WO1997041238A1 - Catalytic macro molecules having cdc25b like activity - Google Patents

Catalytic macro molecules having cdc25b like activity Download PDF

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Publication number
WO1997041238A1
WO1997041238A1 PCT/US1997/005404 US9705404W WO9741238A1 WO 1997041238 A1 WO1997041238 A1 WO 1997041238A1 US 9705404 W US9705404 W US 9705404W WO 9741238 A1 WO9741238 A1 WO 9741238A1
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Prior art keywords
cdc25b
arg
seq
glu
gly
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PCT/US1997/005404
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French (fr)
Inventor
Martin R. Deibel, Jr.
Anthony W. Yem
Cindy L. Wilson
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Pharmacia & Upjohn Company
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Application filed by Pharmacia & Upjohn Company filed Critical Pharmacia & Upjohn Company
Priority to EP97923404A priority Critical patent/EP0907743A1/en
Priority to JP53889297A priority patent/JP2002515742A/en
Priority to AU29216/97A priority patent/AU2921697A/en
Publication of WO1997041238A1 publication Critical patent/WO1997041238A1/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
    • C12N9/10—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

Abstract

This invention discloses novel forms of catalytic macro molecules that are related to cdc25B, a cell cycle specific phosphatase. These special domains of cdc25B, special fusions with GST, and unique peptides and proteins, their utility, and the method of making them are all described.

Description

CATALYTIC MACRO MOLECULES HAVING CDC25B LIKE ACTIVITY
Field of the Invention This invention relates to the field of protein phosphatases, specifically cdc25B like enzymes.
Information PiBrtpsuje
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., vol. 270(23), pp. 14229-34 (1995).
David H. Beach and Konstantin Galaktionov, U.S. Patent 5,441,880, issued Aug. 15, 1995. "Human cdc25 genes, encoded products and uses thereof."
J.M. Denu and J.E. Dixon. "A catalytic mechanism for the dual-specific phosphatases." Proc. Natl. Acad. Sci. U.S.A., vol. 92(13), pp. 5910-4 (1995).
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., vol. 270(8), pp. 3796-803 (1995)..
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, vol. 5, pp. 5-12 (1996).
K.I. Galaktionov, and D.H. Beach, "Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: Evidence for multiple roles of mitotic cyclins," Cell, vol. 67, pp. 1181-1194 (1991). Galaktionov K, Lee AK, Eckstein J., Draetta G., Meckler J., Loda M.,
Beach D., "CDC25 phosphatases as potential human oncogenes." Science, vol. 269(5230), pp. 1575-7 (1995).
Gottlin E.B., Xu X., Epstein D.M., Burke S.P., Eckstein J.W., Ballou D.P., and Dixon J.E. " Kinetic analysis of the catalytic domain of human cdc25B" J. Biol. Chem., vol. 271(44), pp. 27445-9 (1996).
I. Hoffman, P.R. Clarke, M.J. Marcote, E. Karsenti, and G. Draetta, "Phosphorylation and activation of human cdc25C by cdc2-cyclin B and its involvement in the self-amplification of MPP at mitosis." EMBO J, vol.12 pp. 53-63 (1993). I. Hoffman, G. Draetta, and E. Karsenti, "Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the Gl/S trans t on. , vo . , pp. - .
Takashi Horiguchi, et al., "Dnacin Al and Dnacin Bl are antitumor antibiotics that inhibit cdc25b phosphatase activity." Biochemical Pharmacology, vol. 48 pp. 2139-2141, (1994). T. Ishibashi, D.P. Bottaro, P. Michieli, CA. Kelley, and S.A. Aaronson. "A novel dual specificity phosphatase induced by serum stimulation and heat shock." J. Biol. Chem. 1994 Nov 25; 269(47): 29897-902.
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., vol. 89(24), pp. 12170-4 (1992).
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 BRCAl." Genomics. vol.23(l), pp. 163-7 (1992). A. Kumagai and W.G. Dunphy, "The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system." Cell, vol. 64 pp. 903- 914 (1991).
A. Kumagai and W.G. Dunphy, "Regulation of the cdc25 protein during the cell cycle in Xenopus extracts." Cell, vol. 70 pp. 139-151 (1992). 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., vol. 269(5), pp. 3596-604 (1994).
U.K. Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature, vol. 227 pp.680-685 (1970). J.B.A. Millar, CH. McGowan, G. Lenaers, R. Jones, and P. Russell,
"p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast." EMBO J, vol. 10, pp. 4301-4309 (1991).
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.
U. Strausfeld, A. Fernandez, J-P. Capony, F. Girard, N. Lautredou, J. Derancourt, J-C. Labbe, and N.J.C. Lamb, "Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells." Journal of Biological Chemistry, vol. 269 pp. 5989-6000 (1994). Xu Xu and S.P. Burke, "Roles of Active Site Residues and the NH2-terminal
Domain in the Catalysis and Substrate Binding of Human Cdc25." Journal of Biological emistry, vo . 1, no. , pp - .
J. Yuvaniyama, J.M. Denu, J.E. Dixon, and MA. Saper. "Crystal Structure of the Dual Specificity Protein Phosphatase VHR." Science, vol. 272, pp. 1328-1331. Z.Y. Zhang, Y. Wang, L. Wu, E.B. Fauman, J.A. Stuckey, H.L. Schubert, MA. Saper, and J.E. Dixon. "The Cys(X)5Arg catalytic motif in phosphoester hydrolysis." Biochemistry, vol. 33(51), pp. 15266-70 (1994) .
G. Zhou, J.M. Denu, L. Wu, and J.E. Dixon. The catalytic role of Cysl24 in the dual specificity phosphatase VHR. "Results demonstrate that the dual specificity phosphatases and the tyrosine-specific PTPases employ similar catalytic mechanisms." J. Biol. Chem., vol. 269(45), pp. 28084-90 (1994).
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 suggested that 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).
Apparently cdc25 acts as a cdc2 phosphatase which dephosphorylates tyrosine and possibly threonine residues on p34cdc2 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. To study this protein and/or develop a screen us ng s pro e n one requ res e so a on o e ac ve ca a y c oma n o c c . Unfortunately, the native full length cdc25B is difficult to obtain due to its sensitivity to proteolysis and its low abundance in mammalian cells. On the other hand, a GST fusion protein with cdc25B has been described which has activity. The latter protein has partial solubility and measurable activity as a phosphatase, but as a fusion protein is not amenable to structural analyses including crystallographic studies. To date, successful removal of the GST moiety from a full length fusion protein of cdc25B, with subsequent isolation of full length cdc25B without a GST tag, has not been reported. There have been attempts to make smaller recombinant catalytic domains of cdc25B, including one recently described by Horiguchi et al. The latter recombinant form includes a GST fusion partner and codes for amino acids 355-566, Takashi Horiguchi, et al., Biochemical Pharmacology, Vol. 48 pp. 2139-2141, (1994), incorporated by reference, but this construct appears to result in low protein yields and the protein product is poorly soluble with low activity. The observation of low activity of recombinant forms of cdc25B expressed in E. coli is most likely attributable to improper enzyme folding.
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.
Summary of the Invention This invention discloses the fusions shown below,
(DNA)
1138 1740
GST p R 976 , cdc25B like , 1773
GST p R 302 " cdc25B like ! 566
356 556
(peptide)
where, 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, a) the region has about the number of sequences indicated by the numbers shown, b) the region has either the same amino acids as native cdc25B or substituted nucleic or amino acid residues, where the native nucleic or amino acid residues of the cdc 25B like region are those sequences disclosed in the CHARTS and sequence listings. where the 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.
More particularly 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 cdc25B302"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. 14; 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. NO. 16; the fusion protein comprising, GST-Ile- Glu-Gly-Arg-Gly-Ile-Gln302...-Gin566.
Disclosed are 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 cdc25B976"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. NO. 12; comprised of the nucleic acid residues shown in CHART 13 as Mutein3 or SEQ ID. NO. 13. 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.
Many of the 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. 22; the amino acid residues disclosed in CHART 16 as Mutein 3 or SEQ. ID. NO. 23; the amino acid residues, of SEQ. ID. NO. 9, that is produced from the fusion protein that is GST-Ile-Glu-Gly- Arg-Gly-Ile-SEQ. ID. NO. 4. 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. 14; 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,
(DNA)
1162
GST VHR like 1659
GST p R VHR like
364 529
(peptide)
where, 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 as those sequences disclosed in Chart 17, SEQ. ID. NO. 24 and SEQ. ID. NO 25, and 70% homologous and substantially similar sequences thereof.
The process of making any of the fusions, peptides, constructs or molecules, intermediates, intermediate processes, steps and procedures used to created the fusions and peptides for all of the fusions and peptides are described herein.
Brief Description nf the Drawings Figure IA. 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. 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. 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 of the same blot. Columns one (1) show GST-cdc25B(31-566) and columns two (2) show the special domain cdc25B (356-556).
Figure 3. Figure 3 is a gel filtration size exclusion chromatograph of the purified monomeric special minimal domain cdc25B(356-556).
Figure 4. Figure 4 is an agarose gel electrophoresis showing the product of the PCR reaction (0.8Kb).
Figure 5. Figure 5 is an agarose gel electrophoresis of plasmid mini preps obtained from transformed JM109 E coli.
Additional More Petailed Description of the Invention
Definitions Definitions are included throughout this document in addition to the specific definitions and sources of materials noted below.
"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) "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-β-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) are obtained from Bio 101®., LaJolla, California.
"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.
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-nitrophenyl phosphate (PNPP)."
"PVDF" is polyvinylidene difluoride.
"SDS" is sodium dodecyl sulfate.
"TA cloning kit," containing the pCRII plasmid, and INVαF' 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. Described herein are 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. Furthermore, 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. This type of cdc25B is not stable when stored over time. As a consequence of this instability, the kinetics of the full length protein change over time and this changes the binding constant for the substrate, leading to inconclusive results in any screening operation using the full length protein with a GST tag. Few stability problems are detected with the constructs and fusions disclosed by this invention.
Full length cdc25B, is previously disclosed in US 5,441,880 0880), incorporated by reference. It also appears in a paper by K.I. Galaktionov, and D.H. Beach, "Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: Evidence for multiple roles of mitotic cyclins" Cell (1991) 67; 1181-1194. The known cDNA and amino acid sequence of cdc25B is produced in Chart 3 (cDNA) and Chart 4 (amino acids), below. The DNA and protein sequences are numbered and this numbering system is retained throughout this document. For example, the full length protein is numbered from 1 (Met) to 566 (Gin). Another method of referring to this sequence is cdc25B 5 , or cdc25B (1-566). For example, a macromolecule of only 10 amino acids, might be described as "cdc25B556"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 . c s requen y a ac e o g u a one -trans erase (GST). Indeed, this is the only form of the protein that was previously disclosed.
In other situations a few amino acids may be inserted between the GST and the peptide. These amino acid inserts should be understood from the context of the disclosure in general, or they may be specifically delineated. Thus, it is possible the construct may be represented as "GST-Gly-Ile-cdc25B (302-566)."
In addition to amino acids and GST, there may be other linking molecules between the fusion partner and the protein. For example, in some embodiments of this invention an intervening factor Xa cleavage site will be produced, and this may be introduced between the GST and the cdc25B. In these situations 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).
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- cdc25B1 . 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
10 amino acids in Chart 2, i.e. "Arg-Glu-Leu-Cys-Ser-Arg-Leu-Gln-Asp-Gln." An alternative method of describing this sequence would be to refer to the amino acids and a number indicating the relative segment, e.g. "Arg 556 to Gin 566." Another example would be a reference to a GST fusion to an Xa site, fused to Gly-Ile fused to amino acids 302 to 566 of cdc25B, this would be described as "GST-Xa site-Gly-Ile- cdc25B (302-566)."
The following embodiments and characteristics of this invention are described.
The fusion protein (GST-Xa site-Gly-Ile-cdc25B302"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.
When these special constructs, such as, cdc25B(356-556), are evaluated for enzymatic activity, using PNPP as a substrate, 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-cdc25B31-566. The lower 1^ reflects better binding properties to the substrate than any enzymes currently known. The Vmaχ 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.
When the protein cdc25B (356-556) is evaluated for enzymatic activity, using PNPP as a substrate, 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).
Disclosed herein from a previous disclosure is the expression of GST-cdc25B (355-566), a different protein which is closely related to the sequence of the minimal domain being claimed in the present invention, but results in low yields of poorly soluble, low activity GST-cdc25B355"566 product. This form was previously reported in the literature. 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." The reported form contained GST and no attempt to remove the GST was reported. General Discussion of the Methods
The sequence of the full length DNA is available in GenBank, accession number M81934.gb_pr., submission by Beach and Galaktionov. This sequence was also disclosed by Nagata, who used a different numbering system. According to the Nagata 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 Beach and Galaktionov numbering sys em s use roug ou s ocumen . 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 INVαF' 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 Galaktionov); 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 DΝA is isolated using the commercially available GeneClean kit (Bio 101®).
The purified DΝA 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 DΝA 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 Gin302 . The -Gly-Ile- comes from the coding region contributed by part of the restriction site. See CHART 2.
