Mammalian Cell Membrane Proteins; Related Reagents

Abstract

The purification and isolation of various genes which encode mammalian cell surface polypeptides. Nucleic acids, proteins, antibodies, and other reagents useful in modulating development of cells, e.g., lymphoid and myeloid, are provided, along with methods for their use.

1 . A substantially pure or recombinant polypeptide:
 a) exhibiting identity over a length of at least about 12 amino acids to the mature polypeptide from SEQ ID NO: 2 or 6;   b) exhibiting identity over a length of at least about 12 amino acids to the mature SEQ ID NO: 8 or 10; or   c) exhibiting identity over a length of at least about 12 amino acids to the mature SEQ ID NO: 12 or 14.   
     
     
         2 . The polypeptide of  claim 1 , wherein:
 a) said SEQ ID NO: is 2 or 6, and said polypeptide:
 i) is a mature natural sequence DAP12 from Table 1; 
 ii) comprises an ITAM motif; or 
 iii) comprises a charged residue in a transmembrane domain; 
   b) said SEQ ID NO: is 8 or 10, and said polypeptide:
 i) is a mature natural sequence DAP10 from Table 2; 
 ii) comprises an ITIM motif; or 
 iii) comprises a charged residue in a transmembrane domain; or 
   c) said SEQ ID NO: is 12 or 14, and said polypeptide:
 i) is a mature natural sequence MDL-1 of Table 3; or 
 ii) comprises a charged residue in a transmembrane domain. 
   
     
     
         3 . A polypeptide of  claim 1 , which:
 a) comprises a plurality of said lengths; or   b) is a natural allelic variant of DAP12;   c) is a natural allelic variant of DAP10;   d) is a natural allelic variant of MDL-1;   e) has a length at least about 30 amino acids;   f) is a synthetic polypeptide;   g) is attached to a solid substrate;   h) is conjugated to another chemical moiety;   i) is a 5-fold or less substitution from natural sequence; or   j) is a deletion or insertion variant from a natural sequence.   
     
     
         4 . A composition comprising:
 a) a sterile DAP12 polypeptide of  claim 3 ;   b) said DAP12 polypeptide of  claim 3  and a carrier, wherein said carrier is:
 i) an aqueous compound, including water, saline, and/or buffer; and/or 
 ii) formulated for oral, rectal, nasal, topical, or parenteral administration; 
   c) a sterile DAP10 polypeptide of  claim 3 ; or   d) said DAP10 polypeptide of  claim 3  and a carrier, wherein said carrier is:
 i) an aqueous compound, including water, saline, and/or buffer; and/or 
 ii) formulated for oral, rectal, nasal, topical, or parenteral administration; 
   e) a sterile MDL-1 polypeptide of  claim 3 , or   f) said MDL-1 polypeptide of  claim 3  and a carrier, wherein said carrier is:
 i) an aqueous compound, including water, saline, and/or buffer; and/or 
 ii) formulated for oral, rectal, nasal, topical, or parenteral administration. 
   
     
     
         5 . A fusion protein comprising said polypeptide of  claim 1  and:
 a) a detection or purification tag, including a FLAG, His6, or immunoglobulin peptide;   b) bacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor; or   c) sequence of another membrane protein.   
     
     
         6 . A kit comprising said polypeptide of  claim 1 , and:
 a) a compartment comprising said polypeptide; and/or   b) instructions for use or disposal of reagents in said kit.   
     
     
         7 . A binding compound comprising an antigen binding portion from an antibody, which specifically binds to:
 a) a natural DAP12 polypeptide of  claim 2 , wherein said antibody:
 i) is raised against a mature polypeptide of Table 1; 
 ii) is immunoselected; 
 iii) is a polyclonal antibody; 
 iv) binds to a denatured DAP12; 
 v) exhibits a Kd to antigen of at least 30 μM; 
 vi) is attached to a solid substrate, including a bead or plastic membrane; 
 vii) is in a sterile composition; or 
 viii) is detectably labeled, including a radioactive or fluorescent label; 
   b) a natural DAP10 polypeptide of  claim 2 , wherein said antibody:
 i) is raised against a mature polypeptide of Table 2; 
 ii) is immunoselected; 
 iii) is a polyclonal antibody; 
 iv) binds to a denatured DAP10; 
 v) exhibits a Kd to antigen of at least 30 μM; 
 vi) is attached to a solid substrate, including a bead or plastic membrane; 
 vii) is in a sterile composition; or 
 viii) is detectably labeled, including a radioactive or fluorescent label; or 
   c) a natural MDL-1 polypeptide of  claim 2 , wherein said antibody:
 i) is raised against a mature polypeptide of Table 3; 
 ii) is immunoselected; 
 iii) is a polyclonal antibody; 
 iv) binds to a denatured MDL-1; 
 v) exhibits a Kd to antigen of at least 30 μM; 
 vi) is attached to a solid substrate, including a bead or plastic membrane; 
 vii) is in a sterile composition; or 
 viii) is detectably labeled, including a radioactive or fluorescent label. 
   
