WO1999043702A1 - PROTEINE SE LIANT A NE-dlg - Google Patents

PROTEINE SE LIANT A NE-dlg Download PDF

Info

Publication number
WO1999043702A1
WO1999043702A1 PCT/JP1998/003740 JP9803740W WO9943702A1 WO 1999043702 A1 WO1999043702 A1 WO 1999043702A1 JP 9803740 W JP9803740 W JP 9803740W WO 9943702 A1 WO9943702 A1 WO 9943702A1
Authority
WO
WIPO (PCT)
Prior art keywords
nedacin
polynucleotide
protein
amino acid
cdna
Prior art date
Application number
PCT/JP1998/003740
Other languages
English (en)
Japanese (ja)
Inventor
Toshihiko Kishimoto
Shin-Ichiro Niwa
Hiroaki Kuwahara
Hideyuki Saya
Original Assignee
Sumitomo Electric Industries, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Electric Industries, Ltd. filed Critical Sumitomo Electric Industries, Ltd.
Priority to CA002322070A priority Critical patent/CA2322070A1/fr
Publication of WO1999043702A1 publication Critical patent/WO1999043702A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to a protein that binds to NE-d1 g (including a splicing isoform thereof).
  • the present invention also relates to a polynucleotide encoding the above protein.
  • the present invention relates to a homolog of the protein or the polynucleotide.
  • the present invention also relates to an antibody that recognizes the above-mentioned protein.
  • the d1g gene a Drosophila tumor suppressor gene, is a gene that, when deleted, causes hyperplasia of adult disciogenesis.
  • a group of genes having homology with the d1g gene is called MAGUK family, and is considered to have a function of localizing at cell adhesion sites and nerve endings and accumulating specific proteins.
  • the present inventors isolated the NE-d1g gene, which is a human gene and has homology to the Drosophila d1g gene, and further isolated the NE-d1g gene encoded by the NE-d1g gene.
  • NE—d 1 g D 1 g protein (hereinafter abbreviated as NE—d 1 g), and revealed that it interacts with the C-terminal region of the APC tumor suppressor protein. Has a negative control on cell growth (Oncogene, 14, 2425-2433).
  • the present invention provides a protein that binds to the NE-d1g,
  • the present invention provides a protein (named nedacin) which binds to 1 g of NE-d, has no homology to a known mammalian protein, and has a molecular weight of 5 lkD.
  • Nedacin can be obtained by affinity-purifying the crude protein fraction of each tissue using a column on which 1 g of NE-d is immobilized.
  • the present invention provides a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 1, 3, 5, and 7 in the sequence listing. These are nedacins (including sublysing isoformers) in humans.
  • the present invention also provides a protein comprising an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and binding to NE-d1 g. I do. It is a mutant of human nedacin S.
  • the present invention also provides a polynucleotide encoding the above-mentioned nedacin (including a mutant).
  • the present invention also provides an antisense polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide.
  • a method for producing nedacin is disclosed. Specifically, a method is disclosed in which a transformant into which nedacin cDNA is introduced produces nedacin. Also disclosed is a recombinant nedacin produced by the method.
  • the present invention also provides an antisense polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the nedacin or the polynucleotide encoding the nedacin mutant.
  • the antisense polynucleotide is included in the polynucleotide, but in the present application, it is referred to as an antisense polynucleotide particularly when it is specified that the polynucleotide comprises a nucleotide sequence of an antisense strand.
  • Representative Antisense Polynucleotides also provides polynucleotides that are all or part of a nucleotide consisting of 12 or more bases that encode nedacin or a nedacin variant.
  • This polynucleotide for portions of the coding region, can be used to produce partial length proteins of nedasin or nedasin variants, respectively. Ma It can also be used as a probe.
  • the present invention also provides an antisense polynucleotide which is all or a part consisting of 12 or more bases of the antisense polynucleotide of nedacin or a nedacin mutant.
  • the antisense polynucleotide is capable of inhibiting the biosynthesis of nedacin or a nedacin mutant, respectively. It can also be used as a probe.
  • the present invention also provides a polynucleotide obtained by chemically modifying the above-mentioned polynucleotide (including an antisense polynucleotide).
  • the present application discloses that the analysis by the northern blot hybridization method using a DNA probe enables the detection of nedasin mRNA from each tissue and that the nedasin mRNA is naturally expressed. Is what you do.
  • the present invention also provides a nedacin homolog protein encoded by a homolog gene of the nedacin gene.
  • nedacin in each vertebrate is collectively indicated. The above nedacin can be produced in a transformant into which the nedasin gene has been introduced.
  • the present invention uses a polynucleotide encoding human nedasin or a part thereof consisting of at least 12 bases (a part of the coding region) as a probe to obtain nedasin from another vertebrate, preferably mammalian cDNA library. It also includes a method for obtaining cDNA and nedacin cDNA obtained by the method. The present invention also provides a nedacin protein which is a homologue of human nedacin encoded by the nedacin cDNA.
  • the present invention also provides an antibody that recognizes the nedacin and a mutant of the nedacin.
  • the antigenicity of nedacin that is, that an antibody can be produced from nedacin.
  • FIG. 1 is a diagram showing the positional relationship between the cDNA of nedasin S, each fragment obtained during the cloning process, and each primer.
  • FIG. 2 is a diagram showing a positional relationship of each region of NE-d1g.
  • Fig. 3 is an electrophoresis photograph showing the result of SDS-PAGE electrophoresis of a protein extracted from E. coli brain cells binding to 1 g of NE-d and silver staining of the gel as it is.
