Crystals of the Fc region of immunoglobulin epsilon heavy chain protein

Abstract

The present invention includes three-dimensional models of antibodies, such as Fc-Cε3/Cε4 regions of IgE antibodies, as well as methods to produce such models. The present invention also includes muteins having increased stability and/or antibody receptor binding activity, as well as methods to produce such muteins, preferably using information derived from three-dimensional models of the present invention. Also included are nucleic acid sequences encoding muteins of the present invention and use of those sequences to produce such muteins. Also included is the use of the model to identify compounds that inhibit the binding of an antibody receptor protein to an antibody. The present invention also includes uses of such muteins and inhibitory compounds, for example, in methods to diagnose and protect animals from allergy and other abnormal immune responses.

1 - 20 . (canceled)  
     
     
         21 . An isolated crystal comprising a polypeptide which comprises at least the Cε3 or at least the Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein.  
     
     
         22 . The isolated crystal of  claim 21 , wherein said Cε3 or Cε4 domain is from the Fc region of a human immunoglobulin epsilon heavy chain protein.  
     
     
         23 . The isolated crystal of  claim 21 , wherein said crystal belongs to spacegroup P42 1 2, C2 or P2 1 .  
     
     
         24 . The isolated crystal of  claim 22 , wherein said crystal has cell dimensions selected from the group consisting of: 
 (a) cell dimensions of about 105 angstroms by about 105 angstroms by about 47 angstroms;    (b) cell dimensions of about 158 angstroms by about 108 angstroms by about 102 angstroms;    (c) cell dimensions of about 66 angstroms by about 99 angstroms by about 77 angstroms; and,    (d) cell dimensions of about 48 angstroms by about 104 angstroms by about 150 angstroms.    
     
     
         25 . The isolated crystal of  claim 21 , wherein said polypeptide consists of the Cε3 and/or the Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein.  
     
     
         26 . The isolated crystal of  claim 21 , wherein the amino acid sequence of said Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein has been altered to prevent glycosylation.  
     
     
         27 . The isolated crystal of  claim 21 , wherein said Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein comprises an amino acid sequence at least 95% identical to SEQ ID NO:2 or SEQ ID NO:8.  
     
     
         28 . The isolated crystal of  claim 21 , wherein Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8.  
     
     
         29 . An isolated crystal comprising at least the Cε3 or at least the Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein, produced by a method comprising: 
 (a) obtaining an isolated protein comprising at least the Cε3 or at least the Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein;    (b) crystallizing said isolated protein using a precipitant comprising about 25 mM sodium acetate, and about 33% (w/v) polyethylene glycol 4000.    
     
     
         30 . The isolated crystal of  claim 29 , wherein said precipitant has a pH of between 4 and 5.  
     
     
         31 . The isolated crystal of  claim 29 , wherein said crystallization step is performed at about room temperature.  
     
     
         32 . The isolated crystal of  claim 29 , wherein the amino acid sequence of said Cε3 or said Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein has been altered to prevent glycosylation.  
     
     
         33 . The isolated crystal of  claim 29 , wherein said Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein comprises an amino acid sequence at least about 95% identical to SEQ ID NO:2 or SEQ ID NO:8.  
     
     
         34 . The isolated crystal of  claim 29 , wherein said Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8.  
     
     
         35 . A method to produce a protein crystal comprising at least the Cε3 or at least the Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein, said method comprising: 
 (a) obtaining an isolated protein comprising at least the Cε3 or at least the Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein;    (b) crystallizing said isolated protein using a precipitant comprising about 25 mM sodium acetate, and about 33% (w/v) polyethylene glycol 4000.    
     
     
         36 . The method of  claim 35  wherein said precipitant has a pH of between 4 and 5.  
     
     
         37 . The method of  claim 35 , wherein said crystallization step is performed at about room temperature.  
     
     
         38 . The method of  claim 35 , wherein the amino acid sequence of said Cε3 or said Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein has been altered to prevent glycosylation.  
     
     
         39 . The method of  claim 35 , wherein said Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein comprises an amino acid sequence at least about 95% identical to SEQ ID NO:2 or SEQ ID NO:8.  
     
     
         40 . The method of  claim 35 , wherein said Cε3 or Cε4 domain from the Fc region of an immunoglobulin epsilon heavy chain protein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8.