From these general procedures one skilled in the art could practice this invention. The following CHARTS, Analytical Methods, Additional, Special and
Optimal Conditions and Considerations are provided to better illustrate and describe but not limit this invention.
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 Galaktionov, 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. To improve the production of the protein as well as its stability, we engineered mutations at two sites (see vertical dashed lines below), 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.
Visual images of the fusions are shown on the next page.
Figure imgf000019_0001
10
Figure imgf000019_0002
Figure imgf000019_0003
25
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. Other suitable plasmids, restriction sites and biological reagents known to one skilled in the art, can be used. 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. The dashes between sequences below indicate additional sequences not shown here, this Chart is intended to show only insertion points and cleavage sites. The sequences that are shown have been given separate Sequence Identification Numbers, SEQ. ID. NO.s, also provided in CHART 18, for the sake of completeness.
PCR Reaction
{Bam HI
5' - GCG GATC^AGCGGCTCTTCΠGCTCTC 5 ' - CCAGCGGCTCTTCCGCTCTCCGTC AGCCGGCTGCAGGACCAGTGA - 3 '
3 ' - GGTCGCCGAGAAGGCGAGAGGCAG TCGGCCGACGTCCTGGTCACT - 5 '
GCCGACGTCCTGGTCACTGAGCT CCG
Xho It -5'
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).
Bam Hl/Xho I Digestion Ligation into Bam Hl/Xho I sites of pGEX-5X-3
jBam HI txho I
5 'GAAGGTCGTGG GATC C AGCGGCTCTTCCGC CAGGACCAGTGAC TCGA GCGGCCGCAT 3'
3 ' CTTCCAGCACC CTAG G TCGCCGAGAAGGCG GTCCTGGTCACTG AGCT CGCCGGCGTA 5 ' Bam Hit Xho it
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 Galaktionov. 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 Galaktionov system. This sequence was also disclosed by Nagata, who used a different numbering system. According to the Nagata 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.
1 GCCAGCTGTG CCGGCGTTTG TTGGCTGCCC TGCGCCCGGC CCTCCAGCCA
51 GCCTTCTGCC GGCCCCGCCG CGATGGAGGT GCCCCAGCCG GAGCCCGCGC
151 GGCCACCTCC CGGGCCTCCT GCTGGGATCT CATGGCCTCC TGGGGTCCCC
201 GGTGCGGGCG GCCGCTTCCT CGCCGGTCAC CACCCTCACC CAGACCATGC
251 ACGACCTCGC CGGGCTCGGC AGCCGCAGCC GCCTGACGCA CCTATCCCTG
301 TCTCGACGGG CATCCGAATC CTCCCTGTCG TCTGAATCCT CCGAATCTTC
351 TGATGCAGGT CTCTGCATGG ATTCCCCCAG CCCTATGGAC CCCCACATGG
401 CGGAGCAGAC GTTTGAACAG GCCATCCAGG CAGCCAGCCG GATCATTCGA
451 AACGAGCAGT TTGCCATCAG ACGCTTCCAG TCTATGCCGG TGAGGCTGCT
501 GGGCCACAGC CCCGTGCTTC GGAACATCAC CAACTCCCAG GCGCCCGACG
551 GCCGGAGGAA GAGCGAGGCG GGCAGTGGAG CTGCCAGCAG CTCTGGGGAA
601 GACAAGGAGA ATGATGGATT TGTCTTCAAG ATGCCATGGA AGCCCACACA
651 TCCCAGCTCC ACCCATGCTC TGGCAGAGTG GGCCAGCCGC AGGGAAGCCT
701 TTGCCCAGAG ACCCAGCTCG GCCCCCGACC TGATGTGTCT CAGTCCTGAC
751 CGGAAGATGG AAGTGGAGGA GCTCAGCCCC CTGGCCCTAG GTCGCTTCTC
801 TCTGACCCCT GCAGAGGGGG ATACTGAGGA AGATGATGGA TTTGTGGACA
851 1CCTAGACAG TGACTTAAAG GATGATGATG CAGTTCCCCC AGGCATGGAG 951 GGACCTCGTC ATGTACAGCA AGTGCCAGCG GCTCTTCCGC TCTCCGTCCA
1001 TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG
1051 GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC
1101 TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA
1151 AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA
1201 GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG
1251 AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT
1301 TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
1351 AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT
1401 GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC
1451 CCTGTAGCΓT ΠGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA
1501 TCTGAGCGTΠ GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGΓ
1551 TGTCAACGAΠ TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG
1601 GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
1651 GACTACCGGΓ. CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT
1701 CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT
1751 GTAGCCGGCT GCAGGACCAG TGAGGGGCCT GCGCCAGTCC TGCTACCTCC
1801 CTTGCCTTTC GAGGCCTGAA GCCAGCTGCC CTATGGGCCT GCCGGGCTGA
1851 GGGCCTGCTG GAGGCCTCAG GTGCTGTCCA TGGGAAAGAT GGTGTGGTGT
1901 CCTGCCTGTC TGCCCCAGCC CAGATTCCCC TGTGTCATCC CATCATTTTC
1951 CATATCCTGG TGCCCCCCAC CCCTGGAAGA GCCCAGTCTG TTGAGTTAGT
2001 TAAGTTGGGT TAATACCAGC TTAAAGGCAG TATTTTGTGT CCTCCAGGAG
2051 CTTCTTGTTT CCTTGTTAGG GTTAACCCTT CATCTTCCTG TGTCCTGAAA CTCCT T
2151 AGGATGGGTC AGAGCTAAAC TCCTTCCTGG CCTGAGAGTC AGCTCTCTGC
2201 CCTGTGTACT TCCCGGGCCA GGGCTGCCCC TAATCTCTGT AGGAACCGTG
2251 GTATGTCTGC CATGTTGCCC CTTTCTCTTT TCCCCTTTCC TGTCCCACCA
2301 TACGAGCACC TCCAGCCTGA ACAGAAGCTC TTACTCTTTC CTATTTCAGT
2351 GTTACCTGTG TGCTTGGTCT GTTTGACTTT ACGCCCATCT CAGGACACTT
2401 CCGTAGACTG TTTAGGTTCC CCTGTCAAAT ATCAGTTACC CACTCGGTCC
2451 CAGTTTTGTT GCCCCAGAAA GGGATGTTAT TATCCTTGGG GGCTCCCAGG
2501 GCAAGGGTTA AGGCCTGAAT CATGAGCCTG CTGGAAGCCC AGCCCCTACT
2551 GCTGTGAACC CTGGGGCCTG ACTGCTCAGA ACTTGCTGCT GTCTTGTTGC
2601 GGATGGATGG AAGGTTGGAT GGATGGGTGG ATGGCCGTGG ATGGCCGTGG
2651 ATGCGCAGTG CCTTGCATAC CCAAACCAGG TGGGAGCGTT TTGTTGAGCA
2701 TGACACCTGC AGCAGGAATA TATGTGTGCC TATTTGTGTG GACAAAAATA
2751 TTTACACTTA GGGTTTGGAG CTATTCAAGA GGAAATGTCA CAGAAGCAGC
2801 TAAACCAAGG ACTGAGCACC CTCTGGATTC TGAATCTCAA GATGGGGGCA
2851 GGGCTGTGCT TGAAGGCCCT GCTGAGTCAT CTGTTAGGGC CTTGGTTCAA
2901 TAAAGCACTG AGCAAGTTGA GAAAAAAAAA AAAAAAAAAA
The amino acid sequence derived from the full length cdc25B DNA sequence shown in CHART 3. This is Sequence LD. no. 2.
1 MEVPQPEPAP GSALSPAGVC GGAQRPGHLP GLLLGSHGLL GSPVRAAASS
51 PVTTLTQTMH DLAGLGSRSR LTHLSLSRRA SESSLSSESS ESSDAGLCMD
101 SPSPMDPHMA EQTFEQAIQA ASRIIRNEQF AIRRFQSMPV RLLGHSPVLR
151 NITNSQAPDG RRKSEAGSGA ASSSGEDKEN DGFVFKMPWK PTHPSSTHAL
201 AEWASRREAF AQRPSSAPDL MCLSPDRKME VEELSPLALG RFSLTPAEGD
251 TEEDDGFVDI LESDLKDDDA VPPGMESLIS APLVKTLEKE EEKDLVMYSK
301 CQRLFRSPSM PCSVIRPILK RLERPQDRDT PVQNKRRRSV TPPEEQQEAE
351 EPKARVLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSRRELC SRLQDQ
The DNA sequence of the Cdc25B976"1773 segment. The expected theoretical product of Factor Xa cleavage for the expressed GST fusion. This is Sequence LD. no. 3.
976 CAGCG GCTCTTCCGC TCTCCGTCCA
1001 TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG
1051 GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC
1101 TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA
1151 AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA
1201 GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG
1251 AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT
1301 TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
1351 AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT
1401 GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC
1451 CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA
1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
1551 TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG
1601 GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
1651 GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT
1701 CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT
1751 GTAGCCGGCT GCAGGACCAG TGA The peptide sequence of cdc25Boυ'ώ"000 corresponding to the DNA sequence shown in CHART 5. This is Sequence LD. no. 4
302 QRLFRSPSM PCSVIRPILK RLERPQDRDT PVQNKRRRSV TPPEEQQEAE
351 EPKARVLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSRRELC SRLQDQ
The DNA sequence of the GGG-ATC-Cdc25B976"1773 segment. This is a notional sequence describing the expected theoretical product of Factor Xa cleavage for the expressed GST fusion, plus two codons. This is Sequence LD. no. 5.
970 G GGATCCAGCG GCTCTTCCGC TCTCCGTCCA
1001 TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG
1051 GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC
1101 TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA
1151 AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA
1201 GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG
1251 AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT
1301 TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
1351 AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT
1401 GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC
1451 CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA
1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
1551 TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG
1601 GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
1651 GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT
1701 CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT
1751 GTAGCCGGCT GCAGGACCAG TGA CHART 8 The peptide sequence of Gly-Ile-cdc25B corresponding to the DNA sequence shown in CHART 7. This is a notional sequence describing the expected peptide that would be expressed from the nucleic acid sequence in CHART 7. This is Sequence LD. no. 6.
300 G
301 IQRLFRSPSM PCSVIRPILK RLERPQDRDT PVQNKRRRSV TPPEEQQEAE
351 EPKARVLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSRRELC SRLQDQ
The DNA sequence of the ATC-GAA-GGT-CGT-GGG-ATC-Cdc25B976'1773 segment. This is a notional sequence describing a fusion form. This is Sequence LD. no. 7.
958 ATC GAAGGTCGTG GGATCCAGCG GCTCTTCCGC TCTCCGTCCA
1001 TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG
1051 GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC
1101 TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA
1151 AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA
1201 GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG
1251 AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT
1301 TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
1351 AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT
1401 GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC
1451 CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA
1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
1551 TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG
1601 GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
1651 GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT
1701 CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT
1751 GTAGCCGGCT GCAGGACCAG TGA The peptide sequence of [GST](GST not shown below)- — Ile-Glu-Gly-Arg-Gly- Ile-Gln302...-Gln566 corresponding to the DNA sequence shown in CHART 9. When combined with GST this construct would be a fusion protein. This is a notional sequence describing the expected peptide that would be expressed from the nucleic acid sequence in CHART 9. This is Sequence LD. no. 8.
IEGRG
IQRLFRSPSM PCSVIRPILK RLERPQDRDT PVQNKRRRSV TPPEEQQEAE
351 EPKARVLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSRRELC SRLQDQ
The peptide sequence of cdc25B356*556. This is Sequence LD. no. 9.
356 VLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSR
The peptide sequence of cdc25B356"566 . This is Sequence LD. no. 10.
356 VLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSRRELC SRLQDQ
(Advance Form - Nucleotide - Design) This CHART contains sequences disclosed under the section of this invention entitled, "Advanced Forms of the Macromolecule." There are eight different sequences disclosed in this section of the document, including both amino acid and nucleotide sequences. One construct described is the same construct described previously, in CHART 5 (nucleotide, Seq. ID. No. 3) and in CHART 6 (peptide, SEQ. ID. No. 4). Four of the constructs are from the Design section and four from the Results section. The sequences from the 3 Design constructs, nucleotide, from the Advanced Forms, are provided in this CHART, below.
First, the DNA sequence of the Cdc25B976"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.
976 (from CHART 5) CAGCG GCTCTTCCGC TCTCCGTCCA
Muteinl
Mutein2
Mutein3
1001 TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG
Muteinl
Mutein2
Mutein3
1051 GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC
Muteinl
Mutein2
Mutein3
1101 TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA
Muteinl AT.G. ..G
Mutein2
Mutein3 AT .G. ..G
1151 AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA
Muteinl
Mutein2
Mutein3
1201 GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG
Muteinl
Mutein2
Mutein3
1251 AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT Mute n . Mutein2 . Mutein3 .
1301 TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
Muteinl
Mutein2
Mutein3 -.
