACTTCTTTCC AGAATATATG GAACTGTGTG AACCACAGAG CTACTGCCCT 540
ATGCATCATC AGGACCACAA GACTGAGTTG CTGAGG 576 (2) INFORMATION FOR SEQ ID NO : 9 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
( i) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
GGGATCCAGG ACATTACTAT CACTCAGATG CTGGAGGAAG ATTCTAACCA GGGGCACCTG 60
ATTGGTGATT TTTCCAAGGT ATGTGCGCTG CCAACCGTGT CAGGGAAACA CCAAGATCTG 120 AAGTATGTCA ACCCAGAAAC AGTGGCTGCC TTACTGTCGG GGAAGTTCCA GGGTCTGATT 180
GAGAAGTTTT ATGTCATTGA TTGTCGCTAT CCATATGAGT ATCTGGGAGG ACACATCCAG 240
GGAGCCTTAA ACTTATATAG TCAGGAAGAA CTGTTTAACT TCTTTCTGAA GAAGCCCATC 300
GTCCCTTTGG ACACCCAGAA GAGAATAATC ATCGTGTTCC ACTGTGAATT CTCCTCAGAG 360
AGGGGCCCCC GAATGTGCCG CTGTCTGCGT GAAGAGGACA GGTCTCTGAA CCAGTATCCT 420 GCATTGTACT ACCCAGAGCT ATATATCCTT AAAGGCGGCT ACAGAGACTT CTTTCCAGAA 480
TATATGGAAC TGTGTGAACC ACAGAGCTAC TGCCCTATG 519
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGGATCCAGG ACATTACTAT CACTCAGATG CTGGAGGAAG ATTCTAACCA GGGGCACCTG 60 ATTGGTGATT TTTCCAAGGT ATGTGCGCTG CCAACCGTGT CAGGGAAACA CCAAGATCTG 120
AAGTATGTCA ACCCAGAAAC AGTGGCTGCC TTACTGTCGG GGAAGTTCCA GGGTCTGATT 180
GAGAAGTTTT ATGTCATTGA TTGTCGCTAT CCATATGAGT ATCTGGGAGG ACACATCCAG 240
GGAGCCTTAA ACTTATATAG TCAGGAAGAA CTGTTTAACT TCTTTCTGAA GAAGCCCATC 300 GTCCCTTTGG ACACCCAGAA GAGAATAATC ATCGTGTTCC ACTGTGAATT CTCCTCAGAG 360
AGGGGCCCCC GAATGTGCCG CTGTCTGCGT GAAGAGGACA GGTCTCTGAA CCAGTATCCT 420
GCATTGTACT ACCCAGAGCT ATATATCCTT AAAGGCGGCT ACAGAGACTT CTTTCCAGAA 480
TATATGGAAC TGTGTGAACC ACAGAGCTAC TGCCCTATG 519 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
( i) SEQUENCE DESCRIPTION: SEQ ID NO : 11 :
GCGGATCCAG TTAAAGAAGA CAGTCT 26
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GCCTCGAGTC ATGGGCTCAT GTCCTT 26 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GCGGATCCAG GACATTACTA TCACTCA
(2) INFORMATION FOR SEQ ID NO : 14 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
GCCTCGAGTC ACATAGGGCA GTAGCTC
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (ϋi) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ACTGAGTTGC TGGCGTCTGC AAGCCAGAGC AAA
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
AAGACTGAGT TGCTGAGGTG TCGAAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 17 : TTCTGACTCA ACGACTCCAC AGCTTCGGTC TCGTTTCACG TCCTTCCCCT CGCCGTCGAC
(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TGACTCAACG ACCGCAGACG TTCGGTCTCG TTT
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys Pro Met His 1 5 10 15
His Gin Asp His Lys Thr Glu Leu Leu Arg Cys Arg Ser Gin Ser Lys 20 25 30
Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin lie Ala Leu Leu Val 35 40 45
Lys Asp Met Ser Pro 50
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys Pro Met His
1 5 10 15
His Gin Asp His Lys Thr Glu Leu Leu Ala Ser Ala Ser Gin Ser Lys 20 25 30
Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin lie Ala Leu Leu Val 35 40 45
Lys Asp Met Ser Pro 50
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys Pro Met His
1 5 10 15 His Gin Asp His Lys Thr Glu Leu Leu Arg 20 25
( 2 ) INFORMATION FOR SEQ ID NO : 22 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys Pro Met His 1 5 10 15 His Gin Asp His Lys Thr Glu Leu Leu Ala Ser Ala Ser Gin Ser Lys
20 25 30
Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin lie Ala Leu Leu Val 35 40 45
Lys Asp Met Ser Pro 50
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
ACTGAGTTGC TGAGGTGTCG AAGCCAGAGC AAA
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iϋ) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
ACTGAGTTGC TGGCGTCTGC AAGCCAGAGC AAA
(2) INFORMATION FOR SEQ ID NO: 25'
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 223 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(il) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: lie Glu Gly Arg Gly lie Gin Leu Lys Lys Thr Val Ser Leu Cys Asp 1 5 10 15 He Thr He Thr Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu
20 25 30 lie Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys 35 40 45
His Gin Asp Leu Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu 50 55 60
Ser Gly Lys Phe Gin Gly Leu He Glu Lys Phe Tyr Val He Asp Cys 65 70 75 80
Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His He Gin Gly Ala Leu Asn
85 90 95 Leu Tyr Ser Gin Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro He
100 105 110
Val Pro Leu Asp Thr Gin Lys Arg He He He Val Phe His Cys Glu 115 120 125
Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu 130 135 140
