EP0907743A1 - Macromolecules catalytiques presentant une activite semblable a celle de la cdc25b - Google Patents

Macromolecules catalytiques presentant une activite semblable a celle de la cdc25b

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Publication number
EP0907743A1
EP0907743A1 EP97923404A EP97923404A EP0907743A1 EP 0907743 A1 EP0907743 A1 EP 0907743A1 EP 97923404 A EP97923404 A EP 97923404A EP 97923404 A EP97923404 A EP 97923404A EP 0907743 A1 EP0907743 A1 EP 0907743A1
Authority
EP
European Patent Office
Prior art keywords
cdc25b
arg
seq
glu
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97923404A
Other languages
German (de)
English (en)
Inventor
Martin R. Deibel, Jr.
Anthony W. Yem
Cindy L. Wilson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmacia and Upjohn Co
Original Assignee
Pharmacia and Upjohn Co
Upjohn Co
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Filing date
Publication date
Application filed by Pharmacia and Upjohn Co, Upjohn Co filed Critical Pharmacia and Upjohn Co
Publication of EP0907743A1 publication Critical patent/EP0907743A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes

Definitions

  • This invention relates to the field of protein phosphatases, specifically cdc25B like enzymes.
  • 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 a 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 cdc2 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. As a cell cycle specific phosphatase, cdc25B is believed to be crucial for progression from G2 through mitosis.
  • Stable recombinant forms of cdc25B are needed that have improved activity making them suitable for use in enzyme assays with improved solubility characteristics.
  • Stable recombinant forms of the protein that are capable of easy manipulation for crystallography studies in order to better understand and characterize these types of phosphatases by structural analyses and models are also needed. This invention provides macro molecules having these and other desirable characteristics.
  • the different parts of the fusions are shown as different lines in a box, where the figure represents a construct that can be composed of either nucleic or amino acids, where, a) the boxes, lines and numbers are not drawn to scale, b) the GST region, labeled GST, is shown with a straight line down the middle ofthe box, c) the protease cleavage site, labeled P, is shown with a dotted line down the middle of the box, d) the restriction site, labeled R, is shown with a wavey line down the middle of the box, e) the GST region is relatively large, compared to the cleavage and restriction sites f) the cdc25B like region, labeled cdc25B like, is shown as a box with a heavy line down the middle of the box, where the numbers above the box indicating DNA nucleotide residues and the numbers below the box indicating peptide amino acid residues, where, with reference to the cdc25B like region,
  • substituted nucleic or amino acid residues of the cdc 25B like reg on are ose sequences sc ose as su st tut ons n t e an sequence listings or where the subsitituted nucleic or amino acid residues may be obtained by deleting, adding or replacing one to several nucleic or amino acid residues where the fusion, when it is a protein, may optionally be associated with a bacterial polypeptide.
  • the fusion shown above may have a protease cleavage site is created to be responsive to thrombin or Factor Xa, the restriction site may be Bam HI.
  • the fusions may be nucleic acid residue fusions, DNA, or they may be amino acid residue fusions, peptides or proteins.
  • One of the GST fusions is where the fusion is a peptide where the cdc25B like region is comprised of the amino acid residues shown in CHART 6 as cdc25B 302"566 or SEQ. ID. NO. 4. This is also described as a fusion protein comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO. 4.
  • Other specific peptide fusions are comprised of the following: the cdc25B like region is comprised of the amino acid residues shown in CHART 14 as Mutein 1 or SEQ. ID. NO.
  • the fusion protein comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO. 14; the fusion protein where the cdc25B like region is comprised of the amino acid residues shown in CHART 14 as Mutein 2 or SEQ. ID. NO. 15; The fusion protein comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO. 15; the fusion protein where the cdc25B like region is comprised of the amino acid residues shown in CHART 14 as Mutein 3 or SEQ. ID. NO. 16; the fusion protein comprising, GST- Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. 16; the fusion protein comprising, GST-Ile- Glu-Gly-Arg-Gly-Ile-Gln 302 ...-Gin 566 .
  • fusion proteins that are closely associated with a bacterial polypeptide, especially where said polypeptide is a chaperonin. Especially any of the fusion proteins where the chaperonin polypeptides are DnaK and/or GroEL.
  • the fusions are also nucleic acid fusions, the nucleic acid, or DNA residues of course code for the peptides that are expressed from the DNA, but the nucleic acids residues are also rightly considered fusions.
  • Some of the specific DNA or nucleic acid fusions are comprised of nucleic acid residues where the nucleic acid residues of the cdc25B like region are comprised of the nucleic acid residues shown in CHART 5 and CHART 13 as cdc25B 976"1773 or SEQ ID. NO. 4; comprised of the nucleic acid residues shown in CHART 13 as Muteinl or SEQ ID. NO. 11; comprised of the nucleic acid residues shown in CHART 13 as Mutein2 or SEQ ID.
  • ome o ese us ons may e more par cu ar y esc e as: GST-Xa-BamHI restriction site- cdc25B(976-1773)-XhoI restriction site, or as GST- Xa-GGG-ATC-cdc25B(976-1773)-XhoI restriction site, see CHART 2.
  • fragments of the complete fusions described above are also described and claimed. Specific fragments of nucleic acids from, or associated with, the construction of the fusions are disclosed, such as those disclosed in various CHARTS and particularly CHART 5 or SEQ. ID. NO. 3.
  • fusion fragments are peptides or amino acid residues. Proteins, peptides, protein and peptide fragments or as they are also called, amino acid residues covalently linked with amide bonds, are disclosed. More particularly the following peptides are important and may be useful by themselves, or as essential intermediates, the amino acid residues disclosed in CHART 6 or SEQ. ID. NO. 4; the amino acid residues disclosed in CHART 11 or SEQ. ID. NO. 9; the amino acid residues disclosed in CHART 12 or SEQ. ID. NO. 10; the amino acid residues disclosed in CHART 16 as Muteinl or SEQ. NO. 21; the amino acid residues disclosed in CHART 16 as Mutein 2 or SEQ. ID. NO.
  • the peptides and proteins, the nucleic acids or DNA residues may also be called catalytic macromolecules. In some cases these macromolecules are identified precisely as products derived from a particular process, such as, a catalytic macromolecule comprising the amino acid residues, of SEQ. ID. NO. 21, that is produced from the fusion protein that is GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO.