  • 1 is an electrophoretic photograph showing the result of electrophoresis of a protein extracted from a brain cell of a mouse bound to 1 g of a NE-d delivery mutant.
  • FIG. 4B is a diagram showing the relationship between the partial or full length of the NE-dlg delivery mutant.
  • FIG. 5 is an electrophoretic photograph showing the results of analyzing the expression of nedacin mRNA in each tissue by the Northern Protocol hybridization method.
  • FIG. 6 is an electrophoretic photograph showing the results of RT-PCR analysis of the expression of nedacin mRNA in each tissue and each cell.
  • FIG. 7 is an electrophoresis photograph showing the result of electrophoresis of an immunoprecipitate containing nedacin and 1 g of NE-d.
  • the nedacin according to the present invention is obtained by combining a crude protein fraction extracted from cells derived from tissues of various animals as well as humans with 1 g of NE-d, and further dialysis, ammonium sulfate precipitation or gel filtration column, Purification can be performed singly by chromatography, using an exchange column or hydroxyapatite as an adsorbent, or by glycerol density gradient centrifugation.
  • Nedacin can be purified by combining various types of chromatography. The purified nedacin fraction binds 1 g of NE-d.
  • the fraction containing purified nedacin was analyzed by SDS-polyacrylamide electrophoresis (S DS-PAGE ⁇ Ant ibodies A Laboratory Manu a 1, p636-640), and separation by two-dimensional electrophoresis using 0, Farrell, etc. Bands are observed.
  • Nedacin can be isolated from the band obtained by SDS-PAGE or two-dimensional electrophoresis by extracting a protein from a band having a molecular weight of about 51 kDa. That is, nedacin separated and identified by electrophoresis can be transferred to polyvinylidene difluoride (PVDF) or the like.
  • PVDF polyvinylidene difluoride
  • Nedacin after the above-mentioned treatment is subjected to various enzyme treatments such as lysylendopeptidase into a mixture of polypeptide fragments, and the obtained mixture of peptide fragments is separated by high performance liquid chromatography.
  • This synthetic polynucleotide can be used as a probe for searching for a polynucleotide encoding nedasin.
  • a general method for preparing a cDNA library for screening the polynucleotide encoding nedasin of the present invention, a general method can be used, for example, by the following steps.
  • mRNA messenger RNA
  • cDNA single-stranded complementary polynucleotide
  • a plasmid or a plasmid containing the target polynucleotide is obtained from the transformant by an appropriate method, for example, colony hybridization or plaque hybridization. Isolating di,
  • Step (i): mRNA encoding the polypeptide of nedasin can be obtained from various animal tissues, organs, and producer cells, more specifically, from the brain, placenta, liver, kidney, and the like.
  • guanidium / cesium chloride a method (Guanidi umu / Cesi umu Chloride method, Mo lecular Clonin Second Edition, Cold Spring Harbor Laboratory or Pre- ss, p7. 19-7.23, 1989) Guanidimuthiocinate method (Analyt i cal Bio chemi stry, 162, pl 56—159, 1987) is generally used.
  • Separation and purification of mRNA from total RNA obtained by the above operation can be performed, for example, by using oligo dT-cellulose (Collaborative Research Co., Ltd.) or Oligodex-1 dT 30 (Yukara Co., Ltd.). This can be carried out by an adsorption column method or a batch method.
  • the Okayama-Berg method Okayama, H. and Ber, P., Molecular and Cereal
  • l lu l ar B ioloy, 3, p 280, 1983 cDNASynthesize cDNA according to the method of Gubler and V. Hoffman (Gubler, V. and Hoffman, BJ, Gene, 25, p 263-269, 1983) I do.
  • Examples of plasmid vectors incorporating cDNA include PBR322 (Gene, 2, 95, 1977), PBR325 (Gene, 4, 121, 1978), PUC12 or PUC13 (Gene, 19, 259, 1982), PUC 18 or PUC 19 (Gene, 33, 103, 1985), PU C 118 or PUC 119 (Me t ho ds in Enzymology, 153, 3, 1987), Blu scr ip t II (Nucleic Acids. Res., 17, 9494, 1989), etc., but any other one can be used as long as it is replicated and maintained in the host. .
  • phage vector incorporating cDNA examples include, for example, Agt10 (Huynh, TV, Young, RA and Davis, .W., DNA cloning, A Practi ca l App roach, IRL Pre ss, Oxford, 1, 49, 1985), ⁇ gt 11 (Proc. Natl. Ac ad. Sci., USA, 80, 1194, 1983) or human ZAP II (Nucleic Acids. Res., 17, 9494,
  • Examples of a method for incorporating cDNA into plasmid include, for example, the method of Sambrook (J.), et al. (Mo 1 ecu 1 ar Cloning Sect on Ed Ed., Pi. 53— 1.73, 1989).
  • a method of incorporating cDNA into a phage vector for example, the method of Hyunh, T.V., etc. (DNA cloning as described above) can be used.
  • Escherichia coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • Saccharomyces cerevisiae Saccharomyces cerevisiae (Saccharomyces escer evisiae) and the like, and can be transformed.
  • Methods for transforming a host with a plasmid vector include, for example, the electroporation method described in Molecular Cloning (Moe ecu 1 ar Cloning, supra, page 1.74-1.84) or Calcium chloride method and the like can be mentioned.
  • a phage vector can be introduced, for example, into grown Escherichia coli using an in vitro packaging method.
  • Polynucleotide used as a probe in the above hybridization Any kind of polynucleotide can be used as long as it is a polynucleotide that hybridizes with nedacin.
  • a polynucleotide chemically synthesized based on the amino acid sequence of nedacin can be used.
  • nedacin a polynucleotide encoding nedacin can be prepared. (Determination of base sequence)
  • Determination of the nucleotide sequence of the cDNA obtained in accordance with the above can be performed by, for example, the Maxiam-Gilbert method (Methods in Enzymology, 65, 499-560, 1980), the dideoxy method (Messing, J. eta 1., Nucleic Acids Research, 9, 309, 1981), and Taq cycle sequencing using fluorescent dyes (Biotechniques, 7, 494-499, 1989). .
  • a combination of DNAs was selected to cover the coding region, By cutting and joining the DNA with an appropriate restriction enzyme site contained in the overlapping portion of the DNA, the full-length DNA can be obtained. Further, a primer is designed so as to include the entire coding region, and PCR is performed using the primers with a cDNA library of a target vertebrate, preferably a mammal, as a type I, to obtain a full-length primer. DNA can also be obtained.
