EP1045905A2 - Adn codant il-17rh et polypeptides il-17rh - Google Patents

Adn codant il-17rh et polypeptides il-17rh

Info

Publication number
EP1045905A2
EP1045905A2 EP99903032A EP99903032A EP1045905A2 EP 1045905 A2 EP1045905 A2 EP 1045905A2 EP 99903032 A EP99903032 A EP 99903032A EP 99903032 A EP99903032 A EP 99903032A EP 1045905 A2 EP1045905 A2 EP 1045905A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polypeptides
molecular weight
protein
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99903032A
Other languages
German (de)
English (en)
Inventor
Melanie Spriggs
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immunex Corp
Original Assignee
Immunex Corp
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 Immunex Corp filed Critical Immunex Corp
Publication of EP1045905A2 publication Critical patent/EP1045905A2/fr
Withdrawn legal-status Critical Current

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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Definitions

  • the invention is directed to purified and isolated IL-17RH polypeptides, the nucleic acids
  • polypeptides antibodies generated against these polypeptides, fragmented peptides derived from
  • proteins are routinely analyzed using techniques such as electrophoresis, sedimentation,
  • Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein samples (New England Biolabs Inc. Catalog:130-131,
  • Enzymatic fragmentation of a protein can be achieved by incubation of a protein
  • composition of a protein with regard to its specific amino acid
  • peptides possess unique charge characteristics that determine the isoelectric pH of the peptide.
  • Multildent Internet site:
  • Fragmentation of proteins is further employed for the production of fragments for amino acids
  • fragmentation of proteins can be used in the preparation of peptides for mass spectrometry (W.J. Henzel et al.,
  • modification sites e.g. phosphorylation
  • the invention aids in fulfilling this need in the art.
  • the invention encompasses an
  • isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO:l and an isolated
  • nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2.
  • the invention also provides a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2.
  • Double-stranded RNA and DNA variants of IL-17RH encompassed by the invention.
  • a double-stranded RNA and DNA variants of IL-17RH encompassed by the invention.
  • stranded DNA probe allows the detection of nucleic acid molecules equivalent to either strand of
  • nucleic acid molecule Isolated nucleic acid molecules that hybridize to a denatured, double-
  • stranded DNA comprising the DNA sequence of SEQ ID NO:l or an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2 under conditions of moderate
  • the invention further encompasses isolated nucleic acid molecules derived by in vitro
  • the invention also encompasses isolated nucleic acid
  • the invention also encompasses recombinant vectors that direct the expression of these
  • nucleic acid molecules and host cells transformed or transfected with these vectors.
  • the invention also encompasses isolated polypeptides encoded by these nucleic acid
  • polyclonal or monoclonal antibodies that bind to these polypeptides are encompassed by the
  • the invention further encompasses methods for the production of IL-17RH
  • polypeptides including culturing a host cell under conditions promoting expression
  • polypeptides in bacteria, yeast, plant, and animal cells is encompassed by the invention.
  • IL-17RH polypeptides as therapeutic agents for the treatment of diseases mediated by IL-17RH
  • polypeptide counter-structure molecules are encompassed by the invention. Further, methods of using IL-17RH polypeptides in the design of inhibitors thereof are also an aspect of the invention.
  • the invention further encompasses the fragmented peptides produced from IL-17RH
  • the invention also encompasses a method for the visualization of IL-17RH polypeptide
  • fragmented peptides thereof as molecular weight markers that allow the estimation of the
  • IL-17RH polypeptides encompasses methods for using IL-17RH polypeptides and fragmented peptides thereof as
  • invention also encompasses methods for using IL- 17RH polypeptides and fragmented peptides
  • Figure 1 is the nucleotide sequence of IL-17RH DNA, SEQ ID NO:l.
  • Figure 2 is the amino acid sequence of IL-17RH polypeptide, SEQ ID NO:2.
  • a cDNA encoding human IL-17RH polypeptide has been isolated and is disclosed in
  • polypeptides comprising polypeptides; host cells transfected or transformed with the expression vectors; biologically
  • IL- 17RH functions in human IL- 17 receptor (IL- 17R)
  • signaling enables the design of assays to detect inhibitors of IL-17R activity.
  • IL-17RH DNA was originally seen as three partial EST clones in the public databases.
  • the ESTs were re-sequenced and found to be divergent from one another.
  • the consensus cDNA The consensus cDNA
  • SEQ ID NO:l encodes IL-17RH polypeptide (SEQ ID NO:2), which exhibits 24%
  • homo logy occurs within a portion of the IL-17R known to be within the cytoplasmic domain.
  • IL-17RH polypeptide encodes a receptor, which is related to the IL-17R; it can