  • a catalytic macromolecule comprising the amino acid residues of SEQ. ID. NO. 22, that is produced from the fusion protein that is GST-Ile-Glu-Gly-Arg-Gly-Ile- SEQ. ID. NO. 15; a catalytic macromolecule comprising the amino acid residues of SEQ. ID. NO. 23, that is produced from the fusion protein that is GST-Ile-Glu-Gly- Arg-Gly-Ile-SEQ. ID. NO. 16.
  • n a on to e us ons escr e y e gure a ove ere are ot er fusions also related to cdc25B that are disclosed by this invention. Fusions are disclosed here that may be selected from any of the fusions shown below,
  • the different parts of the fusions are shown as different lines in the box, where a) the GST region, is labelled GST, with a straight line in the box, b) the protease cleavage site, is shown as a dotted line in the box, labelled "P,” c) the restriction site is shown as a wavey line in the box, labelled "R,” and d) the VHR like region is shown as a heavy line in the box, labelled "VHR" where the numbers above the box indicating DNA nucleotide residues and the numbers below the box indicating amino acid residues, where the figure, shown above, represents either nucleic acids or amino acids, where the boxes, lines and numbers are not drawn to scale, where the GST is relatively large, the cleavage and restriction sites relatively small and the VHR region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length VHR region of cdc25B.
  • the VHR like region of cdc25B may be more particularly described
  • Figure IA shows the restriction sites, oligonucleotide primers and portions of cdc25B sequence involved in the PCR reaction and plasmid formation of plasmid pGEX-5X-3.
  • Figure IA is an expanded portion of the plasmid shown in Figure IB.
  • Figure IB shows the plasmid construction of plasmid pGEX-5X- 3.
  • Figure I conta ns t e expan e port on o e segmen s own n gure .
  • Figure 2 shows a western blot in two sections.
  • Columns one (1) show GST-cdc25B(31-566) and columns two (2) show the special domain cdc25B (356-556).
  • Figure 3 is a gel filtration size exclusion chromatograph of the purified monomeric special minimal domain cdc25B(356-556).
  • Figure 4 is an agarose gel electrophoresis showing the product of the PCR reaction (0.8Kb).
  • Figure 5 is an agarose gel electrophoresis of plasmid mini preps obtained from transformed JM109 E coli.
  • compound(s) or “macromolecule(s)” means any molecular structure including complex poly residue entities such as covalently linked amino acids like proteins and peptides and covalently linked nucleic acid residues such as a gene or gene fragment or any fusions of nucleic acid residues or the related peptidic like compounds that would result from the expression of nucleic acids.
  • “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 HCI, 0.5 mM EDTA, 300 mM NaCl, 0.2% NP-40, pH 8.0)
  • TEN buffer 50 mM Tris HCI, 0.5 mM EDTA, 300 mM NaCl, 0.2% NP-40, pH 8.0
  • BCIP is 5-bromo-4-chloro-3-indolyl phosphate.
  • JM109 E. coli cells are a strain of cells available from Promega® as competent E. coli cells..
  • IPTG is isopropyl- ⁇ -D-thiogalactopyranoside from Boehringer Mannheim, Indianapolis, Indiana. Many of the kits used in this invention such as “the GeneClean® kit” and "a
  • RPM plasmid isolation kit RPM-rapid pure minipreps
  • MORPH is a site-specific plasmid DNA mutagenesis kit obtained from 5 PRIME->3 PRIME, Inc.®, Boulder, Colorado.
  • LB media is a solution containing tryptone, yeast extract, sodium chloride and water. It is commerically available from Gibco-BRL.®, Gaithersburg, Maryland. " s n tro ue tetrazo um.
  • PAGE is 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-nitrophenyl phosphate (PNPP)."
  • PVDF polyvinylidene difluoride
  • SDS sodium dodecyl sulfate
  • TA cloning kit containing the pCRII plasmid, and INV ⁇ F' 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 ° , 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 cdc25B in addition to having other uses not possible with previously disclosed human cdc25B because of its physical characteristics.
  • novel recombinant fusion constructs that produce macromolecules that are soluble and that perform some similar biochemical functions as full length cdc25B constructs such as phosphatase activity, but these constructs generally have more activity, are more soluble, do not require refolding and in some cases may be crystallized.
  • the crystallizible 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 compounds or macro molecules, usually peptides and nucleic acid sequences, described herein would make superior drug screening tools over previously disclosed cdc25B proteins because of their characteristics including enhanced activity for some of the constructs.
  • the compounds described herein would be superior over known macromolecules, such as other previously described proteins and peptides, for studies of cdc25B enzyme kinetics and mechanistic studies because these novel compounds are monomeric in structure and because these uniquely designed sequences do not display anomalies present in inhibitor kinetics seen with known GST fusion proteins of cdc 25B.
  • the compounds that are pro e ns an pep es, or e va ves ereo , escr e ere n can e crea e 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 cdc25B 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-cdc25B full length enzyme 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. Previously described cdc25B protein is full length protein, usually created as a GST fusion with cdc25B.
  • cdc25B 5 or cdc25B (1-566).
  • a macromolecule of only 10 amino acids might be described as “cdc25B 556"566 " which would describe a macromolecule of 10 amino acids identical to the last 10 amino acids in Chart 4, i.e. "Arg-Glu-Leu-Cys-Ser-Arg-Leu-Gln-Asp-Gln.”
  • the full length protein by itself, without anything attached to the first or last amino acids, is not easily manipulated in the laboratory.
  • the protein is usually attached or “fused” to a "tag” or “fusion partner” creating a “fusion,” or “fusion cons ruc or cons ruc .
  • linking molecules between the fusion partner and the protein.
  • an intervening factor Xa cleavage site will be produced, and this may be introduced between the GST and the cdc25B.
  • the linking molecules or amino acids should be apparent from the text, even though they are not delineated, or the precise construct may be identified, for example as, GST-Xa site- Gly-Ile-cdc25B (302-566).