1351 AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT
Muteinl
Mutein2
Mutein3 1401 GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC
Muteinl
Mutein2
Mutein3 1451 CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA
Muteinl
Mutein2
Mutein3 1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
Muteinl
Mutein2
Mutein3 1551 TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG
Muteinl
Mutein2
Mutein3 1601 GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
Muteinl
Mutein2
Mutein3 1651 GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT
Muteinl
Mutein2
MuteinS 1701 CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT
Muteinl
Mutein2 AA. AA
Mutein3 AA. AA 1751 GTAGCCGGCT GCAGGACCAG TGA
Muteinl
Mutein2
Mutein3
(Advance Form - Peptides- Design) 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. (peptide, SEQ. ID. No. 4). Four of the constructs are from the Design section and four from the Results section. The sequences from the 3 Design constructs, peptides, from the Advanced Forms, are provided in this CHART, below. The following CHART, CHART 15 provides the corresponding nucleotide sequences, beginning with the sequence first disclosed in CHART 5 (nucleotide, Seq. ID. No. 3).
In the sequence below, 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.
302 -QRLFRSPSM PCSVIRPILK RLERPQDRDT PVQNKRRRSV TPPEEQQEAE
Muteinl -
Mutein2 - Mutein3 -
351 EPKARVLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
Muteinl .IEG
Mutein2 Mutein3 . IEG
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
Muteinl
Mutein2 Mutein3
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
Muteinl
Mutein2 Mutein3 501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
Muteinl
Mutein2
Mutein3
551 AGERSRRELC SRLQDQ
Muteinl
Mutein2 KK
Mutein3 KK
(Advance Form - Nucleotide - Results)
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 previous CHART 14 described the DESIGN section and CHARTS 15 and 16 describe residues from constructs from the RESULTS section of this portion of the invention.
First the DNA sequence of the Cdc25B1138-1740 and Cdc25B1138"1773 segments are provided as modified according to the section, Advanced Forms of the Macromolecule. The three forms: Muteinl, Mutein2, and Mutein3, are shown with the appropriate substitutions shown below the full sequence. All other sequences remain the same as in the full sequence, except where shown as changed below, i.e. dots (.) below indicate no changes. The Sequence ID No. for the wild type sequence of Cdc25B976-1773 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.
1138 GTC CTCCGCTCAA
Muteinl
Mutein2
Mutein3
1151 AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA
Muteinl
Mutein2
Mutein3
1201 GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG
Muteinl
Mutein2
Mutein3
1251 AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT
Muteinl
Mutein2
Mutein3
1301 TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC
Muteinl
Mutein2
Mutein3
1351 AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT
Muteinl
Mutein2
Mutein3
1401 GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC
Muteinl
Mutein2
Mutein3
1451 CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA
Muteinl
Mutein2
Mutein3 1501 TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC
Muteinl
Mutein2
5. Mutein3
1551 TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG
Muteinl -
Mutein2 0 Mutein3
1601 GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG
Muteinl
Mutein2 5 Mutein3
1651 GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT
Muteinl
Mutein2 0 Mutein3
1701 CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG (stops)
Muteinl (stops)
Mutein2 AA. AAGGAGCTCT 5 Mutein3 AA. AA
1751
Mutein2 GTAGCCGGCT GCAGGACCAG TGA
Mutein3
CHART 16 (Advance Form - Peptide - Results)
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 Cdc25B356"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. 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. 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
Muteinl
Mutein2
Mutein3 401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
Muteinl
Mutein2
Mutein3 451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
Muteinl
Mutein2
Mutein3 501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
Muteinl
Mutein2
Mutein3 551 AGERSR (stops at 556)
Muteinl (stops at 556)
Mutein2 KKELC SRLQDQ
Mutein3 KKELC SRLQDQ This CHART contains 2 sequences disclosed in the part of the document relating to cdc25B constructs having similarity to human NHR phosphatase, titled VHR-LIKE CONSTRUCTS. Full length human cdc25B was previously provided in CHART 3, it also appears in NHR-Like-CHART A. The underlined portion of NHR- Like-CHART A shows the VHR like construct which is provided here in CHART 17, PART A. This construct is made into a GST fusion construct, the GST the GST and GIQ are shown in parenthesis. The body of the construct begins with residue 364 and ends with residue 529, as shown in NHR-Like-CHART A. The Part A sequence, without (GST)-(XaMGIQ) is Sequence ID No. 24.
Also shown in this CHART 17, 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
CHART 17, PART A 364 (GST) - (Xa) - (GIQ) -HDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPLERDAE
451 SFLLKSPIAP CSLDKRVILI FHCEFSSERG PRMCRFIRER DRAVNDYPSL
501 YYPEMYILKG GYKEFFPQHP NFCEPQDYR CHART 17 Part B
1 MSGSFELSVQ DLNDLLSDGS GCYSLPSQPC NEVTPRIYVG NASVAQDIPK
51 LQKLGITHVL NAAEGRSFMH VNTNANFYKD SGITYLGIKA NDTQEFNLSA
101 YFERAADFID QALAQKNGRV LVHCREGYSR SPTLVIAYLM MRQKMDVKSA 151 LSIVRQNREI GPNDGFLAQL CQLNDRLAKE GKLKP
This CHART contains other miscellaneous sequences disclosed with this invention.
From Figure IA: The DNA sequences shown before the 0.8 Kb cdc25B insert are: ATC-GAA-GGT-CGT-GGG-ATC-C (Sequence LD. no. 26) corresponding to the following amino acids: Ile-Glu-Gly-Arg-Gly-Ile-. (Sequence LD. no. 27.)
From Figure IA: The sequences shown after the 0.8 Kb cdc25B insert are: C-TCG-AGC-GGC-CGC-ATC-GTG-ACT-GAC-TGA- (Sequence LD. no. 28.) corresponding to the following amino acids: Ser-Ser-Gly-Arg-Ile-Val-Thr-Asp. (Sequence LD. no. 29.)
Disclosed as a primers ordered from Genosys® are: 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.)
The following sequences were disclosed from CHART II: The top part of CHART II started with PCR Reaction and showed:
- GCG GATCCAGCGGCTCTTCCGCTCTC (δ'→3') (Sequence LD. no. 30, repeated from just above.) - CCAGCGGCTCTTCCGCTCTCCGTC -- (5'→3') (Sequence LD. no. 32.)
— AGCCGGCTGCAGGACCAGTGA - (5'→3') (Sequence LD. no. 33.)
- GGTCGCCGAGAAGGCGAGAGGCAG — (3'→5') (This sequence is entered in (5'→3') direction as Sequence LD. no. 34.)
— TCGGCCGACGTCCTGGTCACT - (3'→5') (This sequence is entered in (5*→3') direction as Sequence LD. no. 35.)
GCCGACGTCCTGGTCACTGAGCT CCG - (3'→5') (This sequence is entered in (5'→3') direction as Sequence LD. no. 36.)
The bottom part of CHART II had Bam Hl/Xho I Digestion and Ligation into Bam Hl/Xho I sites of pGEX-5X-3 and showed:
- GAAGGTCGTGGJΪ£∑£C. AGCGGCTCTTCCGC -- (5'→3') (Sequence LD. no. 37.)
-— CAGGACCAGTGAC TCGA GCGGCCGCAT - (δ'→3') ( Sequence LD. no. 38.)
- CTTCCAGCACC CTAGG TCGCCGAGAAGGCG — - (3'→5') (This sequence is entered in (5'→3') direction as Sequence LD. no. 39.)
-- GTCCTGGTCACTG AGCT CGCCGGCGTA - (3'→5') (This sequence is entered n '→ rect on as equence . no. .
There were several minor sequences disclosed in the section of the invention titled, Advanced Forms of the Macromolecule. The following oligonucleotide primers are mentioned:
- GCG GAG GAC GCG GCC TTC AAT TTC CTC AGC CTC - (5'→3') (Sequence ID No. 41.)
- GGG GAG CGG AGC AAG AAG GAG CTC TGT AGC - (5'→3') (Sequence ID No. 42.) - GAG GCT GAG GAA ATT GAA GGC CGC GTC CTC CGC - (5'→3') (Sequence ID No. 43
- GCG GAG GAC GCG GCC TTC AAT TTC CTC AGC CTC - (5'→3') (Sequence ID No. 41, repeated from just above.)
There are several minor sequences disclosed in the VHR-LIKE CONSTRUCT portion of this document, relating to VHR like domains, the following two DNA primers, listed in a 5'->3' orientation were prepared/obtainded from Genosys®: -GCG GAT CCA GCA CGA TGA GAT CGA GAA-. (Geno-III) (Sequence ID No. 44.) and -GCC TCG AGT CAC CGG TAG TCC TGG GGT- (Geno-IV) (Sequence ID No. 45.)
na y ca e o s
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. 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.
Western blotting - Transferred blots were blocked with 2% non-fat dried milk in PBS for 5 min. After washing in tris buffered saline [TBS (10 mM Tris HCI, 150 mM NaCl, pH 7.5)] for 5 min, blots were incubated with primary antibody, (rabbit anti-cdc25B polyclonal antibody) for 1 hr on an orbital shaker. The blots were washed in 0.05% Tween 20 in TBS for 5 min followed by a 5 min wash with TBS. 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.
Sequence analysis - Amino terminal sequencing of purified cdc25B 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.
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. 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 K. and Vmaχ, multiple pNPP concentrations are used at a constant enzyme concentration. Rates at each concentration of substrate are determined and the K_ and Vmaχ calculated from line fitting to a Michaelis Menten equation. The extinction coefficient (1 mM= 17.8 absorbance units at 405 nm) for the p-nitrophenyl product has been previously published. See, N.K. Tonks, CD. Diltz, and E.H. Fischer (1988) J. Biol. Chem. 263: 6731-6737. 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. Combining the methods, procedures and analytic tools above with expression and purification procedures generally known to one skilled in the art, the following sequences are created: (Form I) Fusion protein is,
GST-Ile-Glu-Gly-Arg-Gly-Ile-Gln302...-Gln566. (Form II) Final product of cdc25B after factor Xa treatment is, Val356-...-Arg556.
Additional, Special and Optimal Conditions and Considerations Attempts to utilize factor Xa cleavage using solution phase digestion (i.e., displacing GST-Xa-Gly-Ile-cdc25B from a glutathione Sepharose column with reduced glutathione prior to digestion) were shown to be unsuitable. Protein prepared in this way was contaminated with the glutathione S-transferase (GST) protein component, and subsequent purification procedures are unable to purify the cdc25B away from the GST polypeptide and any fragmentation products of GST- containing polypeptides. Most, if not all of the GST-Xa-Gly-Ile-cdc25B (302-566) fusion protein is soluble in the extraction/lysis system. 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. Both 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. A recent observation by Silva et al., using a similar art, showed conclusively that DNA-K did not bind to the GST component of a GST-protein fusion construct. N.L.C.L. Silva, . . awor , . ng , an . ege , e car oxy - erm na reg on o e a exchanger interacts with mammalian heat shock protein", Biochemistry 34: 10412- 10420 (1995). Both chaperonins, DnaK and GroEL, are resolved from the truncated cdc25B protein by the Q fast flow ion exchange step. Both DnaK and GrόEL may be required for both solubility and correct folding of the truncated cdc25B in the E. coli expression system.
Additional references and descriptions of DnaK and GroEL are provided below and incorporated by reference:
R. Hlodan, P. Tempst, and F.U. Hartl, "Binding of defined regions of a polypeptide to GroEL and its implications for chaperonin-mediated protein folding", Nature Structural Biology, vol. 2, pp. 587-595 (1995).
A.M. Fourie, J.F. Sambrook, and M-J H. Gething, "Common and divergent peptide binding specificities of hsp70 molecular chaperones", J. Biol. Chem., vol. 269, pp. 30470-30478 (1994). J. Wild, E. Altman, T. Yura, and CA. Gross, "DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli", Genes and Development, vol. 6, pp. 1165-1172 (1992) and E.A. Craig, "Chaperones: helpers along the pathways to protein folding", Science, vol. 260, pp. 1902-1903 (1993).
L.S. Itzhaki, D.E. Otzen, and A.R. Fersht, "Nature and consequences of GroEL-protein interactions", Biochemistry, vol. 34, pp. 14581-14587 (1995).
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.
Additional cleavage sites downstream from the engineered factor Xa site were observed. A predominant secondary site was observed at residue 355 (Arg), which appears to be the minimal domain defined by factor Xa cleavage. Digestion of the GST-cdc25B construct yields a minimal catalytic domain defined by the sequence of valine 356 through arginine 556. Due to a large number of arginine residues in the region between residues 302 and 356, accessory factor Xa cleavage sites are recognized during production. Another secondary factor Xa site is found between Arg556 and Arg557, near the C-terminus of cdc25B. Others have reported on the ability of factor Xa to cleave at arginines other than in the IEGR motif. See, R. Lottenberg, J.A. Hall, E. Pautler, A. Zupan, U. Christensen, and CM. Jackson, "The action of factor Xa on peptide p-nitroanilide subtrates: substrate selectivity and examination of hydrolysis with different reaction conditions", Biochem. Biophys. Acta 874: 326-336 (1986). ac or a pro e n an ac v y s eas y remove a er e ges on s ep y the Mono Q ion exchange chromatography step. The activity is separated with baseline resolution from the cdc25B protein. However; for work requiring high cdc25B concentrations after this step, we routinely add APMSF and/or pefabloc (serine protease inhibitors) to the cdc25B preparation after the Mono Q step. The complete removal of detectable protease activity is validated using an assay for factor Xa. The substrate for this reaction is N-p-tosyl-Gly-Pro-Arg-p-nitroanilide. See, by R. Lottenberg et al., Biochem. Biophys. Acta 874: 326-336 (1986).