  • the cytoplasmic domain indicates that IL-17RH polypeptide is capable of signaling. Therefore,
  • IL-17RH polypeptide can induce NFkB and regulate the production of heterologous cytokines.
  • polypeptides can be accomplished utilizing fusion of sequences encoding IL-17RH polypeptides
  • a fusion is a fusion of sequences encoding an IL-17RH polypeptide to
  • construction of the insertion contains a termination codon adjoining the carboxyl terminal codon
  • a DNA fragment can be generated by PCR
  • oligonucleotide primers generates a blunt-ended fragment of DNA that can be isolated by
  • This PCR product can be ligated together with pMAL-p2 (digested with
  • Another preferred embodiment of the invention is the use of IL-17RH polypeptides as
  • the IL-17RH polypeptide together with a sample protein, can be
  • the unique amino acid sequence of IL-17RH (SEQ ID NO:2) specifies a
  • molecular weight marker serves particularly well as a molecular weight marker for the estimation
  • Another preferred embodiment of the invention is the use of IL-17RH fragmented peptide
  • Isolated and purified IL-17RH polypeptide can be treated with cyanogen
  • polypeptide molecular weight markers with cyanogen bromide generates a unique set of IL-
  • each peptide determines its molecular weight.
  • treatment of IL-17RH polypeptide with cyanogen bromide comprises 3 fragmented peptides of at
  • the peptide encoded by amino acids 1-112 of SEQ ID NO:2 has a
  • SEQ ID NO:2 has a molecular weight of approximately 10,451 Daltons.
  • cyanogen bromide generates a unique set of IL-17RH fragmented peptide molecular weight
  • IL-17RH fragmented peptide molecular weight markers have
  • the IL-17RH fragmented peptide molecular weight markers can be used as
  • the unique amino acid sequence of IL-17RH specifies a molecular weight of
  • the IL-17RH fragmented peptide molecular weight markers serve as the IL-17RH fragmented peptide molecular weight markers. Therefore, the IL-17RH fragmented peptide molecular weight markers serve as the IL-17RH fragmented peptide molecular weight markers.
  • sample protein and the IL-17RH polypeptide can be any suitable polypeptide.
  • the sample protein and the IL-17RH polypeptide can be any suitable polypeptide.
  • fragmented peptides from both the IL-17RH polypeptide and the sample protein can be any fragmented peptides from both the IL-17RH polypeptide and the sample protein.
  • Fragmented peptides on the gel can be visualized using a conventional
  • the IL-17RH fragmented peptide molecular weight markers can be used as
  • molecular weight markers serve particularly well as molecular weight markers for the estimation
  • markers can be generated from IL-17RH polypeptide using enzymes that cleave the polypeptide
  • An isolated and purified IL-17RH polypeptide can be treated with Achromobacter
  • protease I under conventional conditions that result in fragmentation of the IL-17RH polypeptide
  • Achromobacter protease I generates a unique set of IL-17RH fragmented peptide molecular
  • weight markers The distribution of lysine residues determines the number of amino acids in each peptide and the unique amino acid composition of each peptide determines its molecular
  • treatment of IL-17RH polypeptide with Achromobacter protease I comprises 12 fragmented
  • the peptide encoded by amino acids 15-24 of SEQ ID NO:2 has a molecular weight of
  • the peptide encoded by amino acids 25-45 of SEQ ID NO:2 has a
  • SEQ ID NO:2 has a molecular weight of approximately 1 , 192 Daltons.
  • amino acids 57-69 of SEQ ID NO:2 has a molecular weight of approximately 1 ,404 Daltons.
  • the peptide encoded by amino acids 170-103 of SEQ ID NO:2 has a molecular weight of
  • the peptide encoded by amino acids 109-122 of SEQ ID NO:2 has
  • the peptide encoded by amino acids 179-190 of SEQ ID NO:2 has a molecular
  • ID NO:2 has a molecular weight of approximately 1,223 Daltons.
  • amino acids 207-219 of SEQ ID NO:2 has a molecular weight of approximately 1,416 Daltons.
  • the peptide encoded by amino acids 227-238 of SEQ ID NO:2 has a molecular weight of
  • Achromobacter protease I generates a unique set of IL-17RH fragmented peptide molecular
  • weight markers have molecular weights of approximately 1,167; 2,635; 1,192; 1,404; 4,096;
  • sample protein can be resolved by denaturing polyacrylamide gel electrophoresis by
  • the sample protein The IL-17RH fragmented peptide molecular weight markers serve
  • sample protein and the IL-17RH polypeptide can be any suitable polypeptide.
  • the sample protein and the IL-17RH polypeptide can be any suitable polypeptide.
  • IL-17RH polypeptide The IL-17RH fragmented peptide molecular weight markers and the
  • fragmented peptides derived from the sample protein are resolved by denaturing polyacrylamide
  • molecular weight markers can be used as molecular weight markers in the estimation of the
  • weight markers serve particularly well as molecular weight markers for the estimation of the
  • monoclonal and polyclonal antibodies against IL-17RH are provided.
  • polypeptides can be generated.
  • Balb/c mice can be injected intraperitoneally on two occasions at
  • mice are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Three weeks later, mice are
