Motif-Specific and Context-Independent Antibodies That Specifically Bind to a Cleaved Caspase Motif

Abstract

There is provided a motif-specific, context-independent antibody that specifically binds a recurring, modified motif consisting of (i) at least one sumoylated lysine residue, and (ii) one or more degenerate amino acids bound by a peptide bond to said sumoylated lysine residue, said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs. Also provided is a motif-specific, context-independent antibody that specifically binds a recurring, modified motif consisting of (i) a C-terminal aspartic acid residue, and (ii) one or more degenerate amino acids bound by a peptide bond to said C-terminal aspartic acid residue, said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs.

1 . A motif-specific, context-independent antibody that specifically binds a recurring, modified motif consisting of (i) at least one sumoylated lysine residue, and (ii) one or more degenerate amino acids bound by a peptide bond to said sumoylated lysine residue, said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs. 
     
     
         2 . The antibody of  claim 1 , wherein said motif comprises the sequence xxxxxKxxxxx, where x is any amino acid and K is a sumoylated lysine residue. 
     
     
         3 . The antibody of any of the preceding claims, wherein said motif comprises a sequence selected from the group consisting of Ψ-K-χ-D and Ψ-K-χ-E, where Ψ is a hydrophobic amino acid residue, K is the sumoylated lysine residue, x is any amino acid residue, D is an aspartic acid residue, and E is a glutamic acid residue. 
     
     
         4 . The antibody of any of the preceding claims, wherein the sumoylated lysine residue is a lysine residue attached via an epsilon amine of said lysine residue to a C-terminal G of the fragment of a SUMO protein, wherein said fragment comprises the sequence TGG. 
     
     
         5 . The antibody of any of the preceding claims, wherein the sumoylated lysine residue is a lysine residue attached via an epsilon amine of said lysine residue to a C-terminal G of the fragment of a SUMO protein, wherein said fragment comprises the sequence QTGG. 
     
     
         6 . The antibody of any of the preceding claims, wherein the sumoylated lysine residue is a lysine residue attached via an epsilon amine of said lysine residue to a C-terminal G of the fragment of a SUMO protein, wherein said fragment comprises a sequence selected from the group consisting of VYQEQTGG, YQEQTGG, QEQTGG, EQTGG, DVFQQQTGG, VFQQQTGG, FQQQTGG, QQQTGG, and QQTGG. 
     
     
         7 . The antibody of any of the preceding claims, wherein the antibody is a rabbit antibody. 
     
     
         8 . The antibody of any of the preceding claims, wherein the antibody is a monoclonal antibody. 
     
     
         9 - 18 . (canceled) 
     
     
         19 . A method for producing an antibody that specifically binds a single modified amino acid, said method comprising the steps of:
 (a) constructing a degenerate peptide library comprising (i) a single fixed modified amino acid consisting of a lysine residue attached via an epsilon amine of said lysine residue to a C-terminal G of a fragment of a SUMO protein, wherein said fragment comprises a sequence selected from the group consisting of VYQEQTGG, YQEQTGG, QEQTGG, EQTGG, DVFQQQTGG, VFQQQTGG, FQQQTGG, QQQTGG, and QQTGG, and (ii) a plurality of degenerate amino acids flanking said modified amino acid;   (b) immunizing a host with said peptide library to raise a context-independent antibody specific for said modified amino acid; and   (c) isolating antisera from said host, and purifying the context-independent antibody of step (b) from said antisera, said antibody specifically binding said single modified amino acid independent of the peptide context in which it occurs.   
     
     
         20 . The method of  claim 1 , further comprising the step of utilizing spleen cells from the host of step (b) to generate at least one monoclonal context independent antibody.