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).
Activity and Usefulness of the Macro Molecules The following macromolecules, or constructs of cdc25B, were observed.
Form I is a fusion protein whose sequence is: GST-Ile-Glu-Gly-Arg-Gly-Ile-Gln302.. .-Gin566. See, CHART 10, SEQ. ID NO. 8
Form II is the final product of cdc25B after factor Xa cleavage, Val356-...-Arg556. See, 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 (Kj) and the concentration of inhibitor which causes 50 per cent inhibition (I5ϋ) of an enzymatic reaction. Biochemical Pharmacology Vol. 22, pp. 3099-3108.
Performing the necessary kinetic experiments and data analysis which distinguish between these three classes of enzyme inhibitors is usually straightforward. When an inhibitor exerts its effects over a concentration range smaller than 100-fold the effect is said to be cooperative. Cooperativity occurs in multimeric enzymes and in monomeric enzymes containing multiple binding sites. Many compounds which reversibly inhibit GST-cdc25B fusion proteins do so in a cooperative manner. Because of the added ambiguity in terms of possible kinetic mechanisms and added mathematical complexity which results from cooperativity, it s o en mposs e o c arac e ze e ne c mec an sm y w c suc n ors exert their effects. This is particularly true if the enzyme under investigation is a synthetic fusion protein and the enzyme portion is normally monomeric and contains only a single known binding site, as is the case with the catalytic domain of cdc25B. With near full length cdc25B (GST-cdc25B (31-566), observed cooperativity is probably the result of dimer or higher oligomer formation caused by the GST domain, which is known to form homodimers. The cooperativity seen with inhibitors of GST-cdc25B are absent in inhibitors of minimal domain cdc25B (cdc25B (356-556)). In other words, inhibitors which act in a cooperative manner toward near full length GST-cdc25B often act as competitive inhibitors toward the minimal domains or catalytic macromolecules of cdc25B and they exhibit few cooperative effects.
In addition to complicating the kinetic behavior of cdc25B inhibitors, the GST domain also complicates the purification process. 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). Subsequent chromatography steps utilizing anion or cation exchange, hydroxyapatite, p- chloromecuribenzoate, or gel filtration do not result in any further enhancement of purity. These results indicate that in addition to its probable dimeric behavior there is also a marked tendency for GST-cdc25B fusion proteins to non-specifically aggregate. The minimal domain cdc25B (356-556) protein or cdc25B catalytic macromolecules, on the other hand, are monomeric and have not been observed to non-specifically aggregate. Unlike GST-cdc25B, minimal domain cdc25B (356-556) protein or cdc25B catalytic macromolecules can be purified to homogeneity without the use of strong chaotropic agents. See Figure 3 for a gel filtration size exclusion chromatograph of the purified monomeric special minimal 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.
The following r^ rates, using PNPP as substrate, were determined in a comparison of truncated cdc25B (356-556) protein with near full length GST-cdc25B (31-566). nzyme amp e approx mate m cdc25B (356-556) 2.0 mM
Mutein3 2.0mM
GST-cdc25B (31-566) 12.0 mM Muteinl, and mutein2 also have activity much higher than GST-cdc25B (31-566) and mutein3 has improved stability over GST-cdc25B (31-566). Muteinl, mutein2 and mutein3 are described in the next section.
Vmaχ 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. For example, Dunphey and Kumagai, Cell, (1991) 67, pp. 189-196, report a K^ and Vmaχ 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. Similarly, Horiguchi et al., Biochem Pharmacol, (1994) vol. 48, pp. 2139-2141, report a KJJJ of 16.6 mM for GST-cdc25B (residues 355-566) using PNPP as substrate at
37°C The purity was not reported, the calculated Vmaχ was not listed, nor can it be estimated without knowledge of the purity of the protein. Using the procedures described herein a Vmaχ for cdc25B(356-556) is equivalent to 500 +/- 100 nmoles/min/mg. When comparing our enzyme (tested at 25°C) against other reported cdc25B proteins which were tested at 37°C, we found it necessary to increase our specific activities by 2-3 fold. This resulted from a measured 2-3 fold difference in rates we see at 37°C versus 25°C These data show that our truncated cdc25B protein representing amino acid residues 356-556 is more active than any reported cdc25 protein.
Another observation of this invention involves a comparison of the GST- cdc25B (302-566) parent protein versus the cdc25B (356-556) final product protein: The I^ for PNPP as substrate using our truncated cdc25B (356-556) protein is 2-3- fold lower than for the parent GST-Xa site-Gly-Ile-cdc25B (302-566) protein. Advanced Forms of the Macromolecule
Introduction
Improved forms of cdc25B (356-556) have also been designed. In the region spanning residues 302 to 355, there are many arginines which are attacked by factor Xa during the cleavage step. A new factor Xa site immediately preceding valine 356 was created to facilitate factor Xa processing directly to the fusion protein initiating at val356. Similarly, a second construct was designed with the su s u on o wo ys nes or wo arg n nes a res ues - . was ope a this substitution would prevent the factor Xa processing of this site which occurs during the normal processing of the GST fusion protein. The outcome of this latter digestion, if allowed to occur, is to generate cdc25B (356-556), which contains a 10 amino acid truncation at the C-terminus. The Arg-Arg to Lys-Lys replacement at 556-557 would allow for the generation of a cdc25B molecule with an intact C-terminus. The improvements would be best exemplified by using both changes in the same construct yielding a more homogenous product with improved stability after Xa processing for uses such as crystallization studies. Experimental
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.
ADVANCED FORM SEQUENCE COMPARISON
(Underlining shows changes from the native form.)
DESIGN
Wiiπ.l.eotide (Starts with residue 976. )
976 ... 1123 THROUGH 1146 1732 THROUGH 1749 1773 Wild Type
... GAA CCT AAA GCC CGC GTC CTC CGC .. CGG AGC CGG CGG GAG CTC ... 1773 Muteinl:
... GAA ATT £AA G£C CGC GTC CTC CGC .. CGG AGC CGG CGG GAG CTC ... 1773 Mutein2 :
... GAA CCT AAA GCC CGC GTC CTC CGC .. CGG AGC AAG AAG GAG CTC ... 1773 MuteinS : ... GAA ATT GAA G£C CGC GTC CTC CGC .. CGG AGC AAG AAG GAG CTC ... 1773
Protein (Starts with residue 302. )
...351 352 353 354 355 356 357 358 . 554 555 556 557 558 559 566 NativeO:
...GLU PRO LYS ALA ARG VAL LEU ARG . ARG SER ARG ARG GLU LEU 566 Muteinl:
... GLU TLB GLU GLY ARG VAL LEU ARG . ARG SER ARG ARG GLU LEU 566 Mutein2 :
...GLU PRO LYS ALA ARG VAL LEU ARG . ARG SER LYS LYS GLU LEU ... 566 Mutein3 : ...GLU ILE GLU GLY ARG VAL LEU ARG . ARG SER LYS LYS GLU LEU ... 566
BESIIT,TS (Protein is listed first for comparison with above)
Protein Starts at 356 357 358 554 555 556 557 558 559 ... 566
Wild Type: VAL LEU ARG .. ARG SER ARG (Stops at arg556) Muteinl: VAL LEU ARG .. ARG SER ARG (Stops at arg556) Mutein2 : VAL LEU ARG .. ARG SER
Figure imgf000050_0001
GLU LEU ... GLN Mutein3: VAL LEU ARG .. ARG SER LYS LYS GLU LEU ... GLN
Nucleotide 1138 THROUGH 1146 1732 THROUGH 1749 1773
NativeO: GTC CTC CGC CGG AGC CGG CGG GAG CTC ... 1773 Muteinl: GTC CTC CGC CGG AGC CGG CGG GAG CTC ... 1773 Mutein2 : GTC CTC CGC . . . CGG AGC AAG AAG GAG CTC . . . 1773
Mutein3 : GTC CTC CGC . . . CGG AGC AAG AAG GAG CTC . . . 1773
The sequences above are described in full in CHARTS 13-16. It should be understood that the GST fusion proteins of these contructs are the form most useful as soluble and in some cases crystallizable constructs.
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. Using 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. After Dpnl digestion to eliminate non-mutated plasmid, 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. Using the same template and protocol as that used with Muteinl, we introduced these mutations into the cdc25B gene using 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.
To create Mutein3, a separate method was utilized. The cDNA of mutein 2, which already contained the RR->KK codon changes, was reconstructed using the Stratagene® quickchange system with primers (sense and antisense oligonucleotide primers containing the desired mutation; 5' GAG GCT GAG GAA ATT GAA GGC CGC GTC CTC CGC 3' SEQ. ID. NO. 43 or 5' GCG GAG GAC GCG GCC TTC AAT TTC CTC AGC CTC 3' SEQ. ID. NO. 41. After denaturation of the plasmid and annealing of the oligo primers containing the appropriate mutations, 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.
The process of expressing the proteins encoded by these constructs was similar to that described for the wildtype protein. Each insert was ligated into the plasmid pGEX-5X-3 as described for the wild type system. E. coli strain JM109 was transformed with each plasmid and expression of the resulting GST fusion proteins of cdc25B minimal domains was conducted as described for the wild type enzyme system. The purification of these cdc25B proteins was conducted in a manner similar to that described for the wild type enzyme. Six liters of frozen E. 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. 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-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 following Specific Embodiments, Examples, Procedures and Techniques are provided to further support and illustrate, but not to limit, the invention.
Original Substrate - 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®.
PCR Cloning - A defined region of cdc25B was desired, requiring the isolation of a section of the cdc25B cDNA which codes for residues 302-566 in the protein sequence. The nucleotide sequence representing this truncated cdc25B protein is residues 976-1773. To amplify only this sequence, the following DNA primers were prepared (ordered from 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. 31 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 μM dNTPs, IX PCR Buffer (Perkin-Elmer® GeneAmp®), 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCI, pH 8.3, 0.001% (w/v) gelatin, Sterile water to total volume 50 μl, 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). In Figure 4, column A shows the 100 base pair ladder, column B shows the cdc25B(976-1773) PCR product and 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 INVαF 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 (Pharmacia Biotech®) 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 MgCl2 and 50 mM NaCl, followed by digestion with Bam HI at 37°C in 50 mM Tris-HCI (pH 8.0), 10 mM MgCl2 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 μg/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:
A. PGEX-5X-3 alone / representing a no insert control =250 ng PGEX-5X-3, Bam Hl/Xho I digested
1 Unit T4 DNA ligase (Gibco BRL®), IX ligase buffer (Gibco BRL®) 50 mM Tris-HCI (pH 7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT 5% (w/v) polyethylene glycol - 8000, 1 mM dATP (Gibco BRL®) Sterile H20 to give total volume 10 μl, B. PGEX-5X-3/cdc25B (302-566) insert
=250 ng PGEX-5X-3, Bam Hl/Xho I digested =100 ng 0.8 Kb cdc25B minimal domain from TA cloning,
Bam Hl/Xho I digested and gel purified 1 Unit T4 DNA ligase (Gibco BRL®), IX ligase buffer (Gibco BRL®) 1 mM dATP (Gibco BRL®), Sterile H20 to give total volume 10 μl
Both reactions are ligated overnight at 15°C
Transformation of E. coli Strain JM109 The transformation reaction is conducted as follows:
1. 5 μl ligation reaction is added to 100 μl Promega® JM109 competent E. coli; this suspension is incubated on ice for 1 hour.
2. Incubate at 42°C for 90 seconds.
3. Cool on ice 1 minute.
4. Add 250 μl LB medium. Incubate with shaking for 1 hour.
5. Plate 10, 100 and 200 μl on LB + Agar + 100 μg/ml ampicillin® plates. The agar plates spread with the transformation mixture are incubated overnight at
37°C Colonies are abundant on PGEX-5X-3/cdc25B insert plates and sparse on control plates. For our example, sixteen colonies were picked (1-2 controls; 3-16 cdc25B insert) and grown overnight in 5 ml cultures of LB + 100 μg/ml ampicillin®.
Miniprep Analysis DNA is purified from 1-12 cultures using 1.5 ml of culture in a Bio 101®
RPM® kit. The DNA is eluted in 50 μl sterile H20, and digested with Bam HI and Xho I simultaneously in React 2 buffer at 37°C for 1 hour. Samples are run on a 0.8% agarose gel in IX TAE buffer containing 0.5 μg/ml EtBr. In our analysis, samples 9-12 all yielded an insert of the correct size (=0.8 Kb). See Figure 5, an agarose gel electrophoresis of plasmid mini preps obtained from transformed JM109 E coli. 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.
Purification of 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. A factor Xa cleavage site (IEGR) is situated immediately after the GST polypeptide, and prior to the beginning of the cdc25B sequence. Two 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 cdc25B protein using factor Xa protease.