  • mice Three days later, mice are sacrificed and spleen cells fused with Ag8.653 myeloma cells
  • mice spleen cells serum-free media and fused to mouse spleen cells at a ratio of three spleen cells to one myeloma
  • the fusing agent is 50% PEG: 10% DMSO (Sigma). Fusion is plated out into twenty 96-
  • 17RH polypeptide or peptides are added to each well, incubated for 60 minutes at room
  • antibodies generated against IL-17RH and fragmented peptides are generated against IL-17RH and fragmented peptides.
  • polypeptide or fragmented peptide molecular weight markers can be mixed with a molar excess
  • Polypeptides can be transferred to a suitable protein binding membrane,
  • polypeptides on the membrane can be visualized using two different methods that allow a
  • polypeptide or fragmented peptide molecular weight markers can be visualized using antibodies
  • polypeptide fragments since small peptides may not contain immunogenic epitopes. It is furthermore than immunogenic epitopes.
  • the sample protein is visualized using a conventional staining procedure.
  • the molar ratio of molar fraction is visualized using a conventional staining procedure.
  • markers is such that the conventional staining procedure predominantly detects the sample
  • the level of IL-17RH polypeptide or fragmented peptide molecular weight markers is
  • sample protein between 2 and 100,000 fold. More preferably, the preferred molar excess of sample protein to
  • IL-17RH polypeptide molecular weight markers is between 10 and 10,000 fold and especially
  • the IL- 17RH polypeptide or fragmented peptide molecular weight markers can be used
  • peptide molecular weight markers serve particularly well as molecular weight and isoelectric
  • polypeptide or fragmented peptide molecular weight markers The ability to simultaneously
  • sample protein under identical conditions allows for increased accuracy in the determination of
  • IL-17RH polypeptide or fragmented peptide molecular weight In another embodiment, IL-17RH polypeptide or fragmented peptide molecular weight
  • markers can be used as molecular weight and isoelectric point markers in the estimation of the
  • sample protein with a cleavage agent. It is understood of course that many techniques can be
  • IL- 17RH polypeptide molecular weight markers encompassed by invention can have
  • variable molecular weights depending upon the host cell in which they are expressed.
  • yeast two-hybrid system For example, the yeast two-hybrid system
  • IL- 17RH screen for inhibitors of IL- 17RH as follows.
  • the screen can be modified so that IL-
  • microtiter plate to couple an easily detected indicator to the other component.
  • IL-17RH polypeptides according to the invention are useful for the structure-
  • Antibodies immunoreactive with IL-17RH polypeptides and in particular, monoclonal
  • antibodies can be useful for inhibiting IL-17RH polypeptide activity in vivo and for detecting the
  • IL-17RH polypeptides refers to a genus of polypeptides that
  • IL-17RH polypeptides refers
  • the isolated and purified IL-17RH polypeptide according to the invention has a
  • IL-17RH polypeptides can be varied by fusing additional peptide sequences to both the amino acids
  • IL-17RH polypeptides can be used to enhance the amino and carboxyl terminal ends of IL-17RH polypeptides.
  • IL-17RH polypeptides or aid in the purification of the protein.
  • peptides of IL-17RH polypeptides generated by enzymatic or chemical treatment.
  • isolated and purified means that the IL-17RH polypeptide
  • molecular weight markers or fragments thereof are essentially free of association with other
  • proteins or polypeptides for example, as a purification product of recombinant host cell culture
  • substantially purified refers to a mixture that contains IL-17RH polypeptide molecular weight markers or
  • substantially purified IL-17RH polypeptides or fragments thereof can be used as molecular
  • purified refers to either the “isolated and purified” form of IL-17RH
  • nucleotide sequence refers to a polynucleotide molecule in the form of a separate
  • RNA isolated at least once in substantially pure form i.e., free of contaminating endogenous
  • IL-17RH polypeptide "variant" as referred to herein means a polypeptide substantially
  • variant amino acid sequence preferably
  • IL-17RH polypeptide amino acid sequence is at least 80% identical to a native IL-17RH polypeptide amino acid sequence, most preferably
  • the percent identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et
  • the preferred default parameters for the GAP program include: (1) a unary
  • comparison matrix (containing a value of 1 for identities and 0 for non-identities) for
  • Variants can comprise conservatively substituted sequences, meaning that a given amino
  • conservative substitutions include substitution of one aliphatic residue for another, such as He,
  • Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as
  • Naturally occurring IL-17RH variants are also encompassed by the invention.
  • proteolytic cleavage of the IL-17RH polypeptides Variations attributable to proteolysis include,
  • the invention provides isolated and purified, or homogeneous, IL-17RH
  • polypeptides both recombinant and non-recombinant.