  • Nedacin S having an SSV structure at the C-terminus has a binding property to NE-d 1 g.
  • the nucleotide sequence of cDNA encoding nedasin S consists of 2040 residues, and the open reading frame is composed of 1365 bases from A at 86 to A at 1450. Yes, encoding 454 amino acids. From the structural point of view that it binds to NE-d 1 g, the C-terminus becomes an S SV structure. It is that it is wearing.
  • the nucleotide sequence of the cDNA encoding nedasin V1 consists of 1926 residues, as shown in SEQ ID NO: 4 in the sequence listing, and the open reading frame is from A at position 1 to A at position 16 Up to 1416 bases and encodes 471 amino acids.
  • nucleotide sequence of cDNA coding for nedacin V2 has an open reading frame consisting of 151 and 18 residues and encodes 505 amino acids.
  • nucleotide sequence of cDNA encoding nedacin V3 has an open reading frame consisting of 133 residues and encodes 460 amino acids. I do.
  • the c DNA according to the present invention includes c DNA consisting of the nucleotide sequence ATG is not bound to the 5 5 end of the open reading frame of the sequence.
  • cDNA not only cDNA, but also a DNA having a primary structure of the base sequence described in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing, and an RNA comprising a base sequence corresponding to the base sequence, the human nedasin of the present invention. Included in the encoding polynucleotide.
  • the polynucleotides of the present invention also include DNAs that include 5'-flanking polynucleotides that encode part or all of the signal peptide for human nedacin. Furthermore, in accordance with the degeneracy of the genetic code, substituting at least one base of the polynucleotide for another type of base without changing the amino acid sequence of the polypeptide produced from the polynucleotide. Can be.
  • polynucleotide of the present invention also has an amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7 in the sequence listing by substitution based on the degeneracy of the genetic code. Includes everything that is
  • the amino acid sequence of human nedacin S of the present invention is the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
  • the calculated molecular weight of the protein consisting of the amino acid sequence is 51.0. kD and the isoelectric point is 5.35.
  • the amino acid sequence of human nedacin V1 of the present invention is the amino acid sequence described in SEQ ID NO: 3 in the sequence listing.
  • the calculated molecular weight of the protein consisting of the amino acid sequence is 52.8 kD, and the isoelectric point is 5.48.
  • the amino acid sequence of human nedacin V2 of the present invention is the amino acid sequence described in SEQ ID NO: 5 in the sequence listing.
  • the calculated molecular weight of the protein consisting of the amino acid sequence is 56.8 kD, and the isoelectric point is 5.36.
  • the amino acid sequence of human nedacin V3 of the present invention is the amino acid sequence described in SEQ ID NO: 7 in the sequence listing.
  • the calculated molecular weight of the protein consisting of the amino acid sequence is 52. lkD, and the isoelectric point is 5.90.
  • Human nedacin includes a polypeptide in which methionine is not bound to the N-terminal of the amino acid sequence. Also, an intermediate in which a part or all of a signal peptide for human nedascin is bound or deleted at the N-terminus of the amino acid sequence is included.
  • a DNA mutant is produced in which the structure of the DNA encoding the target protein is changed, and the DNA mutant is introduced into an appropriate host to prepare a transformant.
  • a transformant can produce a mutant of the desired protein.
  • the present invention relates to a mutant of nedacin S, which includes a protein in which the C-terminal S SV structure of the amino acid sequence has been changed while being conserved, and which binds to NE-d1 g. Mutants of the splicing isoforms nedacin V1, nedacin V2 or nedacin V3 are also included.
  • nedacin V1, nedacin V2 or nedacin V3 are also included.
  • usually several amino acids can be substituted, deleted or added by one operation. By repeating this operation a plurality of times, more amino acids can be substituted, deleted or added, and a desired mutant can be produced.
  • the homology between nedacin S of the present invention and its mutant is preferably 75% or more at the amino acid level, more preferably 90% or more, and even more preferably 95% or more.
  • the present invention also includes a polynucleotide consisting of a nucleotide sequence encoding a nedacin mutant.
  • CDNA and genomic DNA encoding nedasin can be used as they are or after being cut with restriction enzymes, depending on the purpose.
  • the present invention also includes an antisense polynucleotide comprising a base sequence complementary to the base sequence.
  • the antisense polynucleotide of the present invention can be used to inhibit the biosynthesis of human nedacin, and can also be used as a probe.
  • an antisense polynucleotide is included in a polynucleotide.
  • the antisense polynucleotide will be described as being included in the polynucleotide.
  • the antisense polynucleotide is specifically referred to as an antisense polynucleotide, it is referred to as an antisense polynucleotide.
  • the antisense polynucleotide for inhibiting the biosynthesis of the polypeptide preferably comprises 15 or more bases.
  • antisense polynucleotides that are too long are unsuitable for uptake into cells.
  • the base is 12 to 30 bases, preferably 15 to 25 bases, and more preferably.
  • an antisense polynucleotide comprising 18 to 22 bases It is better to use 7
  • the antisense polynucleotide to human nedacin of the present invention also comprises 12 to 30 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases. It is preferable to use an antisense polynucleotide.
