Density enhanced protein tyrosine phosphatases
AssigneeCold Spring Harbor Research Laboratory
InventorsNicholas K. Tonks, Arne Ostman
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Abstract
Novel Type III density enhanced protein tyrosine phosphatases are disclosed and exemplified by human DEP-1 enzyme. Polynucleotides encoding huDEP-1 are disclosed, along with methods and materials for production of the same by recombinant procedures. Binding molecules specific for DEP-1 are also disclosed as useful for modulating the biological activities of DEP-1.
Claims39
A purified and isolated density enhanced protein tyrosine phosphatase polypeptide.
A purified and isolated polynucleotide encoding a density enhanced Type III receptor-like protein tyrosine phosphatase polypeptide.
A purified and isolated polynucleotide selected from the group consisting of:
- a) the DNA sequence set out in SEQ ID NO:
- 1, and
- b) a DNA molecule which hybridizes under stringent conditions to the protein coding portion of the DNA of (a).
A DNA encoding a huDEP-1 amino acid sequence set out in SEQ ID NO:
- 2, or a variant thereof.
A polypeptide or peptide capable of specifically binding to a density enhanced protein tyrosine phosphatase polypeptide.
A polypeptide or peptide capable of specifically binding to a density enhanced Type III receptor-like protein tyrosine phosphatase polypeptide.
A method for isolating a polynucleotide encoding a polypeptide that binds to a density enhanced Type III receptor-like protein tyrosine phosphatase polypeptide comprising the steps of:
- a) transforming or transfecting host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain;
- b) expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of a density enhanced Type III receptor-like protein tyrosine phosphatase polypeptide and either the DNA-binding domain or the activating domain of the transcription factor;
- c) expressing in the host cells a library of second hybrid DNA sequences encoding second fusions of part or all of putative density enhanced Type III receptor-like protein tyrosine phosphatase binding polypeptides and either the activating domain or DNA binding domain of the transcription factor which is not incorporated in the first fusion:
- d) transforming or transfecting the host cells with a DNA construct comprising a protein tyrosine kinase gene;
- e) detecting binding of density enhanced Type III receptor-like protein tyrosine phosphatase binding polypeptide(s) to the phosphatase polypeptide in the host cell by detecting the production of reporter gene product in the host cell(s);
- and
- f) isolating the second hybrid DNA sequences encoding the phosphatase binding polypeptide from the host cell(s).
A method for detecting proteins which bind to a density enhanced Type III receptor-like protein tyrosine phosphatase polypeptide isoform comprising the steps of:
- a) transforming or transfecting host cells with a hybrid DNA sequence encoding a fusion between a putative phosphatase binding protein and a ligand capable of high affinity binding to a specific counterreceptor;
- b) expressing the hybrid DNA sequence in the host cells under appropriate conditions;
- c) immobilizing fusion protein from the host cells by exposing the fusion protein to the specific counterreceptor in immobilized form;
- d) contacting the density enhanced Type III receptor-like protein tyrosine phosphatase polypeptide with the immobilized fusion protein;
- and
- e) detecting the phosphatase polypeptide bound to the fusion protein using a reagent specific for the phosphatase polypeptide.
Dependencies are inferred from the claim text.