  • 17RH polypeptides that can be used as molecular weight markers can be obtained by mutations
  • amino acid sequence can be accomplished by any of a number of conventional methods.
  • Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a
  • mutant sequence flanked by restriction sites enabling ligation to fragments of the native
  • oligonucleotide-directed site-specific mutagenesis procedures can be used.
  • IL-17RH polypeptides can be modified to create IL-17RH polypeptide derivatives by
  • PEG polyethylene glycol
  • Covalent derivatives of IL-17RH polypeptides can be prepared by linking the chemical moieties
  • IL-17RH polypeptides within the scope of this invention include covalent or aggregative conjugates of IL-17RH polypeptides or peptide fragments with other proteins or polypeptides,
  • N-terminal or C-terminal fusions such as by synthesis in recombinant culture as N-terminal or C-terminal fusions.
  • the conjugate can comprise a signal or leader polypeptide sequence (e.g. the ⁇ -factor leader of
  • IL-17RH polypeptide conjugates can comprise peptides added to facilitate purification
  • IL-17RH polypeptides include, for example, poly-His or the
  • the invention further includes IL-17RH polypeptides with or without associated native-
  • COS-1 or COS-7 cells can be similar to or significantly different from a native
  • IL-17RH polypeptide in molecular weight and glycosylation pattern, depending upon the choice
  • E. coli provides non-glycosylated molecules. Glycosyl groups can be removed through
  • 17RH polypeptides can be incubated with a molar excess of glycopeptidase (Boehringer
  • N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any
  • nucleotide sequence encoding these triplets will result in prevention of attachment of
  • EP 212,914 discloses
  • KEX2 protease processing sites are inactivated by deleting, adding, or substituting residues to
  • Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion
  • the invention further encompasses isolated fragments and oligonucleotides derived from
  • the invention also encompasses polypeptides
  • Nucleic acid sequences within the scope of the invention include isolated DNA and RNA
  • conditions of moderate or severe stringency and which encode IL-17RH polypeptides.
  • conditions of moderate stringency as known to those having ordinary skill in the art, and
  • nitrocellulose filters 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of
  • Conditions of high stringency are defined as hybridization conditions as above, and with
  • wash solution salt concentration can be adjusted as necessary according to factors such as the
  • a DNA sequence can vary from that shown in SEQ ID NO:l and
  • variant DNA sequences can result from silent mutations (e.g., occurring during PCR
  • amplification or can be the product of deliberate mutagenesis of a native sequence.
  • the invention thus provides equivalent isolated DNA sequences encoding IL-17RH
  • polypeptides selected from: (a) DNA derived from the coding region of a native mammalian IL-
  • polypeptides encoded by such DNA equivalent sequences are encompassed by the invention.
  • 17RH polypeptides encoded by such DNA include, but are not limited to, IL-17RH polypeptide
  • IL-17RH polypeptide-binding proteins such as the anti-IL-17RH polypeptide antibodies
  • a solid phase such as a column chromatography matrix or a
  • phase contacting surface can be accomplished by any means, for example, magnetic
  • microspheres can be coated with IL-17RH polypeptide-binding proteins and held in the
  • polypeptides on their surface bind to the fixed IL-17RH polypeptide-binding protein and
  • releasing positively selected cells from the solid phase are known in the art and encompass, for
  • enzymes are preferably non-toxic and non-injurious to the
  • cells and are preferably directed to cleaving the cell-surface binding partner.
  • cells first can be incubated with a biotinylated IL-17RH polypeptide-binding protein. Incubation
  • periods are typically at least one hour in duration to ensure sufficient binding to IL-17RH
  • the resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the IL-17RH
  • polypeptide-binding cells to the beads.
  • avidin-coated beads Use of avidin-coated beads is known in the art. See
  • the bound cells is performed using conventional methods.
  • IL-17RH polypeptide-binding proteins are anti-semiconductor proteins
  • IL-17RH polypeptide antibodies and other proteins that are capable of high-affinity binding of
  • IL-17RH polypeptides A preferred IL-17RH polypeptide-binding protein is an anti-IL-17RH
  • IL-17RH polypeptides can exist as oligomers, such as covalently linked or non-
  • Oligomers can be linked by disulfide bonds formed between
  • cysteine residues on different IL-17RH polypeptides are cysteine residues on different IL-17RH polypeptides.
  • IL-17RH polypeptide dimer is created by fusing IL-17RH polypeptides to the Fc region of an
  • the Fc polypeptide preferably is fused to the C-terminus of a soluble IL-17RH
  • polypeptide comprising only the extracellular domain.
  • IL-17RH polypeptide:Fc fusion proteins are allowed to assemble much like antibody
  • IL-17RH polypeptides divalent IL-17RH polypeptides. If fusion proteins are made with both heavy and light chains of
