Novel Shc-binding protein

Abstract

Novel Shc-binding protein, oligonucleotides encoding the same, methods of producing and use thereof are disclosed.

What is claimed is:  
     
         1 . An isolated polynucleotide encoding a PAL polypeptide comprising the amino acid sequence set out in SEQ ID NO.: 2 or SEQ ID NO.: 4, and variants including conserved variants, allelic variants, and analogs including biologically active fragments thereof.  
     
     
         2 . The isolated polynucleotide according to  claim 1  wherein said polynucleotide is selected from the group consisting of cDNA, genomic DNA, and chemically synthesized DNA.  
     
     
         3 . An isolated polynucleotide according to  claim 1  that encodes a polypeptide that is at least 70 percent identical to the polypeptide of SEQ ID NO.: 2 or SEQ ID NO.: 4.  
     
     
         4 . An isolated polynucleotide encoding a PAL polypeptide the polynucleotide being selected from the group consisting of: 
 (a) the DNA molecules set out as SEQ ID NOs.: 1 or 3, DNA molecules encoding variants including conserved variants, allelic variants, analogs, and fragments thereof;    (b) DNA molecules which hybridize to the DNA molecules defined in a) or hybridizable fragments thereof; and    (c) DNA molecules that code an expression for the amino acids encoded by any of the foregoing DNA molecules.    
     
     
         5 . A detectably labeled nucleic acid hybridizable to a polynucleotide according to claims  1 ,  2 ,  3 , or  4 .  
     
     
         6 . A cloning vector which comprises a polynucleotide according to claims  1 ,  2 ,  3 , or  4 .  
     
     
         7 . An expression vector which comprises a polynucleotide according to claims  1 ,  2 ,  3 , or  4 .  
     
     
         8 . The expression vector which comprises a polynucleotide according to claims  1 ,  2 ,  3 , or  4 , operatively associated with an expression control sequence.  
     
     
         9 . The expression vector of  claim 8  wherein said expression control sequence is selected from the group consisting of the immediate early promoters of human cytomegalovirus (hCMV), early promoters of SV-40, early promoters of adenovirus, early promoters of polyoma virus, late promoters of SV-40, late promoters of vaccinia virus, late promoters of polyoma virus, retroviral LTR, inducible promoters, promoters of the lac system, promoters of the trp system, promoters of the TAC system, promoters of the YRC system, the major operators and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, promoters of yeast α mating factor.  
     
     
         10 . A unicellular host transformed with a polynucleotide according to claims  1 ,  2 ,  3  or  4 .  
     
     
         11 . The unicellular host of  claim 10  wherein the host cell is selected from the group consisting of  E. coli,  Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, B-W, LM, C051, C057, BSC1, BSC40, BMT10 and SF9 cells.  
     
     
         12 . The unicellular host according to  claim 11  wherein the unicellular host is a yeast selected from the group consisting of Saccharomyces, Pichia, Candida, Hansenula, and Torulopsis.  
     
     
         13 . A mammalian cell containing a PAL polypeptide encoding DNA modified so as to permit higher expression of PAL polypeptide by means of a homologous recombinational event consisting of inserting an expression regulatory sequence in functional proximity to the PAL encoding DNA.  
     
     
         14 . A mammalian cell according to  claim 13  wherein the inserted expression regulatory sequence is not a native PAL expression regulatory sequence.  
     
     
         15 . A method for producing a PAL polypeptide, the method comprising the steps of: 
 (a) culturing a host cell according to claims  10  or  13  under conditions suitable for the expression of the PAL polypeptide; and    (b) recovering the expressed PAL polypeptide.    
     
     
         16 . A purified PAL polypeptide comprising the PAL amino acid sequence of SEQ ID NO.: 2, and variants including conserved variants, allelic variants, and analogs including biologically active fragments thereof.  
     
     
         17 . A substantially purified and isolated polypeptide comprising of the PAL amino acid sequence of SEQ ID NO.: 4, and variants including conserved variants, allelic variants, and analogs including biologically active fragments thereof.  
     
