WO2000012703A2 - Protein transport-associated molecules - Google Patents
Protein transport-associated molecules Download PDFInfo
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- WO2000012703A2 WO2000012703A2 PCT/US1999/019616 US9919616W WO0012703A2 WO 2000012703 A2 WO2000012703 A2 WO 2000012703A2 US 9919616 W US9919616 W US 9919616W WO 0012703 A2 WO0012703 A2 WO 0012703A2
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- polynucleotide
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to nucleic acid and amino acid sequences of protein transport- associated molecules and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and secretory disorders.
- Signal peptides are found on proteins that are targeted to the endoplasmic reticulum (ER). Signal peptides consist of stretches of amino acids enriched in hydrophobic residues. Signal peptides are usually found at the extreme N-terminus of the protein and are recognized by a cytosolic signal-recognition peptide (SRP). The SRP binds to the signal peptide and to an SRP receptor, an integral membrane protein in the ER. Once bound to the SRP receptor, the newly formed protein containing the signal peptide is translocated across the ER membrane. Proteins containing signal peptides may end up inserted into the lipid bilayer, or they may end up in the lumen of an organelle or secreted from the cell.
- SRP cytosolic signal-recognition peptide
- plasma membrane receptors such as the epidermal growth factor (EGF) receptor and the T-cell receptor (TCR) contain targeting motifs which, when ligand binds to the receptor, become phosphorylated. Phosphorylation of the targeting motif results in internalization and delivery of the receptor to the lysosome for degradation.
- EGF epidermal growth factor
- TCR T-cell receptor
- the information provided by the amino acid motifs is used by the trafficking machinery to sequester and package the protein into vesicles, and then deliver it to the appropriate location.
- Sorting nexin-1 (SNX1) is an example of a protein involved in the recognition of motifs involved in lysosomal targeting.
- AP-1 vesicles which form at the Golgi, include cation-independent and cation-dependent mannose-6-phosphate receptors (MPRs). These vesicles are delivered to the lysosome.
- AP-2 vesicles which form at the plasma membrane, include TGN38 (Stephens, supra) and ligand-bound TCR (Dietrich, supra.)
- the invention features substantially purified polypeptides, protein transport-associated molecules, referred to collectively as "PTAM".
- PTAM protein transport-associated molecules
- the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8 (SEQ ID NO: 1-8), and fragments thereof.
- the invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof.
- the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -8, and fragments thereof.
- the invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof.
- the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof.
- the invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof.
- the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 (SEQ ID NO:9-16), and fragments thereof.
- the invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, and fragments thereof.
- the invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, and fragments thereof.
- the invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample.
- the method further comprises amplifying the polynucleotide prior to hybridization.
- the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof.
- the expression vector is contained within a host cell.
- the invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
- the invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
- the invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO: 1 -8, and fragments thereof.
- the invention also provides a purified agonist and a purified antagonist to the polypeptide.
- the invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of PTAM, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
- the invention also provides a method for treating or preventing a disorder associated with increased expression or activity of PTAM, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and fragments thereof.
- Table 1 shows nucleotide and polypeptide sequence identification numbers (SEQ ID NO), clone identification numbers (clone ID), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding PTAM.
- Table 2 shows features of each polypeptide sequence including potential motifs, homologous sequences, and methods and algorithms used for identification of PTAM.
- Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis, diseases or disorders associated with these tissues, and the vector into which each cDNA was cloned.
- Table 4 describes the tissues used to construct the cDNA libraries from which Incyte cDNA clones encoding PTAM were isolated.
- Table 5 shows the programs, their descriptions, references, and threshold parameters used to analyze PTAM.
- PTAM refers to the amino acid sequences of substantially purified PTAM obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
- agonist refers to a molecule which, when bound to PTAM, increases or prolongs the duration of the effect of PTAM.
- Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of PTAM.
- allelic variant is an alternative form of the gene encoding PTAM. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
- altered nucleic acid sequences encoding PTAM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as PTAM or a polypeptide with at least one functional characteristic of PTAM. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding PTAM, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding PTAM.
- the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent PTAM.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of PTAM is retained.
- negatively charged amino acids may include aspartic acid and glutamic acid
- positively charged amino acids may include lysine and arginine
- amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
- amino acid or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
- fragments or “antigenic fragments” refer to fragments of PTAM which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of PTAM.
- amino acid sequence is recited to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
- antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
- Antibodies that bind PTAM polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
- the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
- an animal e.g., a mouse, a rat, or a rabbit
- Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
- antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
- an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
- composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
- the composition may comprise a dry formulation or an aqueous solution.