  • GST-cdc25B When various tag(s) or fusion partners are attached to the protein, the whole complex may simply be referred to as "GST-cdc25B." If this complex (including fusion tag) were to be comprised of the full length protein it may be called, GST- cdc25B 1 . Compounds differing from the full length cdc25B might be described with numbers indicating a different sequence than the full length, but the numbers will always correspond to the full length sequence in Charts 3 and 4. For example, cdc25B would describe a macromolecule of 10 amino acids identical to the last
  • the fusion protein (GST-Xa site-Gly-Ile-cdc25B 302"566 ), which is defined as a GST polypeptide fused to a truncated form of human cdc25B, containing residues 302-566, is disclosed, as well as various forms of the truncated cdc25B protein itself.
  • These fusion proteins and peptide fragments are soluble using native extraction/buffer systems (see definitions).
  • the products of these extractions, using native extraction/buffer systems are enzymatically active in a defined way (see definitions) with the colorimetric substrate, p-nitrophenyl phosphate (PNPP).
  • the special domains can be released from their GST fusion partners by digestion o an engineere actor a c eavage s te etween t e an c c sequences, respectively. These special constructs, or domains, once they are released from their GST fusion partners, are especially suitable for use in enzyme assays, crystallography, and other examples requiring a stable enzyme. All of the factor Xa released minimal domains lacking the GST moiety can be concentrated to greater than 10 milligrams per ml without precipitation, an advantage for crystallography studies.
  • the Km is significantly lower (i.e., improved binding constant for substrate) than published values for other cdc25B constructs, and at least 6-7 fold lower than for GST-cdc25B 31-566 .
  • the lower 1 ⁇ reflects better binding properties to the substrate than any enzymes currently known.
  • the V ma ⁇ we calculate for the minimal domain, cdc25B (356-556), is greater than published values, showing that the cdc25B (356-556) enzyme is a more active enzyme than that previously disclosed.
  • the K ⁇ is 2-3-fold lower than for the parent GST-Xa-Gly-Ile- cdc25B (302-566) protein.
  • This invention also comprises a method for the preparation of active, homogeneous peptide special domains of cdc25B, including cdc25B (356-556) and mutated forms of cdc25B(356-566).
  • n a y p mers con a n ng am HI (5* sense) and Xho I (3' antisense) restriction sites were prepared.
  • Unpurified products of the PCR reaction are ligated into a TA cloning vector (pCRII; InVitroGen®) according to standard procedures.
  • the ligated TA vector is used to transform INV ⁇ F' cells. See, Charts 1 and 2, and Figure IA and Figure IB, for an overview of the procedures described herein and a general description of the essential intermediates and products produced.
  • Chart 1 shows GST fusion proteins compares two other constructs with the construct disclosed herein.
  • Chart 1 shows: Item 1, a GST fusion protein of cdc25B (1-566), (disclosed by Beach and Gal forcingov); Item 2, a GST fusion protein of cdc25B(355-566), (disclosed by Horiguchi) and Item 3, the GST fusion protein of cdc25B(302-566), (disclosed herein) otherwise called, GST-Xa-Gly-Ile-cdc25B (302-566).
  • Chart 2 shows the plasmids, restrictions sites, oligonucleotide primers and portions of cdc25B sequence involved in the PCR reaction and plasmid formation.
  • Figure IA and Figure IB show the plasmid construction including various sites and primers.
  • Figure IA is an expanded portion of the plasmid shown in Figure IB.
  • the E. coli can be grown in minipreps of 5 ml in LB media + ampicillin® overnight at 37 C on a shaker at a minimum of 200 RPM. 1.5 ml of the suspension of cells may be centrifuged and subjected to the reagents from the RPM kit marketed by Bio 101® designed to purify small amounts of plasmid DNA for gel analysis. Use as suggested by manufacturer. Plasmid DNA samples are digested with BamHI and Xhol for 1 hour at 37° C, and then analyzed by 0.8% agarose gel electrophoresis (IX TAE buffer). The gel is first soaked in 0.5 ug/ml ethidium bromide in IX TAE buffer. Then the appropriate insert (0.8 kb) is excised from the agarose gel under long wavelength UN light and the D ⁇ A is isolated using the commercially available GeneClean kit (Bio 101®).
  • the purified D ⁇ A is then ligated into pGEX-5X-3 which has been linearized with BamHI and Xhol, and the products of the ligation are used for transformation of competent E. coli cells, such as JM109.
  • the transformed E. coli are plated on 1% agar plates (LB media) containing ampicillin®, and grown overnight at 37 C Positive colonies are selected and grown in 5 ml cultures of LB media + ampicillin®. After an additional 12-15 hours at 37° C, aliquots of the cultures are collected by centrifugation and subjected to an RPM plasmid isolation kit from Bio 101®.
  • the plasmid D ⁇ A samples are digested with BamHI and Xhol and analyzed by 0.8% agarose gel electrophoresis in IX TAE buffer.
  • E. coli cultures used to prepare t e p asm samp es, w c are c arac er ze y av ng e . insert piece, are either frozen at -80°C in 10% glycerol or are grown up in LB broth at 37°C prior to induction with IPTG.
  • the construct to be expressed in E. coli is designed so that a fusion protein of GST with cdc25B (Gin 302 to Gin 566), with an intervening factor Xa cleavage site, i.e.
  • the IEGR sequence will be produced.
  • the resulting construct contains IEGR, in addition to two amino acids, -Gly-Ile-, between GST and Gin 302 .
  • the -Gly-Ile- comes from the coding region contributed by part of the restriction site. See CHART 2.
  • GST fusions This CHART, shows and compares three different fusions. The different parts of the fusions are shown as different lines in the box; a) the GST portion, labelled GST, with a straight line in the box, b) the protease cleavage site, when there is one, is shown as a dotted line in the box, labelled "P,” c) the restriction site is shown as a wavey line in the box, labelled "R,” and d) the cdc25B like portion is shown as a heavy line in the box, labelled "cdc25B" with the number above the box indicating DNA nucleotides and the numbers below the box indicating amino acids.
  • the boxes are intended to suggest either DNA or protein.
  • the boxes are not drawn to scale, the GST is relatively large, the cleavage and restriction sites relatively small and the cdc25B region has about the number of sequences indicated relative to the full length cdc25B.
  • Construct number one represents the fusion produced by David H. Beach and Konstantin Gal Toov, U.S. Patent 5,441,880 and Cell (1991) 67; 1181-1194.