  • an antibody it is possible to form an IL-17RH polypeptide oligomer with as many as four IL- 17RH polypeptides extracellular regions.
  • polypeptide domains with a peptide linker polypeptide domains with a peptide linker
  • polypeptides can be prepared using well known methods.
  • the expression vectors include an IL-
  • nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, an
  • a promoter functionally relates to the IL-17RH DNA sequence.
  • a promoter functionally relates to the IL-17RH DNA sequence.
  • nucleotide sequence is operably linked to an IL-17RH DNA sequence if the promoter nucleotide
  • the desired host cells usually conferred by an origin of replication, and a selection gene by
  • transformants which transformants are identified can additionally be incorporated into the expression vector.
  • IL-17RH polypeptides can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide (secretory leader) can be fused in-frame to the IL-
  • a signal peptide that is functional in the intended host comprising the signal peptide.
  • the signal peptide can be any amino acid sequence that influences extracellular secretion of the IL-17RH polypeptide.
  • the signal peptide can be
  • Suitable host cells for expression of IL-17RH polypeptides include prokaryotes, yeast or
  • Prokaryotes include gram negative or gram positive organisms, for example, E. coli or
  • Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus
  • subtilis subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas,
  • polypeptide can include an N-terminal methionine residue to facilitate expression of the
  • the N-terminal Met can be cleaved from
  • Expression vectors for use in prokaryotic host cells generally comprise one or more
  • a phenotypic selectable marker gene is, for example, a
  • Examples of useful expression vectors for prokaryotic host cells include those
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides
  • vectors include, for example, pKK223-3 (Pharmacia Fine).
  • proteins these would include pMAL-p2 and pMAL-c2 vectors that are used for the expression of
  • vectors include ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature
  • particularly useful prokaryotic host cell expression system employs a phage ⁇ P L promoter and a
  • thermolabile repressor sequence Plasmid vectors available from the American Type
  • IL-17RH DNA may be cloned in- frame into the multiple cloning site of an ordinary
  • the vector would contain an inducible promoter upstream of
  • fusion partner such as
  • plasmid may be propagated in a variety of strains of E. coli.
  • the bacterial cells are propagated in growth
  • IPTG isopropyl-b-D-thiogalactopyranoside
  • the cells are harvested by pelleting in a centrifuge, e.g. at 5,000 x G for
  • the pelleted cells may be resuspended in ten
  • inclusion bodies can be purified away from the soluble proteins by pelleting in
  • Tris-HCl pH 8)/l% Triton X-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/ 0.1
  • the protein of interest will, in most cases, be the most abundant protein in the
  • This protein may be "refolded" into the active conformation by
  • initial purification may be carried out
  • hexahistidine-tagged fusion proteins may be partially purified
  • IL-17RH polypeptides alternatively can be expressed in yeast host cells, preferably from
  • Saccharomyces genus e.g., S. cerevisiae
  • Other genera of yeast such as Pichia , K. lactis,
  • Yeast vectors will often contain an origin of
  • replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a
  • Suitable promoter sequences for yeast vectors include, among others,
  • isomerase 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
  • phosphoglucose isomerase phosphoglucose isomerase
  • glucokinase phosphoglucokinase
  • yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et. al., Gene,
  • E. coli can be constructed by inserting DNA sequences from pBR322 for selection and
  • yeast -factor leader sequence can be employed to direct secretion of an IL-17RH
  • the ⁇ -factor leader sequence is often inserted between the promoter sequence and
  • yeast hosts suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to
  • a leader sequence can be modified near its 3' end to contain one or more
  • Trp + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine, and
  • Yeast host cells transformed by vectors containing ADH2 promoter sequence can be
  • a rich medium is one
  • Mammalian or insect host cell culture systems could also be employed to express
  • mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)
  • CHO hamster ovary
  • HeLa cells HeLa cells
  • BHK ATCC CRL 10
  • 1/EBNA-l cell line (ATCC CRL 10478) derived from the African green monkey kidney cell line
  • Lipofectamine (Gibco/BRL) or Lipofectamine-
  • electroporation can be used to transfect mammalian cells using conventional procedures
  • DHFR reductase resistance
  • a suitable host strain for DHFR selection can be CHO strain DX-
  • a plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only
  • selectable markers that can be incorporated into an expression vector include cDNAs conferring
  • Cells harboring the vector can be any suitable antibiotics, such as G418 and hygromycin B.