  • the antisense polynucleotide of the present invention includes all of those in which a plurality of nucleotides consisting of bases, phosphates, and sugars are bound, including those that are not naturally occurring. Typical examples are antisense DNA and antisense RNA.
  • the binding strength to the target DNA or mRNA, tissue selectivity, cell permeability, nuclease resistance, or intracellular stability can be improved.
  • High variety of antisense polynucleotide derivatives are obtained.
  • the derivatives generally known at present are preferably derivatives having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability, and avidity. Particularly preferred are derivatives having a phosphorothioate bond as a skeletal structure.
  • the polynucleotides and derivatives thereof of the present invention also include derivatives having these functions or structures.
  • an antisense polynucleotide or a derivative thereof having a base sequence complementary to the base sequence of the region forming the stem loop should be designed.
  • the antisense polynucleotide and the derivative thereof of the present invention can form a stem loop as necessary.
  • an antisense polynucleotide having a sequence complementary to the sequence of the translation initiation codon, the ribosome binding site, the cabling site, and the splice site can generally be expected to have a high expression suppressing effect. Therefore, the antisense polynucleotide of the present invention or a derivative thereof, which is located near the translation initiation codon of DNA or mRNA encoding nedasin, a ribosome binding site, a caving site, Those containing a sequence complementary to the chair site are expected to have a high expression suppressing effect.
  • the antisense polynucleotide of the present invention can be prepared by using a chemical synthesizer or by a PCR method using a gene encoding human nedacin as type III.
  • some derivatives such as a methylphosphonate type and a phosphorothioate type, can be chemically synthesized.
  • the operation can be carried out in accordance with the instructions attached to the chemical synthesizer, and the resulting synthetic product can be purified by the HP LC method using reverse phase chromatography or the like.
  • Antisense polynucleotide or a derivative thereof can be obtained.
  • Polynucleotide encoding nedacin of the present invention or a part thereof polynucleotide comprising 9 or more consecutive bases
  • an antisense polynucleotide of the antisense strand of the polynucleotide or a derivative thereof from 9 or more consecutive bases
  • Antisense polynucleotide or a derivative thereof can be used as a probe for screening a nedasin gene from a cDNA library or the like. At this time, those having a GC content of 30 to 70% can be suitably used.
  • a polynucleotide comprising 12 or more consecutive bases is more preferable
  • a polynucleotide having 15 or more bases is more preferable.
  • the polynucleotide used as a probe may be a derivative. Usually, a sequence having the number of bases or more is recognized as a sequence having specificity.
  • a cDNA library used in screening using the probe a cDNA library prepared from mRNA can be preferably used. A group of cDNAs selected from these cDNA libraries by random sampling can be used as a sample for the search.
  • DNA consisting of 9 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing or a polynucleotide that hybridizes to the DNA (antisense polynucleotide) is obtained by synthesizing the nedasin gene from a cDNA library or the like. It can be used as a probe for cleaning.
  • tissue expressing nedasin-gene-derived mRNA can be identified. It is possible to find out.
  • the DNA and RNA of the present invention include the above-mentioned chemically modified DNA, RNA or antisense polynucleotide within its scope.
  • the chemically modified DNA or RNA referred to here can exert both a protein-encoding function and a probe function, and the chemically-modified antisense polynucleotide can be used for probe or protein biosynthesis. It can exhibit both the function of inhibiting the activity and the function as a probe.
  • the present invention also includes a recombinant nedacin produced by the above-mentioned transformant into which the nedasin gene has been introduced. Further, the disclosure of the present invention makes it possible to produce a nedacin mutant in which one or more amino acids in the amino acid sequence of nedacin are substituted, deleted or added.
  • the vector and host used for the transformation those described in the section on the preparation and screening of the cDNA library can be used.
  • the method for purifying nedacin or a nedacin mutant the method described in the section on purification of nedasin can be used.
  • the nedacin or nedacin mutant to be produced is
  • the transformant may be prepared as a fusion peptide with a peptide.
  • a chemical substance such as bromocyan or an enzyme such as protease to excise nedacin or nedacin mutant.
  • an antibody recognizing nedasin can be obtained by immunizing a non-human animal with a peptide that is a part of nedasin of the present invention, as exemplified in Example 7, as to the antigenicity of nedasin Is to clarify.
  • an antibody that recognizes nedacin can be obtained by immunizing with full-length nedacin. Therefore, an antibody recognizing nedacin of the present invention (hereinafter sometimes referred to as a human nedacin antibody) is an antibody obtained by immunizing a non-human animal with nedacin, and the antibody is the nedacin of the present invention.
  • Antibodies whose recognition is confirmed by Western blotting, ELISA, immunostaining (eg, measurement by FACS), etc. are included in the range.
  • an immunogen obtained by binding a part of the protein to another carrier protein such as serum albumin is a commonly used method to use an immunogen obtained by binding a part of the protein to another carrier protein such as serum albumin.
  • a part of the protein may be synthesized using, for example, a peptide synthesizer.
  • it is preferable that a part of the protein has eight or more amino acid residues.