  • Construct number two represents the fusion produced by Takashi Horiguchi, et al., Biochemical Pharmacology, vol. 48 pp. 2139-2141, (1994).
  • Construct number three represents the fusion disclosed by this invention.
  • the lines and arrows below construct number three indicates the portion of the cdc25B that becomes the active macromolecule.
  • the first of these immediately preceding nucleotide residue 1138 and protein residue 356 was made to improve the factor Xa processing of the fusion protein and the second of these, surrounding nucleotide residues 1740 and amino acid residues 556, was made to prevent undesired factor Xa cleavage of the protein at this position.
  • CHART 2 This CHART shows the PCR cloning strategy and subsequent insertion of the 0.8 Kb DNA fragment into the plasmid pGEX-5X-3.
  • Single underlined sequence indicates PCR primers, the double arrow t and double underline indicates restriction enzymes sites, and the italicized segments show the sequence from the plasmid, in one described embodiment, the pGEX-5X-3 plasmid.
  • the Sequence ID. Numbers for the sequences below are provided in CHART 18. The sequences shown in this CHART show segments of longer sequences. This CHART is intended to show strategy, and details of insertion techniques, not full sequences.
  • Sequence Identification Numbers SEQ. ID. NO.s, for the fragments of the sequences shown, from top and left to right are as follows: SEQ. ID. NO. 30, 32, 33, 34 and 35 (See also CHART 18).
  • Sequence Identification Numbers SEQ. ID. NO.s, for the fragments of the sequences shown, from top and left to right are as follows: SEQ. ID. NO. 37, 38, 39, and 40 (See also CHART 18).
  • Full length human cDNA which codes for the cdc25B sequence. 2940 nucleotides in single stranded DNA are shown.
  • Sequence disclosed by Beach and Gal forcingov. This is Sequence I.D. no. 1.
  • the coding region of the sequence is underlined, below (73-1773).
  • the sequence below is numbered according to the Beach and Gal forcingov system. This sequence was also disclosed by Nagata, who used a different numbering system.
  • the coding sequences would be numbered 241-1941, See, Nagata A., Igarashi M., Jinno S., Suto K., and Okayama H. New Biol . vol. 3(10), pp. 959-68 (1991) . GENBANK/S78187.
  • the DNA sequence of the Cdc25B 976"1773 segment, from CHART 5 is provided, and then as modified according to the section, Advanced Forms of the Macromolecule.
  • the CHART 5, sequence is provided, then the three forms, Muteinl, Mutein2, and Mutein3 are shown with the appropriate substitutions shown below the original sequence. All other sequences remain the same as in the original sequence, except where shown as changed below, i.e. dots (.) below indicate no changes.
  • the Sequence ID No. for the original sequence is SEQ. ID. No. 3.
  • the Muteinl SEQ. ID. No. is No. 11.
  • the Mutein2 SEQ. ID. No. is No. 12.
  • the Mutein3 SEQ. ID. No. is No. 13.
  • Mutein3 1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
  • This CHART contains sequences disclosed under the section of this invention entitled, "Advanced Forms of the Macromolecule.” There are several different constructs disclosed in this section of the document, both amino acid and nucleotides are disclosed. Three CHARTS are devoted to this section. This CHART is from the “DESIGN” section of Advanced Forms of the Macromolecule. It is followed by two CHARTS from the “RESULTS” portion of the same section (nucleotide-CHART 15 and peptide-CHART 16). This CHART only includes amino acid residues, it does not include restriction sites, protease cleavage sites or the GST fusion portion of the fusion molecules. The first sequence described is described previously, in CHART 6.
  • CHART 15 provides the corresponding nucleotide sequences, beginning with the sequence first disclosed in CHART 5 (nucleotide, Seq. ID. No. 3).
  • the peptide sequence of the Cdc25B segment, from CHART 6 is provided, and then as modified according to the section, Advanced Forms of the Macromolecule.
  • the CHART 6, sequence is provided, then the three forms: Muteinl, Mutein2, and Mutein3 are show with the appropriate substitutions shown below the original sequence. All other sequences remain the same as in the original sequence, except where shown as changed below, i.e. dots (.) below indicate no changes.
  • the Sequence ID No. for the original sequence is SEQ. ID. No. 4.
  • the Muteinl SEQ. ID. No. is No. 14.
  • the Mutein2 SEQ. ID. No. is No. 15.
  • the Mutein3 SEQ. ID. No. is No. 16.
  • This CHART presents the 4 nucleotide sequences from the RESULT section of Advanced Forms of the Macromolecule. This CHART only includes nucleic acid residues, it does not include restriction sites, protease cleavage sites or the GST fusion portion of the fusion molecules.
  • the Sequence ID No. for the wild type sequence of Cdc25B 976 - 1773 segment is SEQ. ID. No. 17.
  • the Muteinl SEQ. ID. No. is No. 18.
  • the Mutein2 SEQ. ID. No. is No. 19.
  • the Mutein3 SEQ. ID. No. is No. 20.
  • Mutein3 1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
  • This CHART presents the 4 peptide sequences from the RESULTS section of the invention, Advanced Forms of the Macromolecule.
  • This CHART only includes amino acid residues, it does not include restriction sites, protease cleavage sites or the GST fusion portion of the fusion molecules.
  • This CHART provides the amino acid residues that correspond to the nucleic acid residues provided in CHART 15.
  • First the peptide sequence of the Cdc25B 356"566 segment sequence is provided, and then as modified according to the section, Advanced Forms of the Macromolecule.
  • the wild type sequence is provided, then three forms: Muteinl, Mutein2, and Mutein3, are provided with the appropriate substitutions shown below the original sequence.
  • the Sequence ID No. for the original sequence is SEQ. ID. No. 9.
  • the Muteinl SEQ. ID. No. is No. 21.
  • the Mutein2 SEQ. ID. No. is No. 22.
  • the Mutein3 SEQ. ID. No. is No. 23. 356 VLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
  • Part B is the amino acid sequence of human VHR phosphatase, as disclosed in, Ishibashi T., Bottaro D.P., Michieli P., Kelley C.A., Aaronson S.A., " A novel dual specificity phosphatase induced by serum stimulation and heat shock," J. Biol. Chem., vol. 269(47), pp. 29897-902 (1994).