Specific description of the procedure: i) Methodology: Two liters of JS. coli cell paste prepared by the expression system defined above is lysed in the presence of 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). This procedure is analogous to an earlier method esc e y ar e a . or a yeas -c c runca e pro e n. e lysate is centrifuged to pellet cell debris and insoluble protein, and the supernatant is collected. 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). 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-GI-cdc25B (302-566) is incubated with equilibration buffer containing factor Xa (50 mM Tris HCI, 100 mM NaCl, 1 mM CaCl2, 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). To gauge the amount of time required for the digestion process, at various timed intervals 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. The amount of inhibitor required to completely inactivate all pro ease s e erm ne us ng an assay or e pro ease. ii) Demonstration of homogeneity was accomplished by SDS polyacrylamide gel electrophoresis, C4 reverse phase HPLC, N-terminal sequencing, mass spectroscopy, and Western blotting. Validation of the sequence was completed by N- terminal sequencing, mass spectroscopy, C4 reverse phase HPLC, and Western blotting using a commercially available antibody versus the C-terminus of cdc25B. Both the fusion protein and the minimal domain of cdc25B (356-556) were shown to be enzymatically active as a phosphatase against p-nitrophenylphosphate (PNPP).
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.
Introduction . 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.
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, MA. 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. Experimental Approach. Software tools were used to obtain a tentative sequence alignment of cdc25B with other protein tyrosine phosphatases. The alignment of a structure between the PTPases gave the best correlation with a segment consisting of a region in the PTPases that is similar to the catalytic domain of cdc25B. While the sequence similarity is very low, we did see a reasonable sequence correlation factor between the VHR sequence and the cdc25B minimal domain. Based on this sequence alignment, 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. Using this model, we could further truncate the minimal domain by removing potential random coil regions of the structure that are needed to connect domains (i.e., begin with residue His364 and end with residue Arg529. In following this model, we propose that if a domain over this region is expressed and purified it would be a significantly smaller domain and would be much more amenable for NMR studies, and would also remove what is proposed to be floppy regions that may adversely effect the crystallization studies. Materials and Methods. Materials. Glutathione Sepharose 4B and Q fast flow ion exchange matrix were obtained from Pharmacia Biotech, Factor Xa was purchased from Boehringer Mannheim. The colorimetric substrate, p-Nitrophenyl phosphate (PNPP) was obtained from Sigma Diagnostics.
Original Substrate. 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®).
PCR Cloning. A defined region of cdc25B was desired, requiring the isolation of a section of the cdc25B cDNA (residues 1162 to 1659) which codes for amino acid residues 364 to 529. To amplify only this sequence, the following DNA primers, listed in a 5'->3' orientation were prepared/obtained from 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 μM dNTPs, IX PCR Buffer [(Perkin-Elmer GeneAmp®), 1.5 mM MgCl2, 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. 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 INVαF' cells. 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)®.
Ligation of Bam HI I Xho I 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 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 μg/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-HCI (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 μl.
B. PGEX-5X-3/cdc25B insert. A mixture of =100 ng PGEX-5X-3, which was Bam HI /EcoRI digested, =100 ng cdc25B minimal domain from the TA cloning experiment (Bam Hl/Xho 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 μl. Both ligation reactions were allowed to proceed overnight at 15°C
Transformation of E. coli Strain JM109. 5 μl of the ligation reaction was added to 100 μl 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 μl 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 μl aliquots onto (1%) 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 picked and grown overnight in 5 ml cultures of LB + 100 μg/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 to identify correct clones. DNA sequencing showed the residue sequences were as predicted.
Expression of the human recombinant truncated cdc25B nrotein (GST-cdc25B (364-5291') in E. coli. Seed fermentation: E. coli was inoculated into 100 ml vols. of M9 medium containing thiamin, at 5μg 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
The production fermentation continued at 200 rpm for an additional 3.5 hr until the turbidity reached about 3.0 when harvest was done by centrifugation. Eighty flasks were fermented to achieve an 8L production batch. M9 medium contains dibasic sodium phosphate, 6g; monobasic potassium phosphate, 3g; NaCl, 0.5g, and ammonium chloride 1 g, per L of deionized water. One hundred ml vols. of M9 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 M9 was completed through the aseptic addition of filtered sterilized IM MgSO4, 2ml; 20% glucose, 20 ml, IM CaCl2, 0.1 ml and thiamine, 5 μg.
Affinity purification of GST-cdc25B (VHR-like domain) 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 lOminutes. Supernatant was obtained by centrifugation at 14k RPM using an SS-34 rotor for 35minutes 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-cdc25B (364-529) was incubated with equ rat on u er con a n ng ac or a m r s , m a , m CaC12, pH 8.0) in a manner analogous to that described above. 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 HCI, 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 cdc25B, 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. Electrophoresis and Western Blotting. SDS gel electrophoresis were performed according to Laemmli, supra. 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. For the latter, 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 NaN3 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(Fc) Alkaline Phosphatase conjugate, Promega®) at 1:1000 dilution. 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.
Enzvme assay and Kinetic Analyses. 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 . t 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 Jζ^ and Vmaχ (specific activity), multiple pNPP concentrations are used at a constant enzyme concentration. Rates at each concentration of substrate are determined and the "K^ and Vmaχ calculated from line fitting to a Michaelis Men ten 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 cdc25B 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. Physical characteristics of the purified truncated recombinant human cdc25B
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).
Characterization of the purified truncated recombinant human cdc25B bv enzvmatic activity. Both the fusion protein and the minimal domain of cdc25B (364- 529) were shown to be enzymatically active as a phosphatase against p- nitrophenylphosphate (PNPP). 1^ and Vmaχ determinations of the GST-free catalytic domain of cdc25B were made using the assay conditions defined previously as well as by a 96 well plate assay method. Enzymatically, the catalytic domain represented by residues 364-529 is active as an enzyme with PNPP as substrate, giving an average 1^ of about 28 mM and an average Vmaχ of about 250 nmoles/min/mg.
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.
MEVPQPEPAP GSALSPAGVC GGAQRPGHLP GLLLGSHGLL GSPVRAAASS 101 SPSPMDPHMA EQTFEQAIQA ASRIIRNEQF AIRRFQSMPV RLLGHSPVLR
151 NITNSQAPDG RRKSEAGSGA ASSSGEDKEN DGFVFKMPWK PTHPSSTHAL 201 AEWASRREAF AQRPSSAPDL MCLSPDRKME VEELSPLALG RFSLTPAEGD 251 TEEDDGFVDI LESDLKDDDA VPPGMESLIS APLVKTLEKE EEKDLVMYSK
301 CQRLFRSPSM PCSVIRPILK RLERPQDRDT PVQNKRRRSV TPPEEQQEAE
351 EPKARVLRSK SLCHDEIENL LDSDHRELIG DYSKAFLLQT VDGKHQDLKY
401 ISPETMVALL TGKFSNIVDK FVIVDCRYPY EYEGGHIKTA VNLPT.F.RDAF
451 SFLLKSPIAP CSLDKRVTLI FHCEFSSERG PRMCRFIRER DRAVNDYPST, 501 YYPEMYILKG GYKEFFPQHP NFCEPQDYRP MNHEAFKDEL KTFRLKTRSW
551 AGERSRRELC SRLQDQ
VHR-Like-CHART B The amino acid sequence of human VHR phosphatase. Reference: A novel dual specificity phosphatase induced by serum stimulation and heat shock, Ishibashi T., Bottaro D.P., Michieli P., Kelley C.A., Aaronson S.A., J. Biol. Chem., vol 269(47), pp. 29897-902 (1994). SEQ. ID. NO. 25.
1 MSGSFELSVQ DLNDLLSDGS GCYSLPSQPC NEVTPRIYVG NASVAQDIPK 51 LQKLGITHVL NAAEGRSFMH VNTNANFYKD SGITYLGIKA NDTQEFNLSA
101 YFERAADFID QALAQKNGRV LVHCREGYSR SPTLVIAYLM MRQKMDVKSA
151 LSIVRQNREI GPNDGFLAQL CQLNDRLAKE GKLKP
(1) GENERAL INFORMATION:
(i) APPLICANT: Pharmacia & Upjohn Company
(ii) TITLE OF INVENTION: Catalytic Macro Molecules Having DCD25B Like Activity
(iii) NUMBER OF SEQUENCES: 45 (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: Patentln 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
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 616-833-7914 (B) TELEFAX: 616-833-6897
(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2890 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:l:
GCCAGCTGTG CCGGCGTTTG TTGGCTGCCC TGCGCCCGGC CCTCCAGCCA GCCTTCTGCC 60
GGCCCCGCCG CGATGGAGGT GCCCCAGCCG GAGCCCGCGC CAGGCTCGGC TCTCAGTCCA 120 GCAGGCGTGT GCGGTGGCGC CCAGCGTCCG GGCCACCTCC CGGGCCTCCT GCTGGGATCT 180
CATGGCCTCC TGGGGTCCCC GGTGCGGGCG GCCGCTTCCT CGCCGGTCAC CACCCTCACC 240
CAGACCATGC ACGACCTCGC CGGGCTCGGC AGCCGCAGCC GCCTGACGCA CCTATCCCTG 300 CTCTGCATGG ATTCCCCCAG CCCTATGGAC CCCCACATGG CGGAGCAGAC GTTTGAACAG 420 GCCATCCAGG CAGCCAGCCG GATCATTCGA AACGAGCAGT TTGCCATCAG ACGCTTCCAG 480
TCTATGCCGG TGAGGCTGCT GGGCCACAGC CCCGTGCTTC GGAACATCAC CAACTCCCAG 540
GCGCCCGACG GCCGGAGGAA GAGCGAGGCG GGCAGTGGAG CTGCCAGCAG CTCTGGGGAA 600
TCCCAGCTCC ACCCATGCTC TGGCAGAGTG GGCCAGCCGC AGGGAAGCCT TTGCCCAGAG 660
ACCCAGCTCG GCCCCCGACC TGATGTGTCT CAGTCCTGAC CGGAAGATGG AAGTGGAGGA 720 GCTCAGCCCC CTGGCCCTAG GTCGCTTCTC TCTGACCCCT GCAGAGGGGG ATACTGAGGA 780
AGATGATGGA TTTGTGGACA TCCTAGAGAG TGACTTAAAG GATGATGATG CAGTTCCCCC 840
AGGCATGGAG AGTCTCATTA GTGCCCCACT GGTCAAGACC TTGGAAAAGG AAGAGGAAAA 900
GGACCTCGTC ATGTACAGCA AGTGCCAGCG GCTCTTCCGC TCTCCGTCCA TGCCCTGCAG 960
CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG GACAGGGACA CGCCCGTGCA 1020 GAATAAGCGG AGGCGGAGCG TGACCCCTCC TGAGGAGCAG CAGGAGGCTG AGGAACCTAA 1080
AGCCCGCGTC CTCCGCTCAA AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG 1140
TGACCACCGA GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG 1200
AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT TGACGGGCAA 1260
GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC AGATACCCCT ATGAATATGA 1320 AGGCGGGCAC ATCAAGACTG CGGTGAACTT GCCCCTGGAA CGCGACGCCG AGAGCTTCCT 1380
ACTGAAGAGC CCCATCGCGC CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG 1440
TGAATTCTCA TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC 1500
TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG GCGGCTACAA 1560
GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG GACTACCGGC CCATGAACCA 1620 CGAGGCCTTC AAGGATGAGC TAAAGACCTT CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA 1680
GCGGAGCCGG CGGGAGCTCT GTAGCCGGCT GCAGGACCAG TGAGGGGCCT GCGCCAGTCC 1740