  • the nedacin antibody of the present invention includes a monoclonal antibody within its scope.
  • antibodies also include active fragments.
  • An active fragment refers to a fragment of an antibody having antigen-antibody reaction activity.
  • F (ab ') 2 , Fab', Fab, Fv and the like can be mentioned.
  • F (ab,) 2 is obtained, and when digested with papain, Fab is obtained.
  • Reduction of F (ab,) 2 with reagents such as 2-mercaptoethanol and alkylation with mono-acetic acid gives Fab '.
  • Fv is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker.
  • a chimeric antibody can be obtained by retaining these active fragments and substituting the other parts with fragments of another animal.
  • the immunoassay using the obtained antibody provides a means for quantitatively measuring the change of the subject in various cells.
  • the method using an antibody include a method for detecting nedacin using a labeled nedacin antibody, and a method for detecting nedacin using a nedacin antibody and a labeled secondary antibody of the antibody.
  • the label for example, a radioisotope (RI), an enzyme, avidin or biotin, or a fluorescent substance (FITC, rhodamine, etc.) is used.
  • Examples of the method utilizing an enzymatic reaction include an ELISA method, an immunoagglutination method, a Western blot method, a method for identifying an immune reaction molecule using flow cytometry, or a method similar thereto.
  • the base sequence up to the 1937th A was inserted, and the vector was introduced into Escherichia coli DH5 to prepare a transformant.
  • the From the PDZ1 region consisting of the amino acid sequence from Ala at position 63 to G1u at position 568 of the NE-d1g protein (protein consisting of the amino acid sequence of SEQ ID NO: 9) in the transformant A GST-NE-d1g fusion protein in which GST was bound to the N-terminal of the portion up to the SH3 region was produced.
  • Figure 2 shows the positional relationship of each area of NE-dig.
  • the beads After immobilizing the GST-NE-d1g fusion protein on glutathione agarose beads, the beads were packed in a column to prepare an affinity column.
  • the protein was extracted from P. cerevisiae brain cells, and the extracted crude fraction was applied to the affinity column at 4 ° C. and passed through the affinity column.
  • proteins elution buffer attached to the beads in a column (0. 5M NaC 1, 3 OmM T ris -HC 1 pH 7. 5, 1 mM EDTA, 5 mM M gCl 2, 1 mM DTT) pH 7 Washed out in 5 and heated in Laemli's sample buffer and placed at 100 ° C for 5 minutes.
  • Lane 1 is a lane in which only GST was electrophoresed
  • lane 2 is a lane in which GST-NE-d 1 g fusion protein was electrophoresed.
  • a fusion protein was prepared by combining the following four types of NE-d 1 g of partial protein and full-length protein with GST, and an affinity column in which each protein was packed with beads was prepared. .
  • NE A protein containing the dig SH3 region (amino acid sequence from Ser 503 to G1u at 568 in the amino acid sequence described in SEQ ID NO: 9 in the sequence listing).
  • NE Dig protein deleted from the GUK region (amino acid sequence from Arg at position 628 to Asp at position 803 in the amino acid sequence described in SEQ ID NO: 9 in the sequence listing).
  • FIG. 4 (B) shows the relationship between the part or the full length of NE—d 1 g of 1 to 4 above.
  • a crude protein fraction extracted from the E. coli brain cells was applied in the same manner as in 2 above, and the proteins bound to the fusion proteins immobilized on the beads were electrophoresed in the same manner as in 3 above.
  • the result is shown in Fig. 4 (A).
  • the middle arrow indicates the protein band of 51 kD. This indicates that the 51 kD protein does not bind to the SH3 region, but binds to NE-d1 g lacking the N-terminal and NE-d1 g lacking the GUK region. . Therefore, it was found that the 51 kD protein binds to NE-dig and its PDZ region.
  • Example 2 Determination of amino acid sequence of NE-d1g binding protein and nucleotide sequence of a gene encoding the same
  • the gel which had been subjected to electrophoresis for the 51 kD protein purified in 2 of Example 1 was applied to a Immobilon-1 P VDF membrane (manufactured by Millipore) using a semi-dry electroprototyping apparatus, Sartoblot II-1 S ( (Manufactured by Sartorius).
  • the membrane filter on which the protein was blotted was washed with distilled water, stained with Ponceau S (P onceau S) staining solution (0.1% Ponceau-13, 1% acetic acid solution), and immersed in distilled water.
  • the background was decolorized by shaking.
  • Lys-C protease manufactured by Boehringer Mannheim
  • HP LC Perkin Elmer
  • Peptide fragment 1 Va 1 Phe Leu G 1 u G 1 u A La Ser G in G in G 1 u Lys (from 43 to 53 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) Array)
  • Peptide fragment 2 GluTrpCysPheLys (sequence from the 47th to the 51st amino acid sequence of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)
  • Peptide fragment 3 Thr Arg As LeuHislie Gin Ser H is (sequences 232 to 240 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)
  • Peptide fragment 4 Asn Leu TyrPRoSeyrTyr Lys (sequences from 253 to 259 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)
  • Peptide fragment 5 G1uPheAspAlaiLieLeuLieAsnProLys (sequence from position 394 to position 403 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)
  • Peptide fragment 6 G1nVa1Va1ProPHeSer (sequence from amino acid position 446 to position 451 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)
  • a human fetal brain cDNA library (Clontech) was used for PCR type II.
  • P1 primer 5'-ATGCCTGGGCTG GTTGATACACAC-3, (base sequence described in SEQ ID NO: 11)
  • P2 primer 5'-CATCACTGTCT TATTTGTCAAAAG-3
  • the nucleotide sequence described in SEQ ID NO: 12 in the sequence listing was designed and synthesized using a DNA synthesizer (ABI, model 392).
  • the synthesized primer was adjusted to 10 pmo1 / ⁇ 1 with distilled water (the subsequent primer synthesis was performed in the same manner).
  • PCR was performed using these primers, a cDNA fragment of 609 bp was obtained. This fragment is hereinafter referred to as fragment A.