Asp Arg Ser Leu Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr 145 150 155 160
He Leu Lys Gly Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu
165 170 175 Cys Glu Pro Gin Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr
180 185 190
Glu Leu Leu Arg Cys Arg Ser Gin Ser Lys Val Gin Glu Gly Glu Arg
195 200 205
Gin Leu Arg Glu Gin He Ala Leu Leu Val Lys Asp Met Ser Pro
210 215 220 (2) INFORMATION FOR SEQ ID NO: 26
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH. 216 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY- linear (ii) MOLECULE TYPE, peptide
(ill) HYPOTHETICAL. NO (IV) ANTI -SENSE. NO
(X ) SEQUENCE DESCRIPTION SEQ ID NO 26 Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr Gin Met Leu
1 5 10 15
Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe Ser Lys Val 20 25 30
Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu Lys Tyr Val 35 40 45
Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly Leu 50 55 60
He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu 65 70 75 80 Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu Leu
85 90 95
Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp Thr Gin Lys 100 105 110
Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro 115 120 125
Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin Tyr 130 135 140
Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly Gly Tyr Arg
145 150 155 160 Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys
165 170 175
Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg Cys Arg Ser
180 185 190
Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin He Ala
195 200 205
Leu Leu Val Lys Asp Met Ser Pro 210 215
(2) INFORMATION FOR SEQ ID NO: 27.
(l) SEQUENCE CHARACTERISTICS- (A) LENGTH: 223 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS. single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iϋ) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
He Glu Gly Arg Gly He Gin Leu Lys Lys Thr Val Ser Leu Cys Asp 1 5 10 15
He Thr He Thr Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu 20 25 30
He Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys 35 40 45
His Gin Asp Leu Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu
50 55 60 Ser Gly Lys Phe Gin Gly Leu He Glu Lys Phe Tyr Val He Asp Cys
65 70 75 80
Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His He Gin Gly Ala Leu Asn
85 90 95
Leu Tyr Ser Gin Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro He
100 105 110
Val Pro Leu Asp Thr Gin Lys Arg He He He Val Phe His Cys Glu 115 120 125
Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu
130 135 140 Asp Arg Ser Leu Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr
145 150 155 160
He Leu Lys Gly Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu 165 170 175
Cys Glu Pro Gin Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr 180 185 190
Glu Leu Leu Ala Ser Ala Ser Gin Ser Lys Val Gin Glu Glv Glu Arg 195 200 205
Gin Leu Arg Glu Gin He Ala Leu Leu Val Lys Asp Met Ser Pro 210 215 220 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iϋ) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr Gin Met Leu 1 5 10 15
Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe Ser Lys Val 20 25 30
Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu Lys Tyr Val 35 40 45 Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly Leu
50 55 60
He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu
65 70 75 80
Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu Leu 85 90 95
Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp Thr Gin Lys 100 105 110
Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro
115 120 125 Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin Tyr
130 135 140
Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly Gly Tyr Arg 145 150 155 160
Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys 165 170 175
Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Ala Ser Ala Ser 180 185 190
Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin He Ala 195 200 205 Leu Leu Val Lys Asp Met Ser Pro
210 215
(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Gly He Gin Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr
1 5 10 15 Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe 20 25 30
Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu 35 40 45
Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe 50 55 60 Gin Gly Leu He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr
65 70 75 80
Glu Tyr Leu Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin 85 90 95
Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp 100 105 110
Thr Gin Lys Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu 115 120 125
Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu 130 135 140 Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly
145 150 155 160
Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin
165 170 175
Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg
180 185 190
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 189 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr Gin Met Leu 1 5 10 15
Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe Ser Lys Val 20 25 30
Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu Lys Tyr Val 35 40 45
Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly Leu 50 55 60 He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu
65 70 75 80 Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu Leu 85 90 95
Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp Thr Gin Lys 100 105 110
Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro 115 120 125 Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin Tyr
130 135 140
Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly Gly Tyr Arg
145 150 155 160
Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys
165 170 175
Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg 180 185
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Gly He Gin Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr 1 5 10 15
Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe
20 25 ' 30 Ser Lys val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu
35 40 45
Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe 50 55 60
Gin Gly Leu He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr 65 70 75 80
Glu Tyr Leu Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin 85 90 95
Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp 100 105 110 Thr Gin Lys Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu
115 120 125
Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu 130 135 140
Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly 145 150 155 160 Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin 165 170 175
Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Ala 180 185 190
Ser Ala Ser Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu 195 200 205
Gin He Ala Leu Leu Val Lys Asp Met Ser Pro 210 215
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr Gin Met Leu 1 5 10 15
Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe Ser Lys Val 20 25 30
Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu Lys Tyr Val 35 40 45 Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gin Gly Leu
50 55 60
He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu 65 70 75 80
Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin Glu Glu Leu 85 90 95
Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp Thr Gin Lys 100 105 110
Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro 115 120 125 Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gin Tyr
130 135 140
Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly Gly Tyr Arg
145 150 155 160
Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin Ser Tyr Cys
165 170 175
Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Ala Ser Ala Ser 180 185 190
Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu Gin He Ala 195 200 205
Leu Leu Val Lys Asp Met Ser Pro 210 215
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Gly He Gin Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr 1 5 10 15
Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe 20 25 30
Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu 35 40 45
Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe
50 55 60 Gin Gly Leu He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr 65 70 75 80
Glu Tyr Leu Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin 85 90 95
Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp 100 105 110
Thr Gin Lys Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu 115 120 125
Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu
130 135 140 Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly
145 150 155 160
Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin 165 170 175
Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Ala 180 185 190