TGCTACCTCC CTTGCCTTTC GAGGCCTGAA GCCAGCTGCC CTATGGGCCT GCCGGGCTGA 1800
GGGCCTGCTG GAGGCCTCAG GTGCTGTCCA TGGGAAAGAT GGTGTGGTGT CCTGCCTGTC 1860
TGCCCCAGCC CAGATTCCCC TGTGTCATCC CATCATTTTC CATATCCTGG TGCCCCCCAC 1920 CCCTGGAAGA GCCCAGTCTG TTGAGTTAGT TAAGTTGGGT TAATACCAGC TTAAAGGCAG 1980
TATTTTGTGT CCTCCAGGAG CTTCTTGTTT CCTTGTTAGG GTTAACCCTT CATCTTCCTG 2040
TGTCCTGAAA CGCTCCTTTG TGTGTGTGTC AGCTGAGGCT GGGGAGAGCC GTGGTCCCTG 2100
AGGATGGGTC AGAGCTAAAC TCCTTCCTGG CCTGAGAGTC AGCTCTCTGC CCTGTGTACT 2160
TCCCGGGCCA GGGCTGCCCC TAATCTCTGT AGGAACCGTG GTATGTCTGC CATGTTGCCC 2220 CTTTCTCTTT TCCCCTTTCC TGTCCCACCA TACGAGCACC TCCAGCCTGA ACAGAAGCTC 2280
TTACTCTTTC CTATTTCAGT GTTACCTGTG TGCTTGGTCT GTTTGACTTT ACGCCCATCT 2340
CAGGACACTT CCGTAGACTG TTTAGGTTCC CCTGTCAAAT ATCAGTTACC CACTCGGTCC 2400 AGGCCTGAAT CATGAGCCTG CTGGAAGCCC AGCCCCTACT GCTGTGAACC CTGGGGCCTG 2520
ACTGCTCAGA ACTTGCTGCT GTCTTGTTGC GGATGGATGG AAGGTTGGAT GGATGGGTGG 2580
ATGGCCGTGG ATGGCCGTGG ATGCGCAGTG CCTTGCATAC CCAAACCAGG TGGGAGCGTT 2640
TTGTTGAGCA TGACACCTGC AGCAGGAATA TATGTGTGCC TATTTGTGTG GACAAAAATA 2700
TTTACACTTA GGGTTTGGAG CTATTCAAGA GGAAATGTCA CAGAAGCAGC TAAACCAAGG 2760
ACTGAGCACC CTCTGGATTC TGAATCTCAA GATGGGGGCA GGGCTGTGCT TGAAGGCCCT 2820 GCTGAGTCAT CTGTTAGGGC CTTGGTTCAA TAAAGCACTG AGCAAGTTGA GAAAAAAAAA 2880
AAAAAAAAAA 2890 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 566 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: 2:
Met Glu Val Pro Gin Pro Glu Pro Ala Pro Gly Ser Ala Leu Ser Pro 1 5 10 15 Ala Gly Val Cys Gly Gly Ala Gin Arg Pro Gly His Leu Pro Gly Leu
20 25 30
Leu Leu Gly Ser His Gly Leu Leu Gly Ser Pro Val Arg Ala Ala Ala 35 40 45
Ser Ser Pro Val Thr Thr Leu Thr Gin Thr Met His Asp Leu Ala Gly 50 55 60
Leu Gly Ser Arg Ser Arg Leu Thr His Leu Ser Leu Ser Arg Arg Ala 65 70 75 80
Ser Glu Ser Ser Leu Ser Ser Glu Ser Ser Glu Ser Ser Asp Ala Gly 85 90 95
Leu Cys Met Asp Ser Pro Ser Pro Met Asp Pro His Met Ala Glu Gin 100 105 110
Thr Phe Glu Gin Ala lie Gin Ala Ala Ser Arg lie lie Arg Asn Glu 115 120 125
Gin Phe Ala lie Arg Arg Phe Gin Ser Met Pro Val Arg Leu Leu Gly 130 135 140
His Ser Pro Val Leu Arg Asn lie Thr Asn Ser Gin Ala Pro Asp Gly 145 150 155 160
Arg Arg Lys Ser Glu Ala Gly Ser Gly Ala Ala Ser Ser Ser Gly Glu
165 170 175 Asp Lys Glu Asn Asp G y P e Va P e Lys e ro rp ys ro r
180 185 190
His Pro Ser Ser Thr His Ala Leu Ala Glu Trp Ala Ser Arg Arg Glu 5. 195 200 205
Ala Phe Ala Gin Arg Pro Ser Ser Ala Pro Asp Leu Met Cys Leu Ser 210 -215 220 0 Pro Asp Arg Lys Met Glu Val Glu Glu Leu Ser Pro Leu Ala Leu Gly 225 230 235 240
Arg Phe Ser Leu Thr Pro Ala Glu Gly Asp Thr Glu Glu Asp Asp Gly 245 250 255 5
Phe Val Asp lie Leu Glu Ser Asp Leu Lys Asp Asp Asp Ala Val Pro 260 265 270
Pro Gly Met Glu Ser Leu lie Ser Ala Pro Leu Val Lys Thr Leu Glu 0 275 280 285
Lys Glu Glu Glu Lys Asp Leu Val Met Tyr Ser Lys Cys Gin Arg Leu 290 295 300 5 Phe Arg Ser Pro Ser Met Pro Cys Ser Val lie Arg Pro lie Leu Lys 305 310 315 320
Arg Leu Glu Arg Pro Gin Asp Arg Asp Thr Pro Val Gin Asn Lys Arg 325 330 335 0
Arg Arg Ser Val Thr Pro Pro Glu Glu Gin Gin Glu Ala Glu Glu Pro 340 345 350
Lys Ala Arg Val Leu Arg Ser Lys Ser Leu Cys His Asp Glu lie Glu 5 355 360 365
Asn Leu Leu Asp Ser Asp His Arg Glu Leu lie Gly Asp Tyr Ser Lys
370 375 380 0 Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His Gin Asp Leu Lys Tyr
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
Glu Gly Gly His He Lys Thr Ala Val Asn Leu Pro Leu Glu Arg Asp 0 435 440 445
Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala Pro Cys Ser Leu Asp
450 455 460 5 Lys Arg Val He Leu He Phe His Cys Glu Phe Ser Ser Glu Arg Gly
465 470 475 480
Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp Arg Ala Val Asn Asp 485 490 495 0
Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys Gly Gly Tyr
500 505 510
Lys Glu Phe Phe Pro Gin His Pro Asn Phe Cys Glu Pro Gin Asp Tyr 515 520 525
Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr Phe Arg 530 535 540 eu ys r rg er rp a y u rg er rg Arg u Leu Cys 545 550 555 560
Ser Arg Leu Gin Asp Gin 565
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 798 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: 3: CAGCGGCTCT TCCGCTCTCC GTCCATGCCC TGCAGCGTGA TCCGGCCCAT CCTCAAGAGG 60
CTGGAGCGGC CCCAGGACAG GGACACGCCC GTGCAGAATA AGCGGAGGCG GAGCGTGACC 120
CCTCCTGAGG AGCAGCAGGA GGCTGAGGAA CCTAAAGCCC GCGTCCTCCG CTCAAAATCA 180
CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
TACTCTAAGG CCTTCCTCCT ACAGACAGTA GACGGAAAGC ACCAAGACCT CAAGTACATC 300 TCACCAGAAA CGATGGTGGC CCTATTGACG GGCAAGTTCA GCAACATCGT GGATAAGTTT 360
GTGATTGTAG ACTGCAGATA CCCCTATGAA TATGAAGGCG GGCACATCAA GACTGCGGTG 420
AACTTGCCCC TGGAACGCGA CGCCGAGAGC TTCCTACTGA AGAGCCCCAT CGCGCCCTGT 480
AGCCTGGACA AGAGAGTCAT CCTCATTTTC CACTGTGAAT TCTCATCTGA GCGTGGGCCC 540
CGCATGTGCC GTTTCATCAG GGAACGAGAC CGTGCTGTCA ACGACTACCC CAGCCTCTAC 600 TACCCTGAGA TGTATATCCT GAAAGGCGGC TACAAGGAGT TCTTCCCTCA GCACCCGAAC 660
TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
ACCTTCCGCC TCAAGACTCG CAGCTGGGCT GGGGAGCGGA GCCGGCGGGA GCTCTGTAGC 780
CGGCTGCAGG ACCAGTGA 798 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 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: 4: n rg e rg e 1 5 10 15
He Leu Lys Arg Leu Glu Arg Pro Gin Asp Arg Asp Thr Pro Val Gin 20 25 30
Asn Lys Arg Arg Arg Ser Val Thr Pro Pro Glu Glu Gin Gin Glu Ala 35 40 45 Glu Glu Pro Lys Ala Arg Val Leu Arg Ser Lys Ser Leu Cys His Asp
50 55 60
Glu He Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu He Gly Asp 65 70 75 80
Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His Gin Asp 85 90 95
Leu Lys Tyr He Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys 100 105 110
Phe Ser Asn He Val Asp Lys Phe Val He Val Asp Cys Arg Tyr Pro 115 120 125
Tyr Glu Tyr Glu Gly Gly His He Lys Thr Ala Val Asn Leu Pro Leu 130 135 140
Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala Pro Cys 145 150 155 160
Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe Ser Ser 165 170 175
Glu Arg Gly Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp Arg Ala 180 185 190
Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys 195 200 205
Gly Gly Tyr Lys Glu Phe Phe Pro Gin His Pro Asn Phe Cys Glu Pro 210 215 220
Gin Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys
225 230 235 240
Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser Arg Arg
245 250 255
Glu Leu Cys Ser Arg Leu Gin Asp Gin 260 265
(2) INFORMATION FOR SEQ ID NO:5 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 804 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:5: AAGAGGCTGG AGCGGCCCCA GGACAGGGAC ACGCCCGTGC AGAATAAGCG GAGGCGGAGC 120
GTGACCCCTC CTGAGGAGCA GCAGGAGGCT GAGGAACCTA AAGCCCGCGT CCTCCGCTCA 180
AAATCACTGT GTCACGATGA GATCGAGAAC CTCCTGGACA GTGACCACCG AGAGCTGATT 240
GGAGATTACT CTAAGGCCTT CCTCCTACAG ACAGTAGACG GAAAGCACCA AGACCTCAAG 300
TACATCTCAC CAGAAACGAT GGTGGCCCTA TTGACGGGCA AGTTCAGCAA CATCGTGGAT 360
AAGTTTGTGA TTGTAGACTG CAGATACCCC TATGAATATG AAGGCGGGCA CATCAAGACT 420 GCGGTGAACT TGCCCCTGGA ACGCGACGCC GAGAGCTTCC TACTGAAGAG CCCCATCGCG 480
CCCTGTAGCC TGGACAAGAG AGTCATCCTC ATTTTCCACT GTGAATTCTC ATCTGAGCGT 540
GGGCCCCGCA TGTGCCGTTT CATCAGGGAA CGAGACCGTG CTGTCAACGA CTACCCCAGC 600
CTCTACTACC CTGAGATGTA TATCCTGAAA GGCGGCTACA AGGAGTTCTT CCCTCAGCAC 660
CCGAACTTCT GTGAACCCCA GGACTACCGG CCCATGAACC ACGAGGCCTT CAAGGATGAG 720 CTAAAGACCT TCCGCCTCAA GACTCGCAGC TGGGCTGGGG AGCGGAGCCG GCGGGAGCTC 780
TGTAGCCGGC TGCAGGACCA GTGA 804
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 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: 6:
Gly He Gin Arg Leu Phe Arg Ser Pro Ser Met Pro Cys Ser Val He 1 5 10 15 Arg Pro He Leu Lys Arg Leu Glu Arg Pro Gin Asp Arg Asp Thr Pro
20 25 30
Val Gin Asn Lys Arg Arg Arg Ser Val Thr Pro Pro Glu Glu Gin Gin 35 40 45
Glu Ala Glu Glu Pro Lys Ala Arg Val Leu Arg Ser Lys Ser Leu Cys 50 55 60
His Asp Glu He Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu He 65 70 75 80
Gly Asp Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His 85 90 95 Gin Asp Leu Lys Tyr He Ser Pro Glu Thr Met Val Ala Leu Leu Thr
100 105 110
Gly Lys Phe Ser Asn He Val Asp Lys Phe Val He Val Asp Cys Arg 115 120 125 Tyr Pro Tyr Glu Tyr Glu Gly G y H s e Lys r A a Va Asn Leu 130 135 140
Pro Leu Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala 145 150 155 160
Pro Cys Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe 165 170 175 Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp
180 185 190
Arg Ala Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He 195 200 205
Leu Lys Gly Gly Tyr Lys Glu Phe Phe Pro Gin His Pro Asn Phe Cys 210 215 220
Glu Pro Gin Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu 225 230 235 240
Leu Lys Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser 245 250 255 Arg Arg Glu Leu Cys Ser Arg Leu Gin Asp Gin
260 265
(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 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:
ATCGAAGGTC GTGGGATCCA GCGGCTCTTC CGCTCTCCGT CCATGCCCTG CAGCGTGATC 60
CGGCCCATCC TCAAGAGGCT GGAGCGGCCC CAGGACAGGG ACACGCCCGT GCAGAATAAG 120 CGGAGGCGGA GCGTGACCCC TCCTGAGGAG CAGCAGGAGG CTGAGGAACC TAAAGCCCGC 180
GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC 240
CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC 300
CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC 360
AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG 420 CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG 480
AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC 540
TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC 600
GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC 660
TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 720 A TC ACTCGCA T GCT G GGAGCGGAGC 780 CGGCGGGAGC TCTGTAGCCG GCTGCAGGAC CAGTGA 816 (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ϋi) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
He Glu Gly Arg Gly He Gin Arg Leu Phe Arg Ser Pro Ser Met Pro 1 5 10 15
Cys Ser Val He Arg Pro He Leu Lys Arg Leu Glu Arg Pro Gin Asp 20 25 30
Arg Asp Thr Pro Val Gin Asn Lys Arg Arg Arg Ser Val Thr Pro Pro 35 40 45
Glu Glu Gin Gin Glu Ala Glu Glu Pro Lys Ala Arg Val Leu Arg Ser 50 55 60 Lys Ser Leu Cys His Asp Glu He Glu Asn Leu Leu Asp Ser Asp His 65 70 75 80
Arg Glu Leu He Gly Asp Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val 85 90 95
Asp Gly Lys His Gin Asp Leu Lys Tyr He Ser Pro Glu Thr Met Val 100 105 110
Ala Leu Leu Thr Gly Lys Phe Ser Asn He Val Asp Lys Phe Val He 115 120 125
Val Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly His He Lys Thr 130 135 140
Ala Val Asn Leu Pro Leu Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys 145 150 155 160
Ser Pro He Ala Pro Cys Ser Leu Asp Lys Arg Val He Leu He Phe 165 170 175
His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Phe He 180 185 190
Arg Glu Arg Asp Arg Ala Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro 195 200 205
Glu Met Tyr He Leu Lys Gly Gly Tyr Lys Glu Phe Phe Pro Gin His 210 215 220 Pro Asn Phe Cys Glu Pro Gin Asp Tyr Arg Pro Met Asn His Glu Ala 225 230 235 240
Phe Lys Asp Glu Leu Lys Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala 245 250 255 y u rg er rg rg u er 260 265 270
(2) INFORMATION FOR SEQ ID NO:9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 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:9:
CGATGTATGC GGTAAACCGC AGGCATTAG 29
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 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 : 10 :
Val Leu Arg Ser Lys Ser Leu Cys His Asp Glu He Glu Asn Leu Leu 1 5 10 15 Asp Ser Asp His Arg Glu Leu He Gly Asp Tyr Ser Lys Ala Phe Leu
20 25 30
Leu Gin Thr Val Asp Gly Lys His Gin Asp Leu Lys Tyr He Ser Pro