  • the fragment A obtained above was subjected to mini gel electrophoresis (0.75% agarose gel), and a band (609 bp) of the DNA was cut out from the gel.
  • the cDNA was collected on a dialysis membrane, and the bands were checked by mini gel electrophoresis.
  • cDNA a l y l up was diluted with 99 1 of TE.
  • the absorbance at 260 nm (A260) was measured, and the DNA concentration was calculated (the DNA concentration when the concentration of 260 was 1.0 was 50 l / ml).
  • the DNA was diluted with TE so that the DNA concentration was.
  • Fragment A with P1 sense primer and P2 antisense primer was used to perform a direct sequence.
  • DNA sequencing was performed using the Auto Sequencer (ABI Model 373A) by the Dye-Mine-Mine-One-Duct method to determine the nucleotide sequence of fragment A cDNA.
  • the following primers were synthesized to obtain further DNA fragments from the human cDNA library.
  • P 3 primer on the 3 ′ side from P 1 primer: 5′-TGATTTGCACA TTCAGAGCCATAT—3, (base sequence described in SEQ ID NO: 13 in the sequence listing), P 4 primer on the 5 ′ side from P 2 primer: 5 , -AGGTCTATGCTA CTTCCAGCAAAG-3, (base sequence described in SEQ ID NO: 14 in the sequence listing) was designed and synthesized using a DNA synthesizer (ABI, model 392). The synthesized primer was adjusted to 10 pmo1 / 1/1 with distilled water.
  • cDNA library a cDNA library of human fetal brain (manufactured by Clonetech) was used, and the AP-1 primer set in the linker of the cDNA library: 5'-CCAT CCTAATACGACTCACTATAGGGC-3 ' One step PCR was carried out using P1, P2, P3 and P primers.
  • the P1 primer was used to extend the single-stranded DNA on the 3 side by PCR.
  • Mineral oil 10 ⁇ 1 is overlaid on the liquid having the above composition, left at 95 ° C. for 5 minutes, and XL Polymerase 0.3 / 1 (registered trademark, manufactured by Parkin Elma Co., Ltd.) was added. The cycle of “6 ° C for 30 seconds, 60 ° C for 30 seconds, and then 72 ° C for 2 minutes” was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C for 7 minutes to complete the PCR.
  • the PCR product was transformed into type III and cloned on the 3 side to synthesize double-stranded DNA.
  • fragment B DNA sequence
  • the P2 primer was used to extend the single-stranded DNA on the 3 side by PCR.
  • a mineral oil (10-1) is overlaid on the liquid having the above composition, left at 95 ° C for 5 minutes, added with XL Polymerase 0.3-1 (registered trademark, manufactured by PerkinElmer Co., Ltd.), and added to “96 ° A cycle of 30 seconds at C, 30 seconds at 60 ° C, and then 2 minutes at 72 ° C "was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C for 7 minutes to complete PCR.
  • the PCR product was transformed into type I and the 5 side was cloned to synthesize double-stranded DNA.
  • Mineral oil 10 ⁇ 1 is overlaid on the liquid of the above composition, left at 95 ° C for 5 minutes, and XL Polymerase 0.3 ⁇ 1 (registered trademark, manufactured by PerkinElmer Inc.) is added. A cycle of “30 seconds, 60 seconds for 30 seconds, and then 72 ° C. for 2 minutes” was repeated 40 times. Finally, a fragment extension reaction was performed for 72 minutes at 72 to complete the PCR.
  • fragment C this DNA fragment is referred to as fragment C hereinafter.
  • P GCTCAGATGCCG-3 (base sequence described in SEQ ID No. 17 in the sequence listing) was set as an antisense primer, P6 primer 1: 5, -AAAT AGGAT CC, the base sequence described in SEQ ID No. 18).
  • PCR was performed under the following conditions using human fetal brain cDNA library as a type III.
  • the cDNA was ligated to a protein that binds to NE-d1 g (this protein was nedasin (NEDAS IN, NE-DiAs soci ating protein IN). ) was identified as cDNA encoding).
  • This nucleotide sequence is shown as SEQ ID NO: 2 in the sequence listing. The gene consisting of this nucleotide sequence was named nedacin S gene. No known mammalian genes having homology with the nedacin S gene were found. Nedacin S cDNA and the position of each fragment and each primer obtained during the cloning process. Figure 1 shows the relationship.
  • nedacin S The amino acid sequence of the nedacin S protein encoded by this gene (hereinafter sometimes simply referred to as nedacin S) is shown in SEQ ID NO: 1 in the sequence listing.
  • the C-terminus of nedacin S was an S SV motif that could bind to the PDZ region. No mammalian protein having homology with this nedacin S was found, but it had about 40% homology with the yeast YDL238c protein.
  • the full-length nedasin cDNA obtained above was inserted into a plasmid vector pGEM (registered trademark) — T Easy (Promega) multi-cloning site.
  • This plasmid vector was introduced into E. coli NM522 to prepare a transformant.
  • the Escherichia coli harboring the cDNA of nedacin S was named Ne dasin S, and the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 1-3 Tsukuba East, Ibaraki, Japan (zip code 305- 8566)) and deposited on February 25, 1998.
  • the accession number is FERM P-16663.
  • the Nedas inS was transferred to the Deposit under the Budapest Treaty at the institute (Accession No. FERM BP-6471).
  • nedasin S gene was added to the MoE ecu1arClonin A Laboratory Manual Manual, Edition Ed. According to the description, hybridization was carried out at 43 ° C. for 16 hours, and analysis was carried out by the Northern blot hybridization method.
  • FIG. 5 shows the results of analysis by the Northern Protocol hybridization method. From this, it was found that mRNA of nedacin was strongly expressed in brain, placenta, liver and kidney.
  • MRNA was extracted from each tissue in which nedacin expression was observed using a Microfast Track (registered trademark) kit (manufactured by Invitrogen) mRNA purification kit, and RT-PCR method (new cell experimental engineering protocol, Shujunsha, Published in 1993, listed on pages 175-176).
  • the obtained gene product was sequenced in the same manner as described above by the dye-mine method.
  • three splicing Form was found to exist. These were named Nedacin VI, Nedasin V2, and Nedasin V3.
  • nedacin V1 The amino acid sequence of nedacin V1 is shown in SEQ ID NO: 3 in the sequence listing, and the nucleotide sequence of the nedacin V1 gene is shown in SEQ ID NO: 4 in the sequence listing.