     
     
         8 . A kit comprising said binding compound of  claim 7 , and:
 a) a compartment comprising said binding compound; and/or   b) instructions for use or disposal of reagents in said kit.   
     
     
         9 . A composition comprising:
 a) a sterile binding compound of  claim 7 , or   b) said binding compound of  claim 7  and a carrier, wherein said carrier is:
 i) an aqueous compound, including water, saline, and/or buffer; and/or 
 ii) formulated for oral, rectal, nasal, topical, or parenteral administration. 
   
     
     
         10 . An isolated or recombinant nucleic acid encoding a polypeptide of  claim 1 , wherein said nucleic acid encodes an antigenic peptide sequence of Table 1, 2, or 3. 
     
     
         11 . The nucleic acid of  claim 10 , which encodes a plurality of antigenic peptide sequences of said table. 
     
     
         12 . The nucleic acid of  claim 10 , which:
 a) is an expression vector;   b) further comprises an origin of replication;   c) is from a natural source;   d) comprises a detectable label;   e) comprises synthetic nucleotide sequence;   f) is less than 6 kb, preferably less than 3 kb;   g) is from a mammal, including a primate or rodent;   h) comprises a natural full length coding sequence;   i) is a hybridization probe for a gene encoding DAP12, DAP10, or MDL-1; or   j) is a PCR primer, PCR product, or mutagenesis primer.   
     
     
         13 . A nucleic acid which hybridizes under stringent wash conditions of at least 50° C., less than 400 mM salt, and 50% formamide to:
 a) SEQ ID NO: 1 or 5;   b) SEQ ID NO: 7 or 9; or   c) SEQ ID NO: 11 or 13.   
     
     
         14 . A cell or tissue comprising a recombinant nucleic acid of  claim 10 . 
     
     
         15 . The cell of  claim 14 , wherein said cell is:
 a) a prokaryotic cell;   b) a eukaryotic cell;   c) a bacterial cell;   d) a yeast cell;   e) an insect cell;   f) a mammalian cell;   g) a mouse cell;   h) a primate cell; or   i) a human cell.   
     
     
         16 . A kit comprising said nucleic acid of  claim 10 , and:
 a) a compartment comprising said nucleic acid;   b) a compartment further comprising a DAP12, DAP10, or MDL-1 polypeptide; and/or   c) instructions for use or disposal of reagents in said kit.   
     
     
         17 . The nucleic acid of  claim 13 , which:
 a) exhibits identity over a stretch of at least about 30 nucleotides to a primate DAP12;   b) exhibits identity over a stretch of at least about 30 nucleotides to a primate DAP10;   c) exhibits identity over a stretch of at least about 30 nucleotides to a primate MDL-1; and/or   d) further encodes a KIR, ILT/MIR or CD94/NKG2C receptor.   
     
     
         18 . The nucleic acid of  claim 17 , wherein:
 a) said wash conditions are at 60° C. and/or 200 mM salt; or   b) said stretch is at least 55 nucleotides.   
     
     
         19 . A method of modulating physiology or development of a cell or tissue culture cells comprising contacting said cell with an agonist or antagonist of a DAP12, DAP10, or MDL-1. 
     
     
         20 . A method of screening for a compound which blocks interaction of a DAP12 or DAP10 of  claim 2  with a KIR, ILT/MIR, or CD94/NKG2C receptor, comprising contacting said compound to said DAP12 or DAP10 in the presence of said receptor.