35 40 45
Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe Ser Asn He Val Asp 50 55 60
Lys Phe Val He Val Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly 65 70 75 80
His He Lys Thr Ala Val Asn Leu Pro Leu Glu Arg Asp Ala Glu Ser
85 90 95
Phe Leu Leu Lys Ser Pro He Ala Pro Cys Ser Leu Asp Lys Arg Val
100 105 110
He Leu He Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met 115 120 125
Cys Arg Phe He Arg Glu Arg Asp Arg Ala Val Asn Asp Tyr Pro Ser 130 135 140
Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys Gly Gly Tyr Lys Glu Phe Phe Pro Gin His Pro Asn Phe Cys Glu Pro Gin Asp Tyr Arg Pro Met 165 170 175
Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr Phe Arg Leu Lys Thr 180 185 190
Arg Ser Trp Ala Gly Glu Arg Ser Arg Arg Glu Leu Cys Ser Arg Leu 195 200 205
Gin Asp Gin 210 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 798 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: 11:
CAGCGGCTCT TCCGCTCTCC GTCCATGCCC TGCAGCGTGA TCCGGCCCAT CCTCAAGAGG 60 CTGGAGCGGC CCCAGGACAG GGACACGCCC GTGCAGAATA AGCGGAGGCG GAGCGTGACC 120
CCTCCTGAGG AGCAGCAGGA GGCTGAGGAA ATTGAAGGCC GCGTCCTCCG CTCAAAATCA 180
CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
TACTCTAAGG CCTTCCTCCT ACAGACAGTA GACGGAAAGC ACCAAGACCT CAAGTACATC 300
TCACCAGAAA CGATGGTGGC CCTATTGACG GGCAAGTTCA GCAACATCGT GGATAAGTTT 360 GTGATTGTAG ACTGCAGATA CCCCTATGAA TATGAAGGCG GGCACATCAA GACTGCGGTG 420
AACTTGCCCC TGGAACGCGA CGCCGAGAGC TTCCTACTGA AGAGCCCCAT CGCGCCCTGT 480
AGCCTGGACA AGAGAGTCAT CCTCATTTTC CACTGTGAAT TCTCATCTGA GCGTGGGCCC 540
CGCATGTGCC GTTTCATCAG GGAACGAGAC CGTGCTGTCA ACGACTACCC CAGCCTCTAC 600
TACCCTGAGA TGTATATCCT GAAAGGCGGC TACAAGGAGT TCTTCCCTCA GCACCCGAAC 660 TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
ACCTTCCGCC TCAAGACTCG CAGCTGGGCT GGGGAGCGGA GCCGGCGGGA GCTCTGTAGC 780
CGGCTGCAGG ACCAGTGA 798 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 798 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA ( ) HYPOTH : (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CAGCGGCTCT TCCGCTCTCC GTCCATGCCC TGCAGCGTGA TCCGGCCCAT CCTCAAGAGG 60
CTGGAGCGGC CCCAGGACAG GGACACGCCC GTGCAGAATA AGCGGAGGCG GAGCGTGACC 120
CCTCCTGAGG AGCAGCAGGA GGCTGAGGAA CCTAAAGCCC GCGTCCTCCG CTCAAAATCA 180 CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
TACTCTAAGG CCTTCCTCCT ACAGACAGTA GACGGAAAGC ACCAAGACCT CAAGTACATC 300
TCACCAGAAA CGATGGTGGC CCTATTGACG GGCAAGTTCA GCAACATCGT GGATAAGTTT 360
GTGATTGTAG ACTGCAGATA CCCCTATGAA TATGAAGGCG GGCACATCAA GACTGCGGTG 420
AACTTGCCCC TGGAACGCGA CGCCGAGAGC TTCCTACTGA AGAGCCCCAT CGCGCCCTGT 480 AGCCTGGACA AGAGAGTCAT CCTCATTTTC CACTGTGAAT TCTCATCTGA GCGTGGGCCC 540
CGCATGTGCC GTTTCATCAG GGAACGAGAC CGTGCTGTCA ACGACTACCC CAGCCTCTAC 600
TACCCTGAGA TGTATATCCT GAAAGGCGGC TACAAGGAGT TCTTCCCTCA GCACCCGAAC 660
TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
ACCTTCCGCC TCAAGACTCG CAGCTGGGCT GGGGAGCGGA GCAAGAAGGA GCTCTGTAGC 780 CGGCTGCAGG ACCAGTGA 798
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 798 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: CAGCGGCTCT TCCGCTCTCC GTCCATGCCC TGCAGCGTGA TCCGGCCCAT CCTCAAGAGG 60
CTGGAGCGGC CCCAGGACAG GGACACGCCC GTGCAGAATA AGCGGAGGCG GAGCGTGACC 120
CCTCCTGAGG AGCAGCAGGA GGCTGAGGAA ATTGAAGGCC GCGTCCTCCG CTCAAAATCA 180
CTGTGTCACG ATGAGATCGA GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT 240
TACTCTAAGG CCTTCCTCCT ACAGACAGTA GACGGAAAGC ACCAAGACCT CAAGTACATC 300 TCACCAGAAA CGATGGTGGC CCTATTGACG GGCAAGTTCA GCAACATCGT GGATAAGTTT 360
GTGATTGTAG ACTGCAGATA CCCCTATGAA TATGAAGGCG GGCACATCAA GACTGCGGTG 420
AACTTGCCCC TGGAACGCGA CGCCGAGAGC TTCCTACTGA AGAGCCCCAT CGCGCCCTGT 480 AGCCTGGACA A GAGTCAT CCTCATTTT A TGTGAAT TCTCATCTGA CGTGGGCCC 540
CGCATGTGCC GTTTCATCAG GGAACGAGAC CGTGCTGTCA ACGACTACCC CAGCCTCTAC 600
TACCCTGAGA TGTATATCCT GAAAGGCGGC TACAAGGAGT TCTTCCCTCA GCACCCGAAC 660
TTCTGTGAAC CCCAGGACTA CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG 720
ACCTTCCGCC TCAAGACTCG CAGCTGGGCT GGGGAGCGGA GCAAGAAGGA GCTCTGTAGC 780
CGGCTGCAGG ACCAGTGA 798 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 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: 14:
Gin Arg Leu Phe Arg Ser Pro Ser Met Pro Cys Ser Val He Arg Pro 1 5 10 15
He Leu Lys Arg Leu Glu Arg Pro Gin Asp Arg Asp Thr Pro Val Gin 20 25 30
Asn Lys Arg Arg Arg Ser Val Thr Pro Pro Glu Glu Gin Gin Glu Ala 35 40 45
Glu Glu He Glu Gly Arg Val Leu Arg Ser Lys Ser Leu Cys His Asp 50 55 60
Glu He Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu He Gly Asp 65 70 75 80
Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His Gin Asp 85 90 95
Leu Lys Tyr He Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys 100 105 110
Phe Ser Asn He Val Asp Lys Phe Val He Val Asp Cys Arg Tyr Pro 115 120 125
Tyr Glu Tyr Glu Gly Gly His He Lys Thr Ala Val Asn Leu Pro Leu 130 135 140
Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala Pro Cys 145 150 155 160
Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe Ser Ser 165 170 175
Glu Arg Gly Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp Arg Ala 180 185 190
Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys 195 200 205 y y yr 210 215 220
Gin Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys 225 230 235 240
Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser Arg Arg 245 250 255 Glu Leu Cys Ser Arg Leu Gin Asp Gin
260 265
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 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:15:
Gin Arg Leu Phe Arg Ser Pro Ser Met Pro Cys Ser Val He Arg Pro 1 5 10 15
He Leu Lys Arg Leu Glu Arg Pro Gin Asp Arg Asp Thr Pro Val Gin 20 25 30
Asn Lys Arg Arg Arg Ser Val Thr Pro Pro Glu Glu Gin Gin Glu Ala 35 40 45
Glu Glu Pro Lys Ala Arg Val Leu Arg Ser Lys Ser Leu Cys His Asp 50 55 60
Glu He Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu He Gly Asp 65 70 75 80
Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His Gin Asp 85 90 95
Leu Lys Tyr He Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys 100 105 110
Phe Ser Asn He Val Asp Lys Phe Val He Val Asp Cys Arg Tyr Pro 115 120 125 Tyr Glu Tyr Glu Gly Gly His He Lys Thr Ala Val Asn Leu Pro Leu 130 135 140
Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala Pro Cys
145 150 155 160
Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe Ser Ser
165 170 175
Glu Arg Gly Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp Arg Ala 180 185 190
Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys
195 200 205 y yr ys u e e ro n s ro sn e ys u ro 210 215 220
Gin Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys 225 230 235 240
Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser Lys Lys 245 250 255 Glu Leu Cys Ser Arg Leu Gin Asp Gin
260 265
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 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:16:
Gin Arg Leu Phe Arg Ser Pro Ser Met Pro Cys Ser Val He Arg Pro 1 5 10 15
He Leu Lys Arg Leu Glu Arg Pro Gin Asp Arg Asp Thr Pro Val Gin 20 25 30
Asn Lys Arg Arg Arg Ser Val Thr Pro Pro Glu Glu Gin Gin Glu Ala 35 40 45
Glu Glu He Glu Gly Arg Val Leu Arg Ser Lys Ser Leu Cys His Asp 50 55 60
Glu He Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu He Gly Asp 65 70 75 80
Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His Gin Asp 85 90 95
Leu Lys Tyr He Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys 100 105 110
Phe Ser Asn He Val Asp Lys Phe Val He Val Asp Cys Arg Tyr Pro 115 120 125
Tyr Glu Tyr Glu Gly Gly His He Lys Thr Ala Val Asn Leu Pro Leu 130 135 140
Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala Pro Cys 145 150 155 160
Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe Ser Ser 165 170 175
Glu Arg Gly Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp Arg Ala 180 185 190
Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys 195 200 205 G y y Tyr Lys u e e ro n s ro sn e ys u ro 210 215 220
Gin Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys 225 230 235 240
Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser Lys Lys 245 - 250 255 Glu Leu Cys Ser Arg Leu Gin Asp Gin
260 265
(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 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:17:
GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC 60
CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC 120 CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC 180
AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG 240
CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG 300
AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC 360
TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC 420 GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC 480
TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 540
TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC 600
CGG 603 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 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:18: T TCCGCT AA ACT GTGT A AT A A CCTCCTGGA CAGTGACCAC 60
CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC 120
CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC 180
AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG 240
CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG 300
AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC 360
TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC 420 GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC 4BO
TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 540
TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC 600
CGG 603 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 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: 19:
GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC 60
CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC 120 CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC 180
AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG 240
CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG 300
AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC 360
TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC 420 GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC 480
TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 540
TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC 600
AAGAAGGAGC TCTGTAGCCG GCTGCAGGAC CAGTGA 636
(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear : C (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC 60
CGAGAGCTGA TTGGAGATTA CTCTAAGGCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC 120 CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGGG CAAGTTCAGC 180
AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG 240
CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG 300
AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC 360
TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC 420 GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC 480
TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC 540
TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC 600
AAGAAGGAGC TCTGTAGCCG GCTGCAGGAC CAGTGA 636
(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 201 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 :
Val Leu Arg Ser Lys Ser Leu Cys His Asp Glu He Glu Asn Leu Leu 1 5 10 15
Asp Ser Asp His Arg Glu Leu He Gly Asp Tyr Ser Lys Ala Phe Leu
20 25 30
Leu Gin Thr Val Asp Gly Lys His Gin Asp Leu Lys Tyr He Ser Pro
35 40 45
Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe Ser Asn He Val Asp 50 55 60
Lys Phe Val He Val Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly 65 70 75 80
His He Lys Thr Ala Val Asn Leu Pro Leu Glu Arg Asp Ala Glu Ser 85 90 95
Phe Leu Leu Lys Ser Pro He Ala Pro Cys Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met 115 120 125
5.