  • nedacin V2 The amino acid sequence of nedacin V2 is shown in SEQ ID NO: 5 in the sequence listing, and the nucleotide sequence of the nedacin V2 gene (only the coding region) is shown in SEQ ID NO: 6 in the sequence listing.
  • nedacin V3 The amino acid sequence of nedacin V3 is shown in SEQ ID NO: 7 in the sequence listing, and the nucleotide sequence of the nedacin V3 gene (only the coding region) is shown in SEQ ID NO: 8 in the sequence listing.
  • the nedacin VI gene was introduced into a plasmid vector pGEM (registered trademark) -TEASy (promega) multi-cloning site.
  • the transformant in which this plasmid was introduced into Escherichia coli DH5 was named Ne dasin VI. Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 1-3 Tsukuba East, Ibaraki, Japan) (Postal code 305-8566)) on February 25, 1998.
  • the accession number is FERM P-16664.
  • the Nadasin V1 was transferred to the Deposit under the Budapest Treaty at the same institute (Accession No. FERM BP-6472).
  • RT_PCR was also performed on each cell to examine the expression of the nedasin splicing isoform.
  • FIG. 6 shows the results of RT-PCR for each tissue and each cell. This confirmed that nedacin S is predominantly expressed in human fetal brain and placenta, and nedacin V1 is predominantly expressed in other tissues.
  • nedasin S cDNA and nedasin V1 cDNA were obtained by inserting a 63-mer linker containing the my c tag into the EcoRI-BamHI site of the plasmid vector pBj-1. It was inserted into the BamHI site of my c. Each of these was introduced into COS cells together with a vector-pCGN (having an HA tag) incorporating the NE-d1 g gene in which the GUK region had been deleted, to prepare transformants.
  • the cells are lysed using TNN buffer (15 OmM NaCl, 5 OmM Tris-HC1H7.5, 0.5% NP-40), and the cells are extracted. A liquid was obtained.
  • An anti-myc antibody was added to the cell extract, and the mixture was centrifuged at 3000 rpm for 5 minutes to separate immunoprecipitates.
  • This immunoprecipitate was electrophoresed on 9% SDS-PAGE.
  • the DNA was transferred to a nitrocellulose membrane using a Transplot system (manufactured by Marisol).
  • the nitrocellulose membrane was immersed in 10% skim milk / PBS, 0.1% Tween 20, allowed to stand for 1 hour, and blocked. Thereafter, the plate was washed twice with 0.3% Tween20 / PBS for 5 minutes each.
  • the anti-myc antibody was diluted to 1 g / ml with PBS, it was added to a nitrocellulose membrane and reacted at room temperature for 60 minutes. Thereafter, the plate was washed three times for 5 minutes each with 0.3% Tween20 / PBS.
  • nedacin S whose C-terminal is S SV binds to NE-d 1 g, but C-terminal
  • nedacin V1 of PFP did not bind to NE-d1g.
  • the binding between nedacin and NE-d1 g was demonstrated in vivo, and the binding was found to be controlled by a change in the C-terminal side of nedacin.
  • Chromosomal mating of the full-length gene of nedacin S was performed as follows.
  • the PCR uses the Genebridge 4 Radiation Hybrid Screening Panel (available from Research Genetics, Inc.) as a type II to convert a portion of the Nedacin S gene.
  • the PCR operation was performed as follows. A liquid having the following composition was placed in a test tube and left at 96 ° C for 9 minutes.
  • the section number of the panel to which the primer (S1 or AS1) was hybridized was changed to the “Whitehead Insitute / MIT C enterf or Genome Research (http: www—genome.wi.mit.edu/cgi”) — E-mail to b in / cont ig / rhmap er. Pi.;)
  • Genome Research http: www—genome.wi.mit.edu/cgi”
  • the nedacin S gene was located on the short arm of chromosome 9.
  • Peptide A RNIEEVYVGGKQVVPFSSSV (amino acid sequence from 435 to 454 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)
  • Peptide B LYP S YKNYT S VYDKNNLLT (position 254 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) Amino acid sequence from position 272 to position 272)
  • a reaction solution was prepared by binding 2 mg of the prepared peptide to 2 mg of maleimide KLH (Pierce). The reaction was performed according to the method described in the instruction manual of Pierce.
  • a second immunization was performed. From the second time on, immunization was performed by changing the adjuvant to Freund's complete adjuvant (Difco). Other operations are the same as the first operation. The second and subsequent immunizations were performed at weekly intervals for a total of six immunizations.
  • the antibody was affinity purified using a protein G Sepharose column (registered trademark, manufactured by Pharmacia). As a result, a total of 5 mg of the peptide-specific antibody was obtained.
  • Nedacin (including splicing isoform) or a mutant thereof of the present invention is effective for analyzing the mechanism of NE-d1 g.
  • the polynucleotide encoding the nedacin or a mutant thereof of the present invention a polynucleotide consisting of a continuous base sequence of 12 or more bases, or a polynucleotide (antisense polynucleotide) that hybridizes to the polynucleotide, is a cDNA. It can be used as a probe for screening nedacin or its mutant gene from a library or the like.
  • the antisense polynucleotide of the present invention is characterized in that nedacin or a mutant thereof Biosynthesis can be inhibited.
  • the antibody of the present invention can be used for elucidating functions such as expression of nedacin and elucidating the formation mechanism of malignant tumors.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une protéine (appelée 'Nédasine') qui se lie à NE-dlg, ne présente pas d'homologie avec aucune protéine mammalienne connue et possède un poids moléculaire de 51 kD; des polynucléotides codant pour ladite Nédasine ou pour ses mutants; des polynucléotides antisens comprenant les séquences de base complémentaires des séquences de base des polynucléotides susmentionnées et; un anticorps reconnaissant la Nédasine susmentionnée ou ses mutants.
PCT/JP1998/003740 1998-02-25 1998-08-24 PROTEINE SE LIANT A NE-dlg WO1999043702A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA002322070A CA2322070A1 (fr) 1998-02-25 1998-08-24 Proteine se liant a ne-dlg