Ser Ala Ser Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg Glu 195 200 205
Gin He Ala Leu Leu Val Lys Asp Met Ser Pro 210 215 (2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 220 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: Gly He Gin Leu Lys Lys Thr Val Ser Leu Cys Asp He Thr He Thr
1 5 10 15
Gin Met Leu Glu Glu Asp Ser Asn Gin Gly His Leu He Gly Asp Phe
20 25 30
Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gin Asp Leu 35 40 45
Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe 50 55 60
Gin Gly Leu He Glu Lys Phe Tyr Val He Asp Cys Arg Tyr Pro Tyr 65 70 75 80 Glu Tyr Leu Gly Gly His He Gin Gly Ala Leu Asn Leu Tyr Ser Gin
85 90 95
Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro He Val Pro Leu Asp 100 105 110
Thr Gin Lys Arg He He He Val Phe His Cys Glu Phe Ser Ser Glu 115 120 125
Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu 130 135 140
Asn Gin Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr He Leu Lys Gly
145 150 155 160 Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gin
165 170 175
Ser Tyr Cys Pro Met His His Gin Asp His Lys Thr Glu Leu Leu Arg 180 185 190
Ala Ser Ala Ser Gin Ser Lys Val Gin Glu Gly Glu Arg Gin Leu Arg 195 200 205
Glu Gin He Ala Leu Leu Val Lys Asp Met Ser Pro 210 215 220
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 660 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
GGGATCCAGT TAAAGAAGAC AGTCTCTCTG TGTGACATTA CTATCACTCA GATGCTGGAG 60
GAAGATTCTA ACCAGGGGCA CCTGATTGGT GATTTTTCCA AGGTATGTGC GCTGCCAACC 120 GTGTCAGGGA AACACCAAGA TCTGAAGTAT GTCAACCCAG AAACAGTGGC TGCCTTACTG 180
TCGGGGAAGT TCCAGGGTCT GATTGAGAAG TTTTATGTCA TTGATTGTCG CTATCCATAT 240
GAGTATCTGG GAGGACACAT CCAGGGAGCC TTAAACTTAT ATAGTCAGGA AGAACTGTTT 300
AACTTCTTTC TGAAGAAGCC CATCGTCCCT TTGGACACCC AGAAGAGAAT AATCATCGTG 360
TTCCACTGTG AATTCTCCTC AGAGAGGGGC CCCCGAATGT GCCGCTGTCT GCGTGAAGAG 420 GACAGGTCTC TGAACCAGTA TCCTGCATTG TACTACCCAG AGCTATATAT CCTTAAAGGC 480
GGCTACAGAG ACTTCTTTCC AGAATATATG GAACTGTGTG AACCACAGAG CTACTGCCCT 540
ATGCATCATC AGGACCACAA GACTGAGTTG CTGGCGTCTG CAAGCCAGAG CAAAGTGCAG 600
GAAGGGGAGC GGCAGCTGCG GGAGCAGATT GCCCTTCTGG TGAAGGACAT GAGCCCATGA 660
(2) INFORMATION FOR SEQ ID NO: 36:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 660 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(11) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
( i) SEQUENCE DESCRIPTION: SEQ ID NO : 36 :
GGGATCCAGT TAAAGAAGAC AGTCTCTCTG TGTGACATTA CTATCACTCA GATGCTGGAG 60
GAAGATTCTA ACCAGGGGCA CCTGATTGGT GATTTTTCCA AGGTATGTGC GCTGCCAACC 120 GTGTCAGGGA AACACCAAGA TCTGAAGTAT GTCAACCCAG AAACAGTGGC TGCCTTACTG 180
TCGGGGAAGT TCCAGGGTCT GATTGAGAAG TTTTATGTCA TTGATTGTCG CTATCCATAT 240
GAGTATCTGG GAGGACACAT CCAGGGAGCC TTAAACTTAT ATAGTCAGGA AGAACTGTTT 300
AACTTCTTTC TGAAGAAGCC CATCGTCCCT TTGGACACCC AGAAGAGAAT AATCATCGTG 360
TTCCACTGTG AATTCTCCTC AGAGAGGGGC CCCCGAATGT GCCGCTGTCT GCGTGAAGAG 420 GACAGGTCTC TGAACCAGTA TCCTGCATTG TACTACCCAG AGCTATATAT CCTTAAAGGC 480
GGCTACAGAG ACTTCTTTCC AGAATATATG GAACTGTGTG AACCACAGAG CTACTGCCCT 540
ATGCATCATC AGGACCACAA GACTGAGTTG CTGGCGTCTG CAAGCCAGAG CAAAGTGCAG 600
GAAGGGGAGC GGCAGCTGCG GGAGCAGATT GCCCTTCTGG TGAAGGACAT GAGCCCATGA 660

Claims

(Note, Key to claim code. * in indicates an independent claim. A or a is to part A construct. B or b is to part B construct. P or p is for peptide. D or d is for DNA.)
The fusion comprising the fusion shown below,
Figure imgf000072_0001
where, the fusion may be comprised of DNA or amino acids, where the different parts of the fusions are shown as different lines in the box, where a) the GST portion, is labeled GST, with a straight line in the box, b) the protease cleavage site is shown as a dotted line in the box, labeled "P," c) the restriction site is shown as a wavey line in the box, labeled "R," and d) the cdc25C like portion is shown as a heavy line in the box, labelled
"cdc25C" where the numbers above the box indicate DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either DNA or amino acids, where the boxes, lines and numbers are not drawn, as shown, to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25C region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length cdc25C, where the numbers near the dotted lines show one construct and the numbers at the ends of the cdc25C box with the heavy line shows a different construct, or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues, where the circle indicates that mutations can be made.