  • Cells harboring the vector can be any antibiotics, such as G418 and hygromycin B.
  • Cells harboring the vector can be any suitable antibiotics, such as G418 and hygromycin B.
  • vectors can be excised from viral genomes.
  • Commonly used promoter sequences and enhancers are commonly used promoter sequences and enhancers
  • sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human
  • cytomegalo virus DNA sequences derived from the SV40 viral genome, for example, SV40
  • Viral early and late promoters are particularly useful because both are easily obtained from
  • a viral genome as a fragment which can also contain a viral origin of replication (Fiers et al.,
  • fragments can also be used, provided the approximately 250 bp sequence extending from the
  • Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • mammalian expression vectors include such elements as the expression augmenting sequence
  • EASE EASE element
  • TPL tripartite leader
  • VA gene RNAs from Adenovirus 2
  • DHFR selectable marker
  • Exemplary expression vectors that employ dicistronic mRNAs are pTR-
  • pCAVNOT A useful high expression vector, pCAVNOT, has been described by Mosley et al., Cell
  • the vectors can be derived from retroviruses. In place of the
  • a heterologous signal sequence can be added, such as the signal sequence
  • IL-17RH polypeptides can be substantially purified, as indicated
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • One process for producing IL-17RH polypeptides comprises culturing a host cell
  • 17RH polypeptide is then recovered from culture medium or cell extracts, depending upon the
  • recombinant protein will vary according to such factors as the type of host cells employed and
  • concentrate can be applied to a purification matrix such as a gel filtration medium.
  • an anion exchange resin can be employed, for example, a matrix or substrate
  • the matrices can be acrylamide, agarose,
  • dextran cellulose or other types commonly employed in protein purification.
  • cellulose cellulose or other types commonly employed in protein purification.
  • cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble
  • matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other
  • aliphatic groups can be employed to further purify IL-17RH polypeptides.
  • IL-17RH polypeptides can be removed from an affinity
  • Recombinant protein produced in bacterial culture is usually isolated by initial disruption
  • Microbial cells can be employed for final purification steps.
  • Microbial cells can be disrupted by any convenient
  • Transformed yeast host cells are preferably employed to express IL-17RH polypeptides
  • Urdal et al. (J. Chromatog. 296:111, 1984). Urdal et al. describe two sequential, reversed-phase
  • IL-17RH polypeptide molecular weight markers can be analyzed by methods including
  • polypeptides can serve as molecular weight markers using such analysis techniques to assist in
  • IL-17RH polypeptides can be subjected to fragmentation into peptides by chemical and
  • Chemical fragmentation includes the use of cyanogen bromide to cleave
  • fragmentation includes the use of a protease such as Asparaginylendopeptidase,
  • Endoproteinase Asp-N, or Endoproteinase Lys-C under conventional conditions to result in
  • Asparaginylendopeptidase can cleave specifically on
  • Arginylendopeptidase can cleave specifically on the carboxyl side of the arginine residues
  • Achrombobacter protease I can cleave specifically on the
  • Trypsin can cleave specifically on the carboxyl
  • aureus V8 protease can cleave specifically on the carboxyl side of the aspartic and glutamic acid
  • Endoproteinase Asp-N can cleave specifically on the amino side of the asparagine
  • Endoproteinase Lys-C can cleave specifically on
  • the resultant fragmented peptides can be analyzed by methods including sedimentation,
  • IL-17RH polypeptides can serve as molecular weight markers using such analysis techniques to
  • molecular weight markers are preferably between 10 and 237 amino acids in size. More
  • IL-17RH fragmented peptide molecular weight markers are between 10 and 100
  • markers between 10 and 50 amino acids in size and especially between 10 and 35 amino acids in
  • IL-17RH fragmented peptide molecular weight markers between 10
  • polypeptide and sample protein under identical conditions can allow for a direct comparison of
  • polypeptide can also result in complete fragmentation of the sample protein.
  • IL-17RH polypeptides and fragmented peptides thereof possess unique
  • isoelectric focusing The technique of isoelectric focusing can be further combined with other
  • IL-17RH polypeptides and fragmented peptides thereof can be used in such
  • sample protein can be assembled from IL-17RH polypeptides and peptide fragments thereof.
  • Kits also serve to assess the degree of fragmentation of a sample protein.
  • kits can be varied, but typically contain IL-17RH polypeptide and fragmented peptide
  • kits can contain IL-17RH polypeptides wherein a site
  • kits can contain reagents for
  • Kits can further contain antibodies directed against IL-17RH polypeptides or fragments thereof.
  • Antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence
  • RNA or DNA capable of binding to a target IL-17RH mRNA sequence (forming a
