Muteins of apolipoprotein D

Abstract

A method of generating a mutein of human apolipoprotein D having detectable binding affinity to a given non-natural ligand of apolipoprotein D is disclosed, which comprises the steps of subjecting apolipoprotein D to mutagenesis at the sequence positions 34 to 38, 60, 62 to 66, 68, 89 to 93, 115, 117 to 121, and 123, resulting in a plurality of muteins of apolipoprotein D; and enriching resulting muteins having binding affinity for a given ligand from the plurality of muteins by selection, and/or isolating said mutein. Muteins of apolipoprotein D obtained by this method are also disclosed.

1 . A mutein of human apolipoprotein D having detectable binding affinity to a given non-natural ligand of apolipoprotein D, obtained by method for generating a mutein of human apolipoprotein D having detectable affinity to a given non- natural ligand of apolipoprotein D, comprising: 
 subjecting apolipoprotein D to mutagenesis at the sequence positions 34 to 38, 60, 62 to 66, 68, 89 to 93, 115, 117 to 121, and 123, resulting in a plurality of muteins of apolipoprotein D; and    enriching resulting muteins having binding affinity for a given ligand from the plurality of muteins by selection, and/or isolating said mutein.    
     
     
         2 . A mutein of human apolipoprotein D according to  claim 1 , wherein the mutein carries an amino acid substitution at least one of the sequence positions 38, 60, 62 to 66, 68, 90, 91, 93, 117 to 121 compared with apolipoprotein D.  
     
     
         3 . A mutein of human apolipoprotein D according to  claim 1 , wherein the mutein carries an amino acid substitution at six or more of the sequence positions 35 to 37, 64, 65, 66, 68, 91, 115, 119 and 123 compared with apolipoprotein D.  
     
     
         4 . A mutein of human apolipoprotein D according to  claim 1 , wherein the mutein carries an amino acid substitution at nine or more of the sequence positions 34 to 38, 60, 63 to 66, 68, 89 to 93, 115, 117-119, and 123 compared to apolipoprotein D.  
     
     
         5 . A mutein according to  claim 1  having the amino acid sequence of SEQ ID NO: 21.  
     
     
         6 . A mutein according to  claim 1 , which is conjugated to a label selected from the group consisting of organic molecule, enzyme label, radioactive label, fluorescent label, chromogenic label, luminescent label, a hapten, biotin, digoxigenin, a metal complex, metals and colloidal gold.  
     
     
         7 . A fusion protein comprising a mutein of apolipoprotein D according to  claim 1 , wherein an enzyme, a protein, a protein domain, a peptide, a signal sequence, or an affinity tag is operably fused to the amino terminus or to the carboxy terminus of the mutein.  
     
     
         8 . A nucleic acid molecule comprising a nucleotide sequence encoding a mutein of apolipoprotein D according to  claim 1 .  
     
     
         9 . A pharmaceutical composition comprising a mutein of apolipoprotein D according to  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         10 . A method for producing a mutein of apolipoprotein D according to  claim 1 , wherein the mutein is produced starting from the nucleic acid encoding the mutein by means of genetic engineering methods in a bacterial or eukaryotic host organism and is isolated from this host organism or its culture.  
     
     
         11 . A method for detecting a given ligand, comprising the steps of contacting a mutein of apolipoprotein D according to  claim 1  with a sample suspected of containing the given ligand under conditions effective to allow formation of a complex between the mutein and the given ligand, and determining the complexed mutein by a suitable signal.  
     
     
         12 . The method of  claim 11 , wherein the given ligand is a protein, a protein domain, a peptide, a nucleic acid molecule, an organic molecule, or a metal complex, and the detection is effective to validate the protein as pharmacological drug target.