2. The fusion shown in claim 1 where the protease cleavage site is created to be responsive to Factor Xa.
3. The fusion shown in claim 1 where the restriction site, when expressed, yields the amino acid sequence GIQ.
4. The fusion protein of claim 1 comprising the following, (GST) -(IEGR)- GIQ-LKK TVSLCDITIT QMLEEDSNQG
HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG
LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK
PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ
YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLRCRSQ SKVQEGERQL REQIALLVKD MSP, (SEQ. ID NO 2) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
5. The fusion protein of claim 1 comprising the following sequences, GIQ-LKK TVSLCDITIT QMLEEDSNQG
HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLR , (SEQ. ID. NO. 3) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
6. The fusion protein of claim 1, comprising the following sequences, GIQ-LKK TVSLCDITIT QMLEEDSNQG
HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG
LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK
PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ
YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLRCRSQ SKVQEGERQL REQIALLVKD MSP, (SEQ. ID. NO. 4) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
7. The fusion protein of claim 1, comprising the following,
(GST) - (IEGR) - GIQ-LKK TVSLCDITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLASASQ SKVQEGERQL REQIALLVKD MSP (SEQ. ID. NO. 33) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
8. The fusion protein of claim 1 comprising the following sequences,
GIQ-LKK TVSLCDITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPMHHQDH KTELLASASQ SKVQEGERQL REQIALLVKD MSP (SEQ. ID. NO. 34) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
9. The DNA coding for the fusion protein of claim 1, comprising the following, the DNA that codes for glutathione S-trans- ferase--, linked to the following sequences..
AT CGAAGGTCGT- - GGGATCCAG- -TTAAAGAAG (IEGR) -- (GIQ) --σdc25C...
ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG
AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA
GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC
ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC
CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC
AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC
CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGAGGTG TCGAAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT GA, (SEQ. ID. NO. 7 - portion following IEGR) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
10. The DNA coding for the fusion protein of claim 1 comprising the following sequences,
GGGATCCAG- TTAAAGAAG
ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA
GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC
CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC
CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGAGG, (SEQ. ID. NO. 8) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
11. The The DNA coding for the fusion protein of claim 1 that is the essential nucleic acid intermediate, comprising a sequence comprising the following sequences,
GGG A TCCAG- TTAAAGAAG ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG
AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA
GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC
ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC
CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC
AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC
CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGAGGTG TCGAAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG
CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT GA, (SEQ. ID. NO. 7) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
12. The DNA coding for the fusion protein of claim 1 that is, essential nucleic acid intermediate, comprising the following,
(GST) - ( IEGR) - GGGATCCAG-TTAAAGAAG ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA
GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC
TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT
CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT
TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC
CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC
TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG
AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT
CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC
CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGGCGTC
TGCAAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG
CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT GA, (SEQ. ID. NO.35) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
13. The DNA coding for the fusion protein of claim 1, comprising, GGGATCCAG- TTAAAGAAG
ACAGTCTCTC TGTGTGACAT TACTATCACT CAGATGCTGG
AGGAAGATTC TAACCAGGGG CACCTGATTG GTGATTTTTC
CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA
GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC CTATGCATCA TCAGGACCAC AAGACTGAGT TGCTGGCGTC TGCAAGCCAG AGCAAAGTGC AGGAAGGGGA GCGGCAGCTG CGGGAGCAGA TTGCCCTTCT GGTGAAGGAC ATGAGCCCAT GA (SEQ. ID. NO. 36) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
14. The fusion comprising the fusion shown below,
(DNA)
GST P 1006 cdc25C 1 515
Figure imgf000077_0001
GST p R 266 cdc25C 435
(peptide) where, the fusion may be comprised of DNA or amino acids, where the different parts of the fusions are shown as different lines in the box, where a) the GST portion, is labeled GST, with a straight line in the box, b) the protease cleavage site is shown as a dotted line in the box, labeled "P," c) a stop codon appears in the DNA sequence, but not the amino acid sequence, d) the restriction site is shown as a wavey line in the box, labeled "R," and e) the cdc25C like portion is shown as a heavy line in the box, labelled "cdc25C" where the numbers above the box indicate DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either DNA or amino acids, where the boxes, lines and numbers are not drawn, as shown, to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25C region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length cdc25C, where the numbers near the dotted lines show one construct and the numbers at the ends of the cdc25C box with the heavy line shows a different construct, or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
15. The fusion shown in claim 14 where the protease cleavage site is created to be responsive to Factor Xa.
16. The fusion shown in claim 14 where the restriction site, when expressed, yields the amino acid sequence GIQ.
17. The fusion protein of claim 14 comprising,
(GST) -(IEGR)-GIQ-DITIT QMLEEDSNQG HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG
LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK
PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ
YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPM, (SEQ. ID. NO. 4) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
18. The fusion protein of claim 14 comprising the following sequences,
GIQ-DITIT QMLEEDSNQG
HLIGDFSKVC ALPTVSGKHQ DLKYVNPETV AALLSGKFQG LIEKFYVIDC RYPYEYLGGH IQGALNLYSQ EELFNFFLKK
PIVPLDTQKR IIIVFHCEFS SERGPRMCRC LREEDRSLNQ
YPALYYPELY ILKGGYRDFF PEYMELCEPQ SYCPM, (SEQ. ID. NO.5) or a similar sequence obtained by deleting, adding or replacing one to several amino acid residues.