Cys Arg Phe He Arg Glu Arg Asp Arg Ala Val Asn Asp Tyr Pro Ser 130 135 140
Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys Gly Gly Tyr Lys Glu Phe 0 145 150 155 160
Phe Pro Gin His Pro Asn Phe Cys Glu Pro Gin Asp Tyr Arg Pro Met 165 170 175 5 Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr Phe Arg Leu Lys Thr
180 185 190
Arg Ser Trp Ala Gly Glu Arg Ser Arg 195 200 0
\ 2 ) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 amino acids 5 (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: 22:
Val Leu Arg Ser Lys Ser Leu Cys His Asp Glu He Glu Asn Leu Leu 1 5 10 15
Asp Ser Asp His Arg Glu Leu He Gly Asp Tyr Ser Lys Ala Phe Leu 20 25 30
Leu Gin Thr Val Asp Gly Lys His Gin Asp Leu Lys Tyr He Ser Pro 35 40 45
Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe Ser Asn He Val Asp 50 55 60
Lys Phe Val He Val Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly 65 70 75 80
His He Lys Thr Ala Val Asn Leu Pro Leu Glu Arg Asp Ala Glu Ser 85 90 95
Phe Leu Leu Lys Ser Pro He Ala Pro Cys Ser Leu Asp Lys Arg Val 100 105 110 He Leu He Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met 115 120 125
Cys Arg Phe He Arg Glu Arg Asp Arg Ala Val Asn Asp Tyr Pro Ser 130 135 140
Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys Gly Gly Tyr Lys Glu Phe 145 150 155 160
Phe Pro Gin His Pro Asn Phe Cys Glu Pro Gin Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr Phe Arg Leu Lys Thr 180 185 190
Arg Ser Trp Ala Gly Glu Arg Ser Lys Lys Glu Leu Cys Ser Arg Leu 195 200 205
Gin Asp Gin 210
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 211 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:23: Val Leu Arg Ser Lys Ser Leu Cys His Asp Glu He Glu Asn Leu Leu
1 5 10 15
Asp Ser Asp His Arg Glu Leu He Gly Asp Tyr Ser Lys Ala Phe Leu 20 25 30
Leu Gin Thr Val Asp Gly Lys His Gin Asp Leu Lys Tyr He Ser Pro 35 40 45
Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe Ser Asn He Val Asp 50 55 60
Lys Phe Val He Val Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly 65 70 75 80
His He Lys Thr Ala Val Asn Leu Pro Leu Glu Arg Asp Ala Glu Ser 85 90 95
Phe Leu Leu Lys Ser Pro He Ala Pro Cys Ser Leu Asp Lys Arg Val
100 105 110
He Leu He Phe His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met
115 120 125
Cys Arg Phe He Arg Glu Arg Asp Arg Ala Val Asn Asp Tyr Pro Ser 130 135 140
Leu Tyr Tyr Pro Glu Met Tyr He Leu Lys Gly Gly Tyr Lys Glu Phe 145 150 155 160 Phe Pro Gin His Pro Asn Phe Cys Glu Pro Gin Asp Tyr Arg Pro Met
165 170 175
Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr Phe Arg Leu Lys Thr 180 185 190
Arg Ser Trp Ala Gly Glu Arg Ser Lys Lys Glu Leu Cys Ser Arg Leu 195 200 205
Gin Asp Gin (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 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:24:
His Asp Glu He Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu He 1 5 10 15
Gly Asp Tyr Ser Lys Ala Phe Leu Leu Gin Thr Val Asp Gly Lys His 20 25 30
Gin Asp Leu Lys Tyr He Ser Pro Glu Thr Met Val Ala Leu Leu Thr 35 40 45
Gly Lys Phe Ser Asn He Val Asp Lys Phe Val He Val Asp Cys Arg 50 55 60
Tyr Pro Tyr Glu Tyr Glu Gly Gly His He Lys Thr Ala Val Asn Leu 65 70 75 80
Pro Leu Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro He Ala 85 90 95
Pro Cys Ser Leu Asp Lys Arg Val He Leu He Phe His Cys Glu Phe 100 105 110
Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Phe He Arg Glu Arg Asp 115 120 125 Arg Ala Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr He 130 135 140
Leu Lys Gly Gly Tyr Lys Glu Phe Phe Pro Gin His Pro Asn Phe Cys 145 150 155 160
Glu Pro Gin Asp Tyr Arg 165
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO : : :
Met Ser Gly Ser Phe Glu Leu Ser Val Gin Asp Leu Asn Asp Leu Leu 1 5 10 15
Ser Asp Gly Ser Gly Cys Tyr Ser Leu Pro Ser Gin Pro Cys Asn Glu 20 25 30
Val Thr Pro Arg He Tyr Val Gly Asn Ala Ser Val Ala Gin Asp He 35 40 45
Pro Lys Leu Gin Lys Leu Gly He Thr His Val Leu Asn Ala Ala Glu 50 55 60 Gly Arg Ser Phe Met His Val Asn Thr Asn Ala Asn Phe Tyr Lys Asp 65 70 75 80
Ser Gly He Thr Tyr Leu Gly He Lys Ala Asn Asp Thr Gin Glu Phe 85 90 95
Asn Leu Ser Ala Tyr Phe Glu Arg Ala Ala Asp Phe He Asp Gin Ala 100 105 110
Leu Ala Gin Lys Asn Gly Arg Val Leu Val His Cys Arg Glu Gly Tyr 115 120 125
Ser Arg Ser Pro Thr Leu Val He Ala Tyr Leu Met Met Arg Gin Lys 130 135 140
Met Asp Val Lys Ser Ala Leu Ser He Val Arg Gin Asn Arg Glu He 145 150 155 160
Gly Pro Asn Asp Gly Phe Leu Ala Gin Leu Cys Gin Leu Asn Asp Arg 165 170 175
Leu Ala Lys Glu Gly Lys Leu Lys Pro 180 185
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 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: 26:
ATCGAAGGTC GTGGGATCC 19
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO v - :
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
He Glu Gly Arg Gly He 1 5 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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: 28: CTCGAGCGGC CGCATCGTGA CTGACTGA 2B (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 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: 29:
Ser Ser Gly Arg He Val Thr Asp 1 5
(2) INFORMATION FOR SEQ ID NO: 30:
(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: 30: GCGGATCCAG CGGCTCTTCC GCTCTC 26 (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: 31: GCCTCGAGTC ACTGGTCCTG CAGCCG 26
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 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:32: CCAGCGGCTC TTCCGCTCTC CGTC 24
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 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: 33: AGCCGGCTGC AGGACCAGTG A 21
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 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:34: GACGGAGAGC GGAAGAGCCG CTGG 24
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 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: TCACTGGTCC TGCAGCCGGC T 21
(2) INFORMATION FOR SEQ ID NO:36:
(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:36: GCCTCGAGTC ACTGGTCCTG CAGCCG 26
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 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:37: GAAGGTCGTG GGATCCAGCG GCTCTTCCGC 30 (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: 38: CAGGACCAGT GACTCGAGCG GCCGCAT 27
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 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: 39: GCGGAAGAGC CGCTGGATCC CACGACCTTC 30
(2) INFORMATION FOR SEQ ID NO:40:
(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:40: ATGCGGCCGC TCGAGTCACT GGTCCTG 27
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA ( ) HYPOTH A : NO (iv) ANTI-SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GCGGAGGACG CGGCCTTCAA TTTCCTCAGC CTC 33
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 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:42: GGGGAGCGGA GCAAGAAGGA GCTCTGTAGC 30
(2) INFORMATION FOR SEQ ID NO:43:
(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:43: GAGGCTGAGG AAATTGAAGG CCGCGTCCTC CGC 33
(2) INFORMATION FOR SEQ ID NO:44:
(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: 44: GCGGATCCAG CACGATGAGA TCGAGAA 27 : :
(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:45: GCCTCGAGTC ACCGGTAGTC CTGGGGT 27

Claims

1. A fusion selected from any of the fusions shown below,
(DNA)
1138 1740
GST P R 976 , cdc25B like , 1773
GST p R 302 cdc25B like '• 566
356 556
(peptide)
where, 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 of the 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 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, a) the region has about the number of sequences indicated by the numbers shown, b) the region has either the same amino acids as native cdc25B or substituted nucleic or amino acid residues, where the native nucleic or amino acid residues of the cdc 25B like region are ose sequences sc ose n e an sequence s ngs, where the substituted nucleic or amino acid residues of the cdc 25B like region are those sequences disclosed as substitutions in the CHARTS and 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.
2. The fusion shown in claim 1 where the protease cleavage site is created to be responsive to thrombin or Factor Xa.
3. The fusion shown in claim 1 where the protease cleavage site is created to be responsive to Factor Xa.
4. The fusion shown in claim 1 where the restriction site is Bam HI.
5. The fusions shown in claim 1 that are comprised of amino acid residues.
6. The fusion protein of claim 5 where the cdc25B like region is comprised of the amino acid residues shown in CHART 6 as cdc25B302"566 or SEQ. ID. NO. 4.
7. The fusion protein of claim 6 comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO. 4.
8. The fusion protein of claim 5 where the cdc25B like region is comprised of the amino acid residues shown in CHART 14 as Mutein 1 or SEQ. ID. NO. 14.
9. The fusion protein of claim 8 comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO. 14.
10. The fusion protein of claim 5 where the cdc25B like region is comprised of the amino acid residues shown in CHART 14 as Mutein 2 or SEQ. ID. NO. 15.
11. The fusion protein of claim 10 comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-SEQ. ID. NO. 15. . compr se e amino acid residues shown in CHART 14 as Mutein 3 or SEQ. ID. NO. 16.
13. The fusion protein of claim 12 comprising,
GST-Ile-Glu-Gly-Arg-Gly-rie-SEQ. ID. NO. 16.
14. The fusion protein of claim 5 comprising, GST-Ile-Glu-Gly-Arg-Gly-Ile-Gln302...-Gln566.
15. The fusion protein of claim 5 is closely associated with a bacterial polypeptide, where said polypeptide is a chaperonin.
16. The fusion protein of claim 15 where the chaperonin polypeptides are DnaK and/or GroEL.
17. The fusions shown in claim 1 that are comprised of nucleic acid residues.
18. The nucleic acids of claim 17 where the cdc25B like region is comprised of the nucleic acid residues shown in CHART 5 and CHART 13 as cdc25B976'1773 or SEQ ID. NO. 4.
19. The nucleic acids of claim 17 where the cdc25B like region is comprised of the nucleic acid residues shown in CHART 13 as Muteinl or SEQ ID. NO. 11.
20. The nucleic acids of claim 17 where the cdc25B like region is comprised of the nucleic acid residues shown in CHART 13 as Mutein2 or SEQ ID. NO. 12.
21. The nucleic acids of claim 17 where the cdc25B like region is comprised of the nucleic acid residues shown in CHART 13 as Mutein3 or SEQ ID. NO. 13.
22. The nucleic acids of claim 17, coding for the fusion protein of claim 5, comprising
GST-Xa-BamHI restriction site- cdc25B(976-1773)-XhoI restriction site.
23. The nucleic acids of claim 17 coding for the fusion protein comprising GST-Xa-GGG-ATC-cdc25B(976-1773)-XhoI restriction site. . nuc e c ac ragmen o m , disclosed in CHART 5 or SEQ. ID. NO. 3.
26. A peptide comprising any of the peptides from the cdc25B like region of the fusion of claim 1.
27. A peptide of claim 26, comprising the amino acid residues disclosed in CHART 6 or SEQ. ID. NO. 4.
28. A peptide of claim 26, comprising the amino acid residues disclosed in CHART 11 or SEQ. ID. NO. 9.
29. A peptide of claim 26, comprising the amino acid residues disclosed in CHART 12 or SEQ. ID. NO. 10.
30. A peptide of claim 26, comprising the amino acid residues disclosed in CHART 16 as Muteinl or SEQ. NO. 21.
31. A peptide of claim 26, comprising the amino acid residues disclosed in CHART 16 as Mutein 2 or SEQ. ID. NO. 22.
32. A peptide of claim 26, comprising the amino acid residues disclosed in CHART 16 as Mutein 3 or SEQ. ID. NO. 23.
33. The catalytic macromolecule of claim 26, comprising the amino acid residues, of SEQ. ID. NO. 9, that is produced from the fusion protein that is GST-Ile-Glu-Gly- Arg-Gly-Ile-SEQ. ID. NO. 4.
34. The catalytic macromolecule of claim 26, 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. 14.
35. The catalytic macromolecule of claim 26, 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. . , 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.
37. A fusion selected from any of the fusions shown below,
(DNA)
1162
GST VHR like 1659
GST VHR like
364 529
(peptide)
where, 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 NHR region has about the number of sequences indicated by the numbers, where the numbers correspond to the same residue numbers as full length NHR region of cdc25B.
38. Intermediates and processes used to created the fusions and peptides in any of the claims above. . e process o ma ng any o e usons, pep es, consrucs or moecues disclosed in this application.
40. The inventions disclosed in the application.
PCT/US1997/005404 1996-05-02 1997-05-02 Catalytic macro molecules having cdc25b like activity WO1997041238A1 (en)

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EP97923404A EP0907743A1 (en) 1996-05-02 1997-05-02 Catalytic macro molecules having cdc25b like activity
JP53889297A JP2002515742A (en) 1996-05-02 1997-05-02 Catalytic polymer with CDC25B-like activity
AU29216/97A AU2921697A (en) 1996-05-02 1997-05-02 Catalytic macro molecules having cdc25b like activity

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US1674896P 1996-05-02 1996-05-02
US60/016,748 1996-05-02
US1732396P 1996-05-07 1996-05-07
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