  • the Part B sequence is SEQ. ID NO. 25
  • This CHART contains other miscellaneous sequences disclosed with this invention.
  • Geno-I is -GCG GAT CCA GCG GCT CTT CCG CTC TC (5' ⁇ 3') (Sequence LD. no. 30.) and Geno-II is - GCC TCG AGT CAC TGG TCC TGC AGC CG (5' ⁇ 3') (Sequence LD. no. 31.)
  • CHART II The following sequences were disclosed from CHART II: The top part of CHART II started with PCR Reaction and showed:
  • Electrophoresis One-dimensional analytical SDS polyacrylamide gel electrophoresis was conducted using 10% gels in a mini Protean II system (Bio-Rad Laboratories) according to the method of Laemmli. See, Laemmli, Nature (1970) 227: 680-685, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Samples were diluted with 1 volume of denaturation buffer (2% SDS, 25% glycerol, 0.25 M Tris HCI, pH 6.8, and 1% beta-mercaptoethanol), and heated for at least 2 minutes in a boiling water bath.
  • denaturation buffer 2% SDS, 25% glycerol, 0.25 M Tris HCI, pH 6.8, and 1% beta-mercaptoethanol
  • Electrophoresis was conducted at constant power (5 watts/gel) for 1 hour at room temperature and terminated when the dye front (bromphenol blue) reached the bottom of the gel.
  • the completed gels were fixed in 50% ethanol and 10% acetic acid, and stained with Coomassie Brilliant Blue G-250. Alternatively, proteins in the gels were electroblotted onto PVDF.
  • the processed blots were incubated in TBS with 1% BSA and 1:2000 dilution of anti- rabbit FC alkaline phosphatase conjugate (Promega) for 1 hr. After washing as described above, color was developed using BCIP/NBT as substrates for alkaline phosphatase. This reaction was stopped by rinsing in deionized water, blots were air dried, and stored.
  • Enzvme assay and Kinetic Analyses - 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)). 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.
  • 25 ul of enzyme is added with mixing, and a continuous recording of absorbances at 405 nm is completed over a short time period.
  • K. and V ma ⁇ multiple pNPP concentrations are used at a constant enzyme concentration. Rates at each concentration of substrate are determined and the K_ and V ma ⁇ calculated from line fitting to a Michaelis Menten equation.
  • the method generally used to screen for all produced cdc25B proteins was Western blotting using a commercially available anti- murine cdc25B polyclonal antibody, which recognizes C-terminal residues 547-566 of human cdc25B.
  • DnaK and GroEL bacterial chaperonins having polypeptide sizes of about 70 and about 60 kD respectively, are associated with the cdc25B protein and/or to the peptide tether between the GST and cdc25B polypeptides. This observation is made during the factor Xa cleavage step since these proteins are found together with the truncated cdc25B protein in the eluate.
  • GST remains bound to the glutathione column matrix during factor Xa cleavage as determined by Western blotting of eluates using a commercially available rabbit anti-GST polyclonal antibody.
  • Form I is a fusion protein whose sequence is: GST-Ile-Glu-Gly-Arg-Gly-Ile-Gln 302 .. .-Gin 566 . See, CHART 10, SEQ. ID NO. 8
  • Form II is the final product of cdc25B after factor Xa cleavage, Val 356 -...-Arg 556 .
  • CHART 11 SEQ. ID. NO. 9
  • Reversible inhibitors of single-site monomeric enzymes generally exert their effects over an approximately 100-fold concentration range. Thus, if a given inhibitor concentration results in a 10% inhibition of an enzymatic reaction, then increasing the inhibitor concentration by two orders of magnitude will result in a 90% inhibition of the reaction. This is true for competitive, noncompetitive, and uncompetitive inhibitors. See, Cheng, Y.-C. and Prusoff, W.H. (1973) Relationship between the inhibitor constant (K j ) and the concentration of inhibitor which causes 50 per cent inhibition (I 5 ⁇ ) of an enzymatic reaction. Biochemical Pharmacology Vol. 22, pp. 3099-3108.
  • GST-cdc25B (31-566) can be partially purified through the use of a GST-affinity column.
  • the resultant product is usually less than 50% pure by the criteria of SDS PAGE. See Figure 2.
  • Figure 2 shows a western blot in two sections. The section on the left (A), represents a Coomassie Blue stained PVDF-P blot and the section on the right (B) represents a rabbit anti-cdc25B probing ofthe same blot. Columns one (1) show GST-cdc25B(31- 566) and columns two (2) show the special domain cdc25B (356-556).
  • Figure 3 is a size exclusion chromatograph that shows the special domain acts as a monomer.
  • the Y axis is absorbance at 220nm.
  • the X axis is retention time in minutes. Relative size markers are included.
  • V ma ⁇ was not measured due to impurity of GST-cdc25B fusion proteins.
  • the cdc25B (356-556) enzyme exhibited higher activity per unit weight of protein than any other reported cdc25 phosphatase.
  • Dunphey and Kumagai, Cell, (1991) 67, pp. 189-196 report a K ⁇ and V ma ⁇ of 50 mM and 56 nmoles/min/mg for p35cdc25 at 37°C using PNPP as substrate.
  • This latter protein is the engineered recombinant C-terminal domain of the Drosophila cdc25 protein.
  • Horiguchi et al., Biochem Pharmacol, (1994) vol. 48, pp. 2139-2141 report a K JJJ of 16.6 mM for GST-cdc25B (residues 355-566) using PNPP as substrate at
  • V ma ⁇ for cdc25B(356-556) is equivalent to 500 +/- 100 nmoles/min/mg.
  • a group of constructs was designed based on the mutagenesis of cdc25B sequence initiating with Gln302 and terminating with Gln566. The rationale for these constructs was to engineer in an improvement in stability and ease of isolation of the expressed protein. For this group, three principle constructs of cdc25B were created which when expressed in E. coli would be expected to give the sequence changes shown below, both design and actual results are shown.
  • Muteinl was constructed to introduce a new factor Xa site immediately preceding Val356, to reduce N-terminal microheterogeneity in the region from residue 302 through 355.