     
         18 . An isolated nucleic acid encoding an analog of a PAL polypeptide comprising the amino acid sequence set out as SEQ ID NOs.: 2 or 4 wherein one or more amino acids selected from the group consisting of amino acids 4, 5, 6, 7, 10, 13, 14, 17, 19, 21, 22, 23, 25, 27, 29, 32, 33, 34, 38, 40, 42, 46, 47, 49, 50, 51, 54, 55, 56, 57, 58, 59, 60, 66, 69, 71, 75, 99 103, 104, 116, 125, 130, 132, 136, 137, 148, 149, 150, 151, 153, 154, 159, 160, 161, 162, 171, 172, 182, 186, 187, 189, 191, 203, 241, 288, 292, 321, 324, 326, 327, 331, 332, 340, 341, 348, 349, 352, 354, 356, 357, 358, 361, 364, 369, 372, 377, 378, 384, 395, 429, 431, 439, 443, 450, 452, 477, 478, 484, 499, 503, 532, 535, 553, 559, 560, 562, 563, 564, 565, 570, 571, 573, 574, 575, 577, 580, 582, 583, 588, 589, 590, 592, 593, 594, 598, 599, 601, 602, 604, 605, 608, 609, 615, 619, 621, 623, 627, 628, 645, 652, 653, 655, and 672 is substituted with another amino acid.  
     
     
         19 . The PAL polypeptide of  claim 16  that does not possess an amino terminal methionine.  
     
     
         20 . The PAL polypeptide of  claim 17  that does not possess an amino terminal methionine.  
     
     
         21 . An antibody which specifically binds human PAL.  
     
     
         22 . An antibody which specifically binds murine PAL.  
     
     
         23 . The antibody of  claim 21  that is a monoclonal antibody.  
     
     
         24 . The antibody of  claim 22  that is a monoclonal antibody.  
     
     
         25 . A derivative of PAL polypeptide having one or more chemical moieties attached thereto optionally in a pharmaceutically acceptable carrier wherein the polypeptide is selected from the group consisting of PAL variants including variants, conserved variants, allelic variants, and analogs including biologically active fragments thereof.  
     
     
         26 . The derivative of  claim 25  wherein said one or more chemical moieties is selected from the group consisting of water soluble polymers.  
     
     
         27 . The polymer of  claim 26  wherein said polymer is selected from the group consisting of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymer, polyethylated polyols, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6,-trioxane, ethylene/maleic anhydride copolymers, homopolymers of polyamino acids, random copolymers of polyamino acids, poly(n-vinyl pyrrolidone)-polyethylene glycol, and polyvinyl alcohol.  
     
     
         28 . A method for determining the presence of PAL protein in a biological sample comprising the steps of: 
 (a) obtaining a biological sample;    (b) exposing said biological sample to a PAL specific antibody; and    (c) detecting the binding of PAL specific antibody in said biological sample.    
     
     
         29 . A diagnostic reagent comprising a detectably labeled polynucleotide encoding part or all of the PAL amino acid sequences set out in SEQ ID NO.: 2, including conserved variants, allelic variants, fragments and analogs.  
     
     
         30 . A diagnostic reagent comprising detectably labeled polynucleotide encoding all or part of the PAL amino acids set out in SEQ ID NO.: 4, including conserved variants, allelic variants, fragments and analogs.  
     
     
         31 . The diagnostic reagent of  claim 29  or  30  wherein said labeled polynucleotide is a DNA.  
     
     
         32 . A diagnostic reagent of  claim 29  or  30  wherein said labeled polynucleotide is a first-strand cDNA.  
     
     
         33 . A method for determining the presence of PAL specific polynucleotide molecule in a biological sample comprising the steps of: 
 (a) collecting a biological sample;    (b) isolating polynucleotide molecules from said biological sample;    (c) hybridizing to said polynucleotide molecules a diagnostic reagent according to  claim 29  or  30 ; and    (d) detecting the binding of the PAL specific polynucleotide molecules in said biological samples.    
     