- Compositions comprising polynucleotide sequences encoding PTAM or fragments of PTAM may be employed as hybridization probes.
- the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
- the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
- salts e.g., NaCl
- detergents e.g., sodium dodecyl sulfate; SDS
- other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
- Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW Fragment Assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
- correlates with expression of a polynucleotide indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding PTAM, by northern analysis is indicative of the presence of nucleic acids encoding PTAM in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding PTAM.
- derivative refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
- a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
- a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
- similarity refers to a degree of complementarity. There may be partial similarity or complete similarity.
- the word "identity” may substitute for the word “similarity.”
- a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
- the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
- a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction.
- the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
- the phrases "percent identity” or “% identity” refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Madison WI). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the clustal method.
- the clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
- the percentage similarity between two amino acid sequences e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity.
- Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
- Human artificial chromosomes are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
- humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
- Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
- a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
- insertion or “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
- Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
- factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
- microarray refers to an arrangement of distinct polynucleotides on a substrate.
- element or “array element” in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
- modulate refers to a change in the activity of PTAM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PTAM.
- nucleic acid or “nucleic acid sequence,” as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
- PNA peptide nucleic acid
- fragments refers to those nucleic acid sequences which comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:9-16, for example, as distinct from any other sequence in the same genome.
- a fragment of SEQ ID NO:9-16 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:9-16 from related polynucleotide sequences.
- a fragment of SEQ ID NO:9-16 is at least about 15-20 nucleotides in length. The precise length of the fragment of SEQ ID NO:9-16 and the region of SEQ ID NO:9-16 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
- a fragment when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
- operably associated or “operably linked” refer to functionally related nucleic acid sequences.
- a promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
- oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
- Oligonucleotide is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.
- PNA protein nucleic acid
- PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
- sample is used in its broadest sense.
- a sample suspected of containing nucleic acids encoding PTAM, or fragments thereof, or PTAM itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
- binding refers to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
- stringent conditions refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides.
- Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell.
- the method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- the term "transformed” cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
- a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
- SNPs single nucleotide polymorphisms
- nucleotide sequences which encode PTAM and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring PTAM under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PTAM or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-natural ly occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
- polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:9-16 and fragments thereof under various conditions of stringency.
- polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:9-16 and fragments thereof under various conditions of stringency.
- dhfr confers resistance to methotrexate
- neo confers resistance to the aminoglycosides neomycin and G-418
- als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
- Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
- marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- sequence encoding PTAM is inserted within a marker gene sequence
- transformed cells containing sequences encoding PTAM can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding PTAM under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells that contain the nucleic acid sequence encoding PTAM and that express PTAM may be identified by a variety of procedures known to those of skill in the art.
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PTAM include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- sequences encoding PTAM, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with nucleotide sequences encoding PTAM may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode PTAM may be designed to contain signal sequences which direct secretion of PTAM through a prokaryotic or eukaryotic cell membrane.
- a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
- CHO, HeLa, MDCK, HEK293, and WI38 Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities
- ATCC American Type Culture Collection
- natural, modified, or recombinant nucleic acid sequences encoding PTAM may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
- a chimeric PTAM protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PTAM activity.
- FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
- a fusion protein may also be engineered to contain a proteolytic cleavage site located between the PTAM encoding sequence and the heterologous protein sequence, so that PTAM may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10.) A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
- Fragments of PTAM may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton. supra pp. 55-60.)
- PTAM protein transport-associated molecules.
- the expression of PTAM is closely associated with cell proliferation. Therefore, in cell proliferative and secretory disorders where the expression or activity of PTAM is low, it is desirable to increase the expression or activity of PTAM. In cell proliferative and secretory disorders where the expression or activity of PTAM is high, it is desirable to decrease the expression or activity of PTAM.
- a vector capable of expressing PTAM or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder including, but not limited to, those described above.
- the polynucleotides encoding PTAM may be used for therapeutic pu ⁇ oses.
- the complement of the polynucleotide encoding PTAM may be used in situations in which it would be desirable to block the transcription of the mRNA.
- cells may be transformed with sequences complementary to polynucleotides encoding PTAM.
- complementary molecules or fragments may be used to modulate PTAM activity, or to achieve regulation of gene function.
- sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding PTAM.
- any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
- An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
- Such pharmaceutical compositions may consist of PTAM, antibodies to PTAM, and mimetics, agonists, antagonists, or inhibitors of PTAM.
- the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
- the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
- compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
- Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
- Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
- compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
- Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
- Non-lipid polycationic amino polymers may also be used for delivery.