  • pGEX-5X-3/cdc25B(302-566) as a template in the MORPH® mutagenesis system from 5 Prime 3 Prime, we used a single mutagenic oligonucleotide primer (5* GCG GAG GAC GCG GCC TTC AAT TTC CTC AGC CTC 3' SEQ. ID. NO. 41) to introduce the mutation.
  • the potential muteins were transformed into the repair- deficient E. coli BMH 71-18mutS.
  • the mutation introduced a new Tsp509I restriction site which permitted us to screen for the desired mutants using restriction mapping.
  • Circular plasmid DNA from a selected clone was then transformed into Promega JM109 cells after which DNA sequence analysis confirmed the desired nucleotide changes.
  • Mutein2 was constructed to substitute two residues in the C-terminus of the protein to decrease the incidence of factor Xa cleavage of these residues during protein workup. Thus, we desired to substitute two Lys groups for the two Arg groups at 556 and 557.
  • a single mutagenic oligonucleotide primer (5' GGG GAG CGG AGC AAG AAG GAG CTC TGT AGC 3' SEQ. ID. NO. 42). In this case, successful mutagenesis was identified by the elimination of a Mwol restriction site. After transformation in JM109 cells, DNA sequence was confirmed to be identical to that predicted.
  • Mutein3 a separate method was utilized.
  • the product was treated with Pfu DNA polymerase to extend and incorporate the mutagenic primers, resulting in nicked circular strands. This product was then used o rans orm . co - ue supercompe en ce s, w c repa r t e n c s n t e mutated plasmid. DNA from the appropriate colonies was identified by restriction analysis with Tsp509I and then used to transform JM109. After preliminary restriction analysis with Tsp509I, isolated DNA was analyzed to confirm predicted nucleotide sequence.
  • coli cell paste were 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 lOminutes. 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-cdc25B) was incubated with equilibration buffer containing factor Xa. After a period of time, the released protein is collected and concentrated by Amicon ultrafiltration.
  • 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-cdc25B antibody.
  • the purified proteins were analyzed by N-terminal sequencing and by mass spectrometry. The resulting proteins derived from the new constructs were shown to have the sequence shown above.
  • the cDNA encoding the entire sequence of cdc25B was obtained from Nagata, (see, Nagata A., Igarashi M., Jinno S., Suto K, and Okayama H. "An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells.” New Biol. vol. 3(10), pp. 959-68 (1991). GENBANK/S78187), then the full length (residues 1-2940) cdc25B DNA in a pCD2 vector was linearized with Hind III to make the cDNA suitable as a template for the po ymerase c a n reac on . e c was pu e y ge e ec rop ores s. The identified linearized cDNA was subsequently purified using a Geneclean kit® commercially available from Bio 101®.
  • Genosys® Geno-I - GCG GAT CCA GCG GCT CTT CCG CTC TC - (5' ⁇ 3') SEQ. ID. NO. 30 Geno-II - GCC TCG AGT CAC TGG TCC TGC AGC CG (5' ⁇ 3') SEQ. ID. NO.
  • the following reagents/system are used in the PCR reaction to generate the desired invention: 10 pmol Geno-I, 10 pmol Geno-II, 6 ng cdc25B template in pCD2, 200 ⁇ M dNTPs, IX PCR Buffer (Perkin-Elmer® GeneAmp®), 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCI, pH 8.3, 0.001% (w/v) gelatin, Sterile water to total volume 50 ⁇ l, 2.5 units Amplitaq DNA Polymerase.
  • PCR cycling conditions are as follows: 8 minutes at 95°, followed by 25 cycles of the following, in order, (1 minute at 95°, 90 seconds at 50°, 2 minutes at 72°% after 25 cycles followed by 5 minutes at 72° followed by a change to 4°C and hold.
  • the PCR reaction yields a 0.8 Kilobase product, see Figure 4, which is ligated immediately (without purification) into the TA Cloning vector pCRII (Invitrogen TA Cloning Kit) according to the manufacturer's directions.
  • Figure 4 is an agarose gel electrophoresis showing the product of the PCR reaction (0.8Kb).
  • column A shows the 100 base pair ladder
  • column B shows the cdc25B(976-1773) PCR product
  • column C shows the PCR control.
  • the unlabeled arrow on the right side of the figure points to the PCR product.
  • the product of the ligation is transformed into INV ⁇ F cells. After overnight incubation of the cells at 37 C, the resulting DNA of selected colonies is isolated using a BiolOl® DNA isolation kit, digested with Bam HI and Xho I, and purified by agarose gel electrophoresis.
  • Ligation of 0.8 Kb Bam Hl/Xho I TA cloning product into PGEX-5X-3- PGEX-5X-3 is prepared for ligation by sequential digestion with the restriction enzyme Xho I at 37°C in 50 mM Tris-HCL (pH 8.0), 10 mM MgCl 2 and 50 mM NaCl, followed by digestion with Bam HI at 37°C in 50 mM Tris-HCI (pH 8.0), 10 mM MgCl 2 and 100 mM NaCl.
  • the resultant double-digested plasmid is subjected to electrophoresis on a 0.8% agarose gel in IX TAE (Tris- acetate-EDTA buffer). After electrophoresis, the gel is soaked in IX TAE with 0.5 ⁇ g/ml ethidium bromide (EtBr) so that the DNA could be visualized under long-wave UV.
  • T e 0.8 pro uct is excise rom t e ge an is puri ed using Geneclean III ® from Bio 101® as recommended by the manufacturer. The following ligation conditions are utilized:
  • Lanes 9-11 contain the desired insert
  • lane A shows the 100 bp ladder
  • lane s ows t e p - - nea ze anes an s ow p - - w no nser s, i.e. controls
  • the arrow on the right side of the figure labeled "a” shows the linearized plasmid and the other arrow labeled "b” shows the insert.
  • Expression of the human recombinant cdc25B protein (GST-Xa site-Glv-Ile-cdc25B f302-566) in E. coli).
  • Escherichia coli strain JM109 containing the expression vector pGEX-5x-3 (a vector which also contains the coding region for the factor Xa site) with the cdc25B insert (976-1773), was grown on Luria broth and/or M9 medium containing 0.5% yeast extract (M9YE) as seed and production media.