     
         34 . A method for determining the presence of PAL specific polynucleotide molecule in a tissue or cellular sample comprising the steps of: 
 (a) collecting tissue or cellular sample;    (b) hybridizing said tissue or cellular sample to a diagnostic reagent according to  claim 29  or  30 ; and    (c) detecting the binding of the PAL specific polynucleotide molecules in the tissue or cellular sample to said diagnostic reagent.    
     
     
         35 . The method of  claim 33  wherein said polynucleotide molecule is DNA.  
     
     
         36 . The method of  claim 34  wherein said polynucleotide molecule is DNA.  
     
     
         37 . The method of  claim 33  wherein said polynucleotide acid molecule is RNA.  
     
     
         38 . The method of  claim 34  wherein said polynucleotide acid molecule is RNA.  
     
     
         39 . A method of identifying a candidate inhibitor of PAL binding to a PAL binding protein comprising the steps of: 
 (a). exposing PAL to a PAL binding protein under conditions which permit binding of PAL to the PAL binding protein in the presence or absence of a candidate inhibitor;    (b) measuring the binding of PAL to the PAL binding protein in the presence or absence of the candidate inhibitor;    (c) comparing the level of binding observed in step (a); and    (d) identifying the compound as an inhibitor of PAL binding by its ability to prevent binding of PAL to the PAL binding protein.    
     
     
         40 . A method for inhibiting cell proliferation in vitro comprising contacting a cell with an antiproliferative amount of the inhibitor compound of  claim 39 .  
     
     
         41 . A method for inhibiting cell proliferation in vivo comprising introducing into a subject an antiproliferative amount of the inhibitor compound of  claim 39 .  
     
     
         42 . A method for assaying the effect of an inhibitor of PAL binding to a PAL binding protein on cell proliferation in vitro comprising the steps of: 
 (a) growing eukaryotic cell lines transfected with PAL nucleic molecules according  claim 15  in a manner allowing the host cell to express said polypeptide;    (b) contacting said cell line with one inhibitor of PAL binding to a protein; and    (c) measuring the effect of said inhibitor on cell proliferation.    
     
     
         43 . A method of stimulating hematopoietic progenitor cell proliferation in a mammal, the method comprising administering to the mammal a hematopoietically effective dose of a PAL, the PAL polypeptide selected from the polypeptides set out in SEQ ID NO.: 2 or 4 including conserved variants, allelic variants and analogs including biologically active fragments thereof; thereby stimulating proliferation of said hematopoietic progenitor cells.  
     
     
         44 . A method of stimulating hematopoietic progenitor cell proliferation the method comprising the steps of: 
 (a) obtaining hematopoietic progenitor cells from a mammal;    (b) culturing the cells obtained in step (a) in vitro;    (c) introducing PAL polynucleotides in an eukaryotic expression vector into the hematopoietic progenitor cells in culture;    (d) selecting producer cells expressing the PAL molecule prepared in step (b);    (e) expanding PAL producing cells in vitro; and    (f) introducing said PAL producing hematopoietic progenitor cells into a mammal.    
     
     
         45 . A method of stimulating hematopoietic progenitor cell proliferation in vitro with PAL protein comprising: 
 (a) obtaining hematopoietic progenitor cells from a donor;    (b) exposing the hematopoietic progenitor cells obtained in step (a) to hematopoietically effective dose of PAL polypeptide, the PAL polypeptide selected from the polypeptide set out in SEQ ID NO. 2 and including conserved variants, allelic variants, and analogs including biologically active fragments thereof; and    (c) administering to said donor the early hematopoietic progenitor cells obtained in step (b).    
     
     
         46 . A method of stimulating hematopoietic progenitor cell proliferation in vitro with PAL protein comprising: 
 (a) obtaining hematopoietic progenitor cells from a donor;    (b) exposing the hematopoietic progenitor cells obtained in step (a) to a hematopoietically effective dose of PAL polypeptide, the PAL polypeptide selected from polypeptide set out in SEQ ID NO. 4 and including conserved variants, allelic variants, and analogs including biologically active fragments thereof, and    (c) administering to said donor the early hematopoietic progenitor cells obtained in step (b).