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10043552A JPH11239483A (ja) 1998-02-25 1998-02-25 ＮＥ−ｄｌｇと結合する蛋白質
JP10/43552 1998-02-25

Publications (1)

Publication Number Publication Date
WO1999043702A1 true WO1999043702A1 (fr) 1999-09-02

Family

ID=12666928

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/003740 WO1999043702A1 (fr) 1998-02-25 1998-08-24 PROTEINE SE LIANT A NE-dlg

Country Status (3)

Country Link
JP (1) JPH11239483A (fr)
CA (1) CA2322070A1 (fr)
WO (1) WO1999043702A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006132701A2 (fr) * 2005-04-04 2006-12-14 Rutgers, The State University Procedes et trousses permettant de reguler l'assemblage des microtubules et la croissance et la ramification des dendrites
US7790843B2 (en) * 2004-01-12 2010-09-07 Firestein-Miller Bonnie L Cypin polypeptide and fragments thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ONCOGENE, Vol. 14, No. 20, (1997), MAKINO K. et al., "Cloning and Characterization of NE-dlg: A Novel Homolog of the Drosophila Disc Large (dlg) Tumor Suppressor Protein Interacts with the APC Protein", p. 2425-2433. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7790843B2 (en) * 2004-01-12 2010-09-07 Firestein-Miller Bonnie L Cypin polypeptide and fragments thereof
WO2006132701A2 (fr) * 2005-04-04 2006-12-14 Rutgers, The State University Procedes et trousses permettant de reguler l'assemblage des microtubules et la croissance et la ramification des dendrites
WO2006132701A3 (fr) * 2005-04-04 2007-06-28 Univ Rutgers Procedes et trousses permettant de reguler l'assemblage des microtubules et la croissance et la ramification des dendrites
US8283440B2 (en) 2005-04-04 2012-10-09 Firestein-Miller Bonnie L Snapin and methods for regulation of microtubule assembly and dendrite growth and branching

Also Published As

Publication number Publication date
CA2322070A1 (fr) 1999-09-02
JPH11239483A (ja) 1999-09-07

Similar Documents

Publication Publication Date Title
JP3255699B2 (ja) ヒトＩＬ−２レセプターγ鎖分子
CA2479498A1 (fr) Compositions et procedes destines au traitement de tumeur
WO2001048189A1 (fr) Nouveaux recepteurs couples a une proteine de liaison au guanosine triphosphate, genes de ces derniers, et production et utilisation de ces derniers
JP4984160B2 (ja) 抗体の作製方法
US7601349B2 (en) GIPs, a family of polypeptides with transcription factor activity that interact with Goodpasture antigen binding protein
WO1999054505A1 (fr) Proteine ameliorant l'expression des canaux potassium a la surface des cellules et acides nucleiques codant cette proteine
WO2006082826A1 (fr) PROCEDE D’IDENTIFICATION D’UN GENE Corl2 CIBLANT LES CELLULES DE PURKINJE
WO1999043702A1 (fr) PROTEINE SE LIANT A NE-dlg
JPH11235186A (ja) 神経組織のナトリウムチャンネルをコードする核酸
JP4127861B2 (ja) シナプス活性化タンパク質組成物および方法
AU714793B2 (en) Novel stress proteins
EP1197554A1 (fr) Facteur de differenciation de proliferation
JP3920573B2 (ja) トランスフェリン受容体様蛋白質をコードする核酸、及びその関連産物
JPH1075781A (ja) ヒトプロテアソームサブユニットｐ５８蛋白質及びそれに特異的な抗体
US7427666B2 (en) Antibody directed against a ubiquitin-specific protease occurring in the brain
JP4348504B2 (ja) ヒトｔｓｃ４０３遺伝子及びヒトｉｎｇ１ｌ遺伝子
KR100427645B1 (ko) 인간 멜라노코틴-4 수용체에 대한 항체와 그 제조방법
JP4499926B2 (ja) 腫瘍抑制遺伝子
CN107056950A (zh) 一种包含钥孔血蓝蛋白片段的融合蛋白及其应用
US20040115682A1 (en) Novel scavenger receptor class a protein
WO1999057269A1 (fr) Gene hrpi humain
WO1999009060A1 (fr) PROTEINE 1(3)mbt, POLYNUCLEOTIDE CODANT POUR LADITE PROTEINE, SON POLYNUCLEOTIDE ANTISENSE ET ANTICORPS RECONNAISSANT LADITE PROTEINE
KR100191125B1 (ko) 마우스에서 분리한 신규의 swi3계 유전자 및 그로부터 발현되는 단백질
JPH11222499A (ja) プロテアソーム抑制蛋白質およびそれをコードするポリヌクレオチド
CA2304793A1 (fr) Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2322070

Country of ref document: CA

Ref country code: CA

Ref document number: 2322070

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 09622828

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: CA