- the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
- the determination of an effective dose is well within the capability of those skilled in the art.
- the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs.
- An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity.
- the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
- Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
- Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
- antibodies which specifically bind PTAM may be used for the diagnosis of cell proliferative and secretory disorders characterized by expression of PTAM, or in assays to monitor patients being treated with PTAM or agonists, antagonists, or inhibitors of PTAM.
- Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for PTAM include methods which utilize the antibody and a label to detect PTAM in human body fluids or in extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non- covalent attachment of a reporter molecule.
- a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding PTAM or closely related molecules may be used to identify nucleic acid sequences which encode PTAM.
- the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding PTAM, allelic variants, or related sequences.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
- Polynucleotide sequences encoding PTAM may be used for the diagnosis of cell proliferative and secretory disorders associated with expression of PTAM.
- disorders include, but are not limited to, cell proliferative disorders such as cancers, including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; immune disorders such as actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis,
- polynucleotide sequences encoding PTAM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered PTAM expression. Such qualitative or quantitative methods are well known in the art.
- the nucleotide sequences encoding PTAM may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
- the nucleotide sequences encoding PTAM may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding PTAM in the sample indicates the presence of the associated disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
- hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
- the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
- the presence of an abnormal amount of transcript (either under- or over-expressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
- a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
- Additional diagnostic uses for oligonucleotides designed from the sequences encoding PTAM may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro.
- Oligomers will preferably contain a fragment of a polynucleotide encoding PTAM, or a fragment of a polynucleotide complementary to the polynucleotide encoding PTAM, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
- Methods which may also be used to quantitate the expression of PTAM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
- radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
- the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
- oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
- the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymo ⁇ hisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
- nucleic acid sequences encoding PTAM may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
- the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA libraries.
- HACs human artificial chromosomes
- YACs yeast artificial chromosomes
- BACs bacterial artificial chromosomes
- PI constructions or single chromosome cDNA libraries.
- Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
- FISH Fluorescent in situ hybridization
- Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding PTAM on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
- the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
- In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
- any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
- PTAM in another embodiment, can be used for screening libraries of compounds in any of a variety of drug screening techniques.
- the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between PTAM and the agent being tested may be measured.
- Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
- This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with PTAM, or fragments thereof, and washed. Bound PTAM is then detected by methods well known in the art. Purified PTAM can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
- the nucleotide sequences which encode PTAM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- RNA was purchased from Clontech or isolated from tissues described in Table 4. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods. Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase.
- RNA was provided with RNA and constructed the corresponding cDNA libraries.
- cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
- cDNA was size-selected (300-1000 bp) using SEPHACRYL SI 000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
- cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
- Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
- Plasmids were recovered from host cells by in vivo excision, using the UNIZAP vector system (Stratagene) or cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep 96 plasmid kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
- the cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific, Sunnyvale CA) or MICROLAB 2200 (Hamilton, Reno NV) systems in combination with the PTC-200 thermal cyclers (MJ Research, Watertown MA).
- the cDNAs were sequenced using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols, base calling software, and kits.
- cDNAs were sequenced using the MEGABACE 1000 capillary electrophoresis sequencing system (Molecular Dynamics, Sunnyvale CA).
- cDNAs were amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
- cDNAs were sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were determined using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7.) Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V.
- the polynucleotide sequences derived from cDNA, extension, and shotgun sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art.
- Table 5 summarizes the software programs, descriptions, references, and threshold parameters used. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides a brief description thereof, the third column presents the references which are inco ⁇ orated by reference herein, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the probability the greater the homology). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, S. San Francisco CA) and LASERGENE software (DNASTAR).
- the polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation, using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
- the full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
- nucleic acid sequences of Incyte ESTs 12033, 1209687, 1717058, 1749964, 1856357, 1871275, 2645806, and 3437773 were used to design oligonucleotide primers for extending partial nucleotide sequence to full length.
- one primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide.
- Primers were used to facilitate the extension of the known sequence "outward" which generates amplicons containing new unknown nucleotide sequence for the region of interest.
- the initial primers were designed from the cDNA using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
- Step 3 68° C for 6 min
- Step 4 94° C for 15 sec
- Step 5 65° C for 1 min
- Step 6 68° C for 7 min
- Step 7 Repeat steps 4-6 for an additional 15 cycles
- Step 8 94° C for 15 sec
- Step 10 68° C for 7:15 min
- Step 11 Repeat steps 8-10 for an additional 12 cycles
- nucleotide sequences of SEQ ID NO:9-16 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
- a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
- An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
- a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
- nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
- PTAM expression and purification of PTAM is achieved using bacterial or virus-based expression systems.
- cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
- promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
- Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
- Antibiotic resistant bacteria express PTAM upon induction with isopropyl beta- D-thiogalactopyranoside (IPTG).
- PTAM is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
- GST glutathione S-transferase
- FLAG or 6-His a peptide epitope tag
- GST a 26-kilodalton enzyme from Schistosoma japonicum. enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
- the GST moiety can be proteolytically cleaved from PTAM at specifically engineered sites.
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry. Oxford, New York NY.
- Phred A base-calling algorithm that examines automated Ewing, B. et al. ( 1998) Genome sequencer traces with high sensitivity and probability. Res. 8: 175-185; Ewing, B. and P. Green ( 1998) Genome Res. 8: 186- 194.
- Motifs A program that searches amino acid sequences for patterns Bairoch et al. svipia; Wisconsin that matched those defined in Prosite. Package Program Manual, version 9, page M51 -59, Genetics Computer Group, Madison, WI.
Abstract
Description
Claims
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JP2000567690A JP2002523089A (en) | 1998-08-27 | 1999-08-26 | Protein transport-related molecules |
US10/986,822 US20050130204A1 (en) | 1998-08-27 | 2004-11-15 | Protein-transport-associated molecules |
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Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002002610A2 (en) * | 2000-06-29 | 2002-01-10 | Incyte Genomics, Inc. | Secretion and trafficking molecules |
JP2003259872A (en) * | 2002-03-07 | 2003-09-16 | Japan Science & Technology Corp | Human esophageal carcinoma antigenic peptide and method for diagnosing esophageal carcinoma |
EP1895017A3 (en) * | 2002-10-03 | 2008-03-12 | Genentech, Inc. | Use of A33 antigens and JAM-IT |
US7419663B2 (en) | 1998-03-20 | 2008-09-02 | Genentech, Inc. | Treatment of complement-associated disorders |
WO2008036135A3 (en) * | 2006-06-01 | 2008-10-02 | Genentech Inc | Crystal structure of crig and c3b: crig complex |
US7776573B2 (en) | 2006-06-01 | 2010-08-17 | Genentech, Inc. | Crystal structure of CRIg and C3b:CRIg complex |
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US8007798B2 (en) | 1997-11-21 | 2011-08-30 | Genentech, Inc. | Treatment of complement-associated disorders |
US8067002B2 (en) | 2006-11-02 | 2011-11-29 | Genentech, Inc. | Humanized anti-factor D antibodies |
US8088386B2 (en) | 1998-03-20 | 2012-01-03 | Genentech, Inc. | Treatment of complement-associated disorders |
US8273352B2 (en) | 2008-04-28 | 2012-09-25 | Genentech, Inc. | Humanized anti-factor D antibodies and uses thereof |
US8753625B2 (en) | 2005-11-04 | 2014-06-17 | Genentech, Inc. | Use of complement inhibitors to treat ocular diseases |
US9234024B2 (en) | 2008-05-06 | 2016-01-12 | Genentech, Inc. | Affinity matured CRIg variants |
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-
1999
- 1999-08-26 EP EP99942518A patent/EP1108019A2/en not_active Withdrawn
- 1999-08-26 WO PCT/US1999/019616 patent/WO2000012703A2/en not_active Application Discontinuation
- 1999-08-26 JP JP2000567690A patent/JP2002523089A/en active Pending
- 1999-08-26 CA CA002340794A patent/CA2340794A1/en not_active Abandoned
- 1999-08-26 AU AU55877/99A patent/AU5587799A/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
Database EMBLhum1 SEQ ID AF044924: Homo sapiens hook2 protein (HOOK2) mRNA, complete cds. 6 April 1998. XP002125427 * |
KAIN R ET AL: "Molecular cloning and expression of a novel human trans-Golgi network glycoprotein, TGN51, that contains multiple tyrosine-containing motifs" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 2, 9 January 1998 (1998-01-09), pages 981-988, XP002125426 MD US * |
PONNAMBALAM S ET AL.: "Primate homologues of rat TGN38: primary structure, expression and functional implications" JOURNAL OF CELL SCIENCE, vol. 109, no. 3, 1996, pages 675-685, XP000857634 * |
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WO2000012703A3 (en) | 2000-06-22 |
AU5587799A (en) | 2000-03-21 |
CA2340794A1 (en) | 2000-03-09 |
EP1108019A2 (en) | 2001-06-20 |
JP2002523089A (en) | 2002-07-30 |
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