WO2001016313A1 - Gene de regulation de la proteine gs humaine - Google Patents

Gene de regulation de la proteine gs humaine Download PDF

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Publication number
WO2001016313A1
WO2001016313A1 PCT/GB2000/003372 GB0003372W WO0116313A1 WO 2001016313 A1 WO2001016313 A1 WO 2001016313A1 GB 0003372 W GB0003372 W GB 0003372W WO 0116313 A1 WO0116313 A1 WO 0116313A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
sequence
polynucleotide
seq
isolated
Prior art date
Application number
PCT/GB2000/003372
Other languages
English (en)
Inventor
David Malcolm Duckworth
Paul Russell Murdock
Tania Tamson Testa
Christopher Geoffrey Carson Larminie
Original Assignee
Smithkline Beecham P.L.C.
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
Priority claimed from GBGB9920746.6A external-priority patent/GB9920746D0/en
Priority claimed from GB0006577A external-priority patent/GB0006577D0/en
Application filed by Smithkline Beecham P.L.C. filed Critical Smithkline Beecham P.L.C.
Priority to EP00956718A priority Critical patent/EP1127118A1/fr
Publication of WO2001016313A1 publication Critical patent/WO2001016313A1/fr

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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO: 1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR).
  • Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SEQ ID NO: 1 , typically at least 95% identity.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification. Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene.
  • the polynucleotide sequences of the present invention are valuable for chromosome localisation studies.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. Mc usick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • a further aspect of the present invention relates to antibodies.
  • the polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997).
  • Fusion proteins such as those made from Fc portion and RGS 17LIKE polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).
  • polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the- effect of added compounds on the production of mRNA and polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • transgenic animals are so-called "knock-out" animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled.
  • the gene knockout may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal.
  • Screening kits for use in the above described methods form a further aspect of the present invention.
  • Such screening kits comprise: (a) a polypeptide of the present invention.
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Polynucleotide generally refers to any polyribo ⁇ ucleotide (RNA) or polydeoxribonucleotide (DNA), which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post- translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide.
  • “Fragment” of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO: 1.
  • “Variant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide.
  • Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination.
  • polypeptides having one or more post-translational modifications for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like.
  • Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines.
  • Allele refers to one of two or more alternative forms of a gene occuring at a given locus in the genome.
  • SNP Single Nucleotide Polymo ⁇ hism
  • SNPs can be assayed using Allele Specific Amplification (ASA).
  • ASA Allele Specific Amplification
  • a common primer is used in reverse complement to the polymo ⁇ hism being assayed. This common primer can be between 50 and 1500 bps from the polymo ⁇ hic base.
  • the other two (or more) primers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymo ⁇ hism. Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • BESTFIT and GAP may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences.
  • BESTFIT uses the "local homology" algorithm of Smith and Waterman (J Mol Biol, 147,195- 197, 1981, Advances in Applied Mathematics, 2, 482-489, 1981) and finds the best single region of similarity between two sequences.
  • BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These differences may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • an average of up to 5 in every 100 of the amino acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described.
  • n a is the number of nucleotide or amino acid differences
  • x a is the total number of nucleotides or amino acids in SEQ ID NO: 1 or SEQ ID NO:2, respectively
  • an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232262].
  • RGS17LIKE The expression pattern of RGS17LIKE was investigated using Sybr-Green fluorescent PCR (Perkin Elmer) and human cDNAs prepared from various brain areas and peripheral tissues. All Sybr-Green analysis was carried out according to the manufacturers instructions using the following oligonucleotides:

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des ploypeptides et des polynucleotides de type RGS 17 ainsi que des procédés de production desdits polypeptides au moyen de techniques de recombinaison. Cette invention concerne également des procédés d'utilisation des polypeptides et des polynucleotides du type RGS 17 dans des doses diagnostiques.
PCT/GB2000/003372 1999-09-02 2000-09-01 Gene de regulation de la proteine gs humaine WO2001016313A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP00956718A EP1127118A1 (fr) 1999-09-02 2000-09-01 Gene de regulation de la proteine gs humaine

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB9920746.6A GB9920746D0 (en) 1999-09-02 1999-09-02 Novel compounds
GB9920746.6 1999-09-02
GB0006577.1 2000-03-17
GB0006577A GB0006577D0 (en) 2000-03-17 2000-03-17 Novel compounds

Publications (1)

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WO2001016313A1 true WO2001016313A1 (fr) 2001-03-08

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EP (1) EP1127118A1 (fr)
WO (1) WO2001016313A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020932A1 (fr) * 2001-09-03 2003-03-13 Takeda Chemical Industries, Ltd. Nouvelles proteines de secretion et adn associe

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JORDAN J DEDRICK ET AL: "Modulation of Rap activity by direct interaction of Galphao with Rap1 GTPase-activating protein.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 31, 30 July 1999 (1999-07-30), pages 21507 - 21510, XP002151346, ISSN: 0021-9258 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020932A1 (fr) * 2001-09-03 2003-03-13 Takeda Chemical Industries, Ltd. Nouvelles proteines de secretion et adn associe

Also Published As

Publication number Publication date
EP1127118A1 (fr) 2001-08-29

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