19. The DNA coding for the fusion protein of claim 14, comprising, the DNA that codes for glutathione S-transferase-l inked to the following . . .
....AT CGAAGGTCGT- - GGGATC (IEGR) - - G I
CAG- -GACAT TACTATCACT CAGATGCTGG AGGAAGATTC Q --cdc25C... TAACCAGGGG CACCTGATTG GTGATTTTTC CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA GATCTGAAGT ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG CCTTAAACTT ATATAGTCAG GAAGAACTGT TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC CTATG , (SEQ. ID. NO.9) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
20. The DNA coding for the fusion protein of claim 14 that is,
GGG AT
CCCA-GACAT TACTATCACT CAGATGCTGG AGGAAGATTC
TAACCAGGGG CACCTGATTG GTGATTTTTC CAAGGTATGT GCGCTGCCAA CCGTGTCAGG GAAACACCAA GATCTGAAGT
ATGTCAACCC AGAAACAGTG GCTGCCTTAC TGTCGGGGAA
GTTCCAGGGT CTGATTGAGA AGTTTTATGT CATTGATTGT
CGCTATCCAT ATGAGTATCT GGGAGGACAC ATCCAGGGAG
CCTTAAACTT ATATAGTCAG GAAGAACTGT TTAACTTCTT TCTGAAGAAG CCCATCGTCC CTTTGGACAC CCAGAAGAGA
ATAATCATCG TGTTCCACTG TGAATTCTCC TCAGAGAGGG
GCCCCCGAAT GTGCCGCTGT CTGCGTGAAG AGGACAGGTC
TCTGAACCAG TATCCTGCAT TGTACTACCC AGAGCTATAT
ATCCTTAAAG GCGGCTACAG AGACTTCTTT CCAGAATATA TGGAACTGTG TGAACCACAG AGCTACTGCC CTATG, (SEQ. ID. NO. 10) or a similar sequence obtained by deleting, adding or replacing one to several nucleic acid residues.
PCT/US1998/000018 1997-01-13 1998-01-09 Special catalytic domains of cdc25c phosphatase WO1998030680A2 (en)

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US60/035,188 1997-01-13

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WO2001021771A2 (en) * 1999-09-22 2001-03-29 Canbas Co., Ltd. Compositions and methods for inhibiting g2 cell cycle arrest and sensitizing cells to dna damaging agents
WO2001044467A2 (en) * 1999-12-14 2001-06-21 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
B. BARATTE ET AL.: "Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis inducing p34cdc2/cyclin Bcdc13 protein kinase" ANTICANCER RESEARCH, vol. 12, 1992, pages 873-880, XP002069498 *
J.B.A MILLAR ET AL.: "p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast" EMBO JOURNAL, vol. 10, no. 13, 1991, pages 4301-4309, XP002069499 *
M.S. LEE ET AL: "cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2" MOLECULAR BIOLOGY OF THE CELL, vol. 3, 1992, pages 73-84, XP002069497 cited in the application *
T. ORIGUCHI ET AL.: "Dnacin A1 and Dnacin B1 are antitmor antibiotics that inhibit cdc25B phosphatase activity" BIOCHEMICAL PHARMACOLOGY, vol. 48, no. 11, 1994, pages 2139-2141, XP002038482 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021771A2 (en) * 1999-09-22 2001-03-29 Canbas Co., Ltd. Compositions and methods for inhibiting g2 cell cycle arrest and sensitizing cells to dna damaging agents
WO2001021771A3 (en) * 1999-09-22 2002-02-14 Canbas Co Ltd Compositions and methods for inhibiting g2 cell cycle arrest and sensitizing cells to dna damaging agents
JP2003518368A (en) * 1999-09-22 2003-06-10 株式会社キャンバス Compositions and methods for inhibition of G2 phase cell cycle arrest and cell sensitization to DNA damaging agents
WO2001044467A2 (en) * 1999-12-14 2001-06-21 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators
WO2001044467A3 (en) * 1999-12-14 2001-12-27 Sod Conseils Rech Applic Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators

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