  • E. coli stored at -80°C in 20% glycerol was used as the primary inoculum for the seed stage fermentation which was carried out at 37°C in 100 ml volumes contained in 500 ml wide mouth fermentation flasks shaken at 200 rpm for 12 hr.
  • the seed media (LB or M9YE) contained ampicillin® at 100 mg/L.
  • the mature seed fermentations were used to inoculate production fermentations at a 2 % rate; in all cases the same medium containing ampicillin® at 100 mg/L was used for the seed and production fermentations.
  • the production fermentations were carried out identically to the seed fermentations for ca. 2.5 hr when the turbidity at 660 nm reached 1.0 unit (+/- 0.2). At this time, IPTG was added to a final concentration of 0.4 mM.
  • the induced fermentations were carried out for another 3.5 hr when the turbidity reached about 3 units. The completed fermentation was harvested by centrifugation.
  • E. coli Expressed Catalytic cdc25B macromolecules The strategy of affinity purification of GST-Xa site-GI-cdc25B (302-566) and subsequent processing and purification of truncated cdc25B:
  • the recombinant fusion protein, produced in E. coli is designed to have a glutathione S-transferase (GST) tag linked to the cdc25B protein encompassing amino acid residues 302-566 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 cdc25B sequence.
  • the factor Xa cleavage site allows for cleavage of the GST tag from the truncated cdc25B protein using factor Xa protease.
  • This solution is mixed with 20 ml of glutathione-Sepharose (Pharmacia Biotech®) which has been pre-equilibrated with factor Xa digestion buffer (50 mM Tris HCI, 100 mM NaCl, 1 mM CaC12, pH 8.0).
  • factor Xa digestion buffer 50 mM Tris HCI, 100 mM NaCl, 1 mM CaC12, pH 8.0.
  • the fusion protein is purified away from E. coli proteins by affinity chromatography on the glutathione Sepharose affinity column.
  • the resin which contains bound GST-GI-cdc25B (302-566) is incubated with equilibration buffer containing factor Xa (50 mM Tris HCI, 100 mM NaCl, 1 mM CaCl 2 , pH 8.0) in a manner analogous to that described by Abeliovich and Schlomai (Anal. Biochem. 228: 351- 354 (1995) ("Reversible oxidative aggregation obstructs specific proteolytic cleavage of glutathione S-transferase fusion proteins") for the factor Xa cleavage of glutathione Sepharose bound GST-UMSBP (universal njinicircle sequence binding protein).
  • factor Xa 50 mM Tris HCI, 100 mM NaCl, 1 mM CaCl 2 , pH 8.0
  • the suspension is centrifuged and an aliquot of the supernatant is removed. This aliquot is assayed using the PNPP method described above. Increasing amounts of cdc25B phosphatase activity versus PNPP are observed until a plateau in activity is found. Similarly, aliquots may also be subjected to SDS polyacrylamide gel electrophoresis to determine levels and purity of the cdc25B product.
  • the method by which a bound GST fusion protein containing the desired protein partner is cleaved by factor Xa while still bound to the glutathione resin is also detailed in the Pharmacia Biotech® protocol booklet for pGEX vector expression ("GST Gene Fusion System, 2nd Edition, Revision 2, Pharmacia Biotech® , p. 17-18, 1996).
  • the eluate from this step is pooled and concentrated by ultrafiltration (YM10 filter, Amicon), and exchanged into a buffer system used for equilibration of a Q fast flow anion exchange column (25 mM Tris HCI, 10 mM DTT, pH 8.0).
  • the equilibrated pool concentrate is loaded onto a Q fast flow column (10 ml bed volume, 1.6 cm diameter) and the non-bound fraction is removed.
  • the truncated cdc25B (356-556) protein product is resolved from contaminants using a linear gradient of NaCl (100-190 mM) over a 36 minute period. Appropriate fractions are collected and concentrated by ultrafiltration (Amicon) to a minimum of 2 mg/ml. Finally, glycerol is added to provide a 50% solution, with storage at -20 C. APMSF and/or pefabloc serine protease inhibitors are also added prior to storage if residual factor Xa activity is measured.
  • VHR-LIKE CONSTRUCTS The cloning, expression, and purification of an active and soluble truncated form of cdc25B based on the alignment and modelling of cdc25 with a family of dual specificity phosphatases.
  • CDC25 phosphatases are responsible for the dephosphorylation and activation of cyclin-dependent protein kinases. The latter events result in cell cycle progression. Blockade of cell cycle progression could be pursued as a strategy for design of novel anti-cancer drug templates.
  • This portion of this invention is based upon the design of cdc25 proteins that are aligned with a family of dual specificity phosphatases. Here we model a cdc25 protein sequence against a known and published 3D structure of a similar phosphatase called VHR (vaccinia Hl- related phosphatase). The latter phosphatase was recently crystallized (J.
  • 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, MA. Saper, and J.E. Dixon.
  • the cdc25B minimal domain was rea e roug e nown crys a ograp c s ruc ure o o a n a reasona e model for cdc25B minimal domain.
  • Glutathione Sepharose 4B and Q fast flow ion exchange matrix were obtained from Pharmacia Biotech, Factor Xa was purchased from Boehringer Mannheim.
  • the cDNA encoding the entire sequence of human cdc25B in pCD2 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. This is followed by purification using a Geneclean® kit (Bio 101®).
  • Genosys® Geno- III (GCG GAT CCA GCA CGA TGA GAT CGA GAA) SEQ. ID. NO. 44, and Geno-TV (GCC TCG AGT CAC CGG TAG TCC TGG GGT) SEQ. ID. NO. 45, (the italicized letters indicate the positions of engineered restriction sites).
  • a reaction (final volume of 100 ul) containing 20 pmol Geno-III, 20 pmol Geno-IV, 12 ng cdc25B template, 200 ⁇ M dNTPs, IX PCR Buffer [(Perkin-Elmer GeneAmp®), 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCI 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 KCl, 10 mM Tris-HCI pH 8.3, 0.001% (w/v) gelatin]
  • Isolated colonies were se ec e on a as s o amp c n res s ance, an a er overn g ncu a on o 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 GeneCleah Spin Kit (Bio 101)®.
  • 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 MgC-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). After electrophoresis, the gel was soaked in IX TAE with 0.5 ⁇ g/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 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-HCI (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 ⁇ l.