  • Antisense or sense oligonucleotides can be made according to the invention.
  • Antisense or sense oligonucleotides according to the invention.
  • present invention comprise a fragment of the coding region of IL-17RH cDNA (SEQ ID NO:l).
  • Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to
  • cDNA sequence for a given protein is described in, for example, Stein and Cohen, Cancer Res.
  • RNA translation
  • DNA transcription
  • antisense oligonucleotides thus can be used
  • Antisense or sense oligonucleotides further include
  • oligonucleotides having modified sugar-phosphodiester backbones or other sugar
  • oligonucleotides with resistant sugar linkages are stable in vivo
  • oligonucleotides that are covalently linked to organic moieties, such as those
  • target nucleic acid sequence such as poly-(L- lysine).
  • intercalating agents such as
  • ellipticine, and alkylating agents or metal complexes can be attached to sense or antisense
  • oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the
  • Antisense or sense oligonucleotides can be introduced into a cell containing the target
  • nucleic acid sequence by any gene transfer method, including, for example, CaPO 4 -mediated
  • DNA transfection electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
  • Antisense or sense oligonucleotides are preferably introduced into a cell containing the target
  • nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable
  • Suitable retroviral vectors include, but are not limited to, the
  • DCT5 A vectors designated DCT5 A, DCT5B and DCT5C (see PCT Application US 90/02656).
  • Sense or antisense oligonucleotides also can be introduced into a cell containing the
  • Suitable ligand binding molecules include, but are not limited to,
  • cell surface receptors include growth factors, other cytokines, or other ligands that bind to cell surface
  • conjugation of the ligand binding molecule does not substantially interfere
  • a sense or an antisense oligonucleotide can be introduced into a cell
  • oligonucleotide- lipid complex containing the target nucleic acid sequence by formation of an oligonucleotide- lipid complex
  • the sense or antisense oligonucleotide-lipid complex is preferably
  • Isolated and purified IL-17RH polypeptides or a fragment thereof can also be useful itself
  • IL-17RH polypeptides can be
  • IL-17RH DNA, IL-17RH polypeptides, and antibodies against IL-17RH polypeptides can be any suitable IL-17RH DNA, IL-17RH polypeptides, and antibodies against IL-17RH polypeptides.
  • these reagents can serve as markers for cell
  • RNA or proteins specific or tissue specific expression of RNA or proteins.
  • these reagents can be used
  • IL-17RH IL-17RH RNA or polypeptides.
  • DNA can be used to determine the chromosomal location of IL-17RH DNA and to map genes in
  • IL-17RH DNA can also be used to examine genetic
  • IL-17RH DNA can be further used to
  • IL-17RH DNA and polypeptides can be used to select for those
  • IL-17RH polypeptides can also be used as a reagent to identify (a) any protein that IL-
  • polypeptides could be used by coupling recombinant protein to an affinity matrix, or by using
  • IL-17RH polypeptides When used as a therapeutic agent, IL-17RH polypeptides can be formulated into
  • IL-17RH polypeptides can be any suitable polypeptide.
  • IL-17RH polypeptides can be any suitable polypeptide.
  • diluents e.g., Tris-HCl, acetate, phosphate
  • preservatives e.g., sodium bicarbonate
  • compositions can contain IL-17RH
  • polypeptides complexed with polyethylene glycol (PEG), metal ions, or incorporated into PEG
  • PEG polyethylene glycol
  • polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated
  • IL-17RH polypeptides and peptides based on the
  • amino acid sequence of IL-17RH can be utilized to prepare antibodies that specifically bind to
  • IL-17RH polypeptides IL-17RH polypeptides.
  • antibodies is meant to include polyclonal antibodies,
  • Antibodies are defined to be specifically binding if
  • binding partners or antibodies can be readily determined using conventional techniques, for example
  • Polyclonal antibodies can be readily generated from a variety of sources, for example,
  • polypeptides can be enhanced through the use of an adjuvant, for example, Freimd's complete or
  • CIEP immuno-electrophoresis
  • ELISA immuno-sorbent assays
  • dot blot assays dot blot assays
  • sandwich assays see U.S. Patent Nos.
  • Monoclonal antibodies can be readily prepared using well-known procedures, see for
  • mice are injected intraperitoneally at least once, and preferably at least twice at about 3 week
  • Mouse sera are then assayed by conventional dot blot technique or antibody capture (IL-17RH) to determine which animal is best to fuse.
  • IL-17RH antibody capture
  • mice are given an intravenous boost of IL-17RH
  • the myeloma cells are washed several times in media and fused to
  • mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • PEG polyethylene glycol
  • the fused cells can then be allowed to grow for approximately eight days.
  • Supernatants from the fused cells can then be allowed to grow for approximately eight days.
  • resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse

Landscapes

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

Abstract

ADN codant Ies polypeptides IL-17RH et procédés d'utilisation desdits polypeptides codés.
EP99903032A 1998-01-09 1999-01-08 Adn codant il-17rh et polypeptides il-17rh Withdrawn EP1045905A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US7107398P 1998-01-09 1998-01-09
US71073P 1998-01-09
PCT/US1999/000515 WO1999035263A2 (fr) 1998-01-09 1999-01-08 Adn codant il-17rh et polypeptides il-17rh

Publications (1)

Publication Number Publication Date
EP1045905A2 true EP1045905A2 (fr) 2000-10-25

Family

ID=22099081

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99903032A Withdrawn EP1045905A2 (fr) 1998-01-09 1999-01-08 Adn codant il-17rh et polypeptides il-17rh

Country Status (5)

Country Link
EP (1) EP1045905A2 (fr)
JP (1) JP2003502006A (fr)
AU (1) AU2315099A (fr)
CA (1) CA2317817A1 (fr)
WO (1) WO1999035263A2 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6482923B1 (en) 1997-09-17 2002-11-19 Human Genome Sciences, Inc. Interleukin 17-like receptor protein
EP1015488B1 (fr) * 1997-09-17 2009-09-09 Human Genome Sciences, Inc. Proteine de type recepteur d'interleukine 17
US6849719B2 (en) 1997-09-17 2005-02-01 Human Genome Sciences, Inc. Antibody to an IL-17 receptor like protein
US8133734B2 (en) 1999-03-16 2012-03-13 Human Genome Sciences, Inc. Kit comprising an antibody to interleukin 17 receptor-like protein
TWI322154B (en) * 2000-03-16 2010-03-21 Amgen Inc Il-17 receptor like molecules and uses thereof
US7094566B2 (en) 2000-03-16 2006-08-22 Amgen Inc., IL-17 receptor like molecules and uses thereof
WO2002008285A2 (fr) * 2000-06-22 2002-01-31 Amgen, Inc. Molecules il-17 et leurs utilisations
GB0417487D0 (en) 2004-08-05 2004-09-08 Novartis Ag Organic compound

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5449758A (en) * 1993-12-02 1995-09-12 Life Technologies, Inc. Protein size marker ladder
JP4373495B2 (ja) * 1995-03-23 2009-11-25 イミュネックス・コーポレーション Ｉｌ−１７受容体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9935263A3 *

Also Published As

Publication number Publication date
AU2315099A (en) 1999-07-26
CA2317817A1 (fr) 1999-07-15
WO1999035263A2 (fr) 1999-07-15
WO1999035263A3 (fr) 1999-09-10
JP2003502006A (ja) 2003-01-21

Similar Documents

Publication Publication Date Title
US20050233355A1 (en) Human cDNAs encoding polypeptides having kinase functions
EP1045905A2 (fr) Adn codant il-17rh et polypeptides il-17rh
WO1999033983A1 (fr) Adn et polypeptides v201
US20090017492A1 (en) SVPH1-26 DNA and polypeptides
EP1045914B1 (fr) Svph1-8 proteinase humaine specifique aux testicules
US20050101768A1 (en) H14 DNA and polypeptides
EP1105485B1 (fr) Adn humain il-1 epsilon et polypeptides
AU745623B2 (en) V196 DNA and polypeptides
US7217553B2 (en) Nucleic acids encoding human adamalysin SVPH1-8
WO2000008178A2 (fr) Molecules d'adn murin codant pour des polypeptides presentant des fonctions kinases
US7250276B2 (en) Human IL-1 epsilon DNA and polypeptides
WO1999033984A1 (fr) Adn et polypeptides v197

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000807

AK Designated contracting states

Kind code of ref document: A2

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

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

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

18D Application deemed to be withdrawn

Effective date: 20020801