  • E. coli Strain JM109 5 ⁇ l of the ligation reaction was added to 100 ⁇ l 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 ⁇ l 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 ⁇ l aliquots onto (1%) agar plates containing LB medium and ampicillin (100 ⁇ g/ml).
  • E. coli was inoculated into 100 ml vols. of M9 medium containing thiamin, at 5 ⁇ g per ml, contained in 500 ml large mouth fermentation flasks. The medium contained 100 mg of ampicillin/L. The inoculated flasks were incubated for 20 hr at 37C while shaking at 200 rpm. M9 was prepared as described below.
  • Production fermentation Production flasks (as above) containing M9 with filter sterilized thiamin (as above) were inoculated with the mature seed fermentation at a 3% rate. This fermentation was continued for 3.25 hr at 37C while shaking at 200 rpm until the turbidity at 660 nm reached about 0.6. At this time, filter sterilized IPTG was added to a final concentration of 0.2 mM, and the temperature was shifted to 30C
  • M9 medium contains dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, and ammonium chloride 1 g, per L of deionized water.
  • M9 contains dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, and ammonium chloride 1 g, per L of deionized water.
  • 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 HCI, 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 HCI, 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.
  • blots were first blocked in 2% nonfat dried milk in lx phosphate buffered saline for 5 min, washed in tris (20 mM) buffered saline (TBS) and exposed to anti-cdc25B primary antibody (1:500 dilution in 1% BSA in TBS supplemented with NaN 3 as a preservative) and incubated for 1 hr.
  • the blots are washed in 0.5% Tween-20 in TBS for 10 min followed by 5 min in TBS and then exposed to secondary antibody (anti-rabbit(F c ) Alkaline Phosphatase conjugate, Promega®) at 1:1000 dilution.
  • secondary antibody anti-rabbit(F c ) Alkaline Phosphatase conjugate, Promega®
  • the location of cdc25B on blots was identified using an alkaline phosphatase NBT/BCIP substrate system (BioRad®). Following development, blots were then air dried prior to storage.
  • Assays of PNPP hydrolase activity associated with cdc25B are conducted using the reagents described earlier. 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 na vo ume o u , nc u ng e a on o res y prepare o re to .
  • the purified cdc25B protein was immunodetected by an antibody as demonstrated by unpublished western blots.
  • the purified protein exhibits a single peak when analyzed by C4 reverse phase HPLC.
  • the amino terminus of the purified cdc25B protein was determined to be Gly-Ile-Gln-His364....
  • a mass ion representing the major product of the cdc25B protein preparation has been identified at about 19,772 daltons. This mass corresponds directly to the sequence of GIQ- cdc25B (364-529).
  • VHR-Like-CHART A The amino acid sequence of human cdc25B showing the VHR like region, underlined, from residue 364 to 529. SEQ. ID. NO. 24.
  • VHR-Like-CHART B The amino acid sequence of human VHR phosphatase.
  • CAGACCATGC ACGACCTCGC CGGGCTCGGC AGCCGCAGCC GCCTGACGCA CCTATCCCTG 300 CTCTGCATGG ATTCCCCCAG CCCTATGGAC CCCCACATGG CGGAGCAGAC GTTTGAACAG 420 GCCATCCAGG CAGCCAGCCG GATCATTCGA AACGAGCAGT TTGCCATCAG ACGCTTCCAG 480
  • AAAAAAAAAA 2890 INFORMATION FOR SEQ ID NO: 2:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • 385 390 395 400 lie Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe Ser Asn 405 410 415 5 lie Val Asp Lys Phe Val lie Val Asp Cys Arg Tyr Pro Tyr Glu Tyr 420 425 430
  • CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
  • TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
  • AAGTTTGTGA TTGTAGACTG CAGATACCCC TATGAATATG AAGGCGGGCA CATCAAGACT 420 GCGGTGAACT TGCCCCTGGA ACGCGACGCC GAGAGCTTCC TACTGAAGAG CCCCATCGCG 480
  • CTCTACTACC CTGAGATGTA TATCCTGAAA GGCGGCTACA AGGAGTTCTT CCCTCAGCAC 660
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC 660
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
  • TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
  • CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
  • TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 540
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 540

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Abstract

Nouvelles formes de macromolécules catalytiques apparentées à la cdc25B, une phosphatase spécifique du cycle cellulaire. On décrit également ces domaines particuliers de la cdc25B, des fusions particulières avec le GST, des peptides et protéines uniques, leur utilisation, et leur procédé de fabrication.
EP97923404A 1996-05-02 1997-05-02 Macromolecules catalytiques presentant une activite semblable a celle de la cdc25b Withdrawn EP0907743A1 (fr)

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US1674896P 1996-05-02 1996-05-02
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US1732396P 1996-05-07 1996-05-07
US17323P 1996-05-07
PCT/US1997/005404 WO1997041238A1 (fr) 1996-05-02 1997-05-02 Macromolecules catalytiques presentant une activite semblable a celle de la cdc25b

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US9562012B2 (en) 2013-07-22 2017-02-07 Metabolic Solutions Development Company, Llc PPAR-sparing compounds for the treatment of metabolic diseases

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CA2394152A1 (fr) * 1999-12-14 2001-06-21 Francoise Goubin-Gramatica Methode d'obtention de phosphatases humaines cdc25 et methode d'identification de modulateurs de phosphatases humaines cdc25
CN1312284A (zh) * 2000-03-07 2001-09-12 上海博德基因开发有限公司 一种新的多肽——人cdc4类似蛋白12和编码这种多肽的多核苷酸

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US5294538A (en) * 1991-11-18 1994-03-15 Cold Spring Harbor Labs. Method of screening for antimitotic compounds using the CDC25 tyrosine phosphatase
US6037136A (en) * 1994-10-24 2000-03-14 Cold Spring Harbor Laboratory Interactions between RaF proto-oncogenes and CDC25 phosphatases, and uses related thereto

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See references of WO9741238A1 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9562012B2 (en) 2013-07-22 2017-02-07 Metabolic Solutions Development Company, Llc PPAR-sparing compounds for the treatment of metabolic diseases

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JP2002515742A (ja) 2002-05-28
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