Method for Production and Use of Mite Group 1 Proteins

Abstract

The present invention includes a method to produce a recombinant mite Group 1 protein in a methyltrophic yeast or an Escherichia coli microorganism. The present invention also relates to a recombinant mite Group 1 protein obtained by such a method, such a recombinant protein being able to selectively bind IgE or cause proliferation of a T cell that proliferates in response to a native mite Group 1 protein. Also included in the present invention is the use of such a recombinant mite Group 1 protein to detect mite allergy or to reduce an allergic response to a mite Group 1 protein. The present invention also includes novel mite Group 1 nucleic acid molecules, proteins, recombinant molecules, and recombinant cells, as well as uses thereof.

1 . A method to produce a recombinant protein comprising a recombinant mite Group 1 protein or a fragment thereof comprising the steps of:
 (a) culturing a methyltrophic yeast microorganism transformed with a nucleic acid molecule encoding said recombinant mite Group 1 protein or fragment; and   (b) recovering said recombinant mite Group 1 protein or fragment from said methyltrophic yeast microorganism;   
       wherein
 said recombinant mite Group 1 protein or fragment is selected from the group consisting of:  Dermatophagoides farinae, Dermatophagoides pteronyssinus , and  Euroglyphus maynei  recombinant mite Group 1 proteins (Der f 1, Der p 1 and Eur m 1) or a fragment thereof and has a function selected from the group consisting of: selectively binding IgE and causing proliferation of a T cell that proliferates in response to a native mite Group 1 protein selected from the group consisting of Der f 1, Der p 1 and Eur m 1 and 
 said nucleic acid molecule consists of a nucleic acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:81, SEQ ID NO:86, and SEQ ID NO:91 or a fragment thereof and wherein said nucleic acid molecule is optionally fused to a signal sequence. 
 
     
     
         2 . The method of  claim 1 , wherein said methyltrophic yeast microorganism is of a genus selected from the group consisting of a  Pichia  microorganism, a  Hansenula  microorganism, a  Torulopsis  microorganism, and a  Candida  microorganism. 
     
     
         3 . The method of  claim 1 , wherein said methyltrophic yeast microorganism is of a species selected from the group consisting of  Pichia pastoris, Pichia acaciae, Pichia anomala, Pichia augusta, Pichia capsulata, Pichia fabianii, Pichia farinosa, Pichia guilliermondii, Pichia methanolica, Pichia norvegensis, Pichia pinus, Pichia stipitis , and  Hansenula polymorpha.    
     
     
         4 . The method of  claim 1 , wherein said methyltrophic yeast microorganism is  Pichia pastoris.    
     
     
         5 .- 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein said nucleic acid molecule encodes a recombinant mite Group 1 protein selected from the group consisting of a pro-form of a recombinant mite Group 1 protein and a mature form of a recombinant mite Group 1 protein. 
     
     
         8 . The method of  claim 1 , wherein said nucleic acid molecule encodes a recombinant mite Group 1 protein fused to a  S. cerevisiae  alpha factor signal sequence. 
     
     
         9 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said recombinant mite Group 1 protein is selected from the group consisting of:
 (a) a recombinant mite Group 1 protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, and SEQ ID NO:92; and   (b) a recombinant mite Group 1 protein that is a fragment of any of said proteins of (a), wherein said fragment has a function selected from the group consisting of selectively binding IgE and causing proliferation of a T cell that proliferates in response to a native mite Group 1 protein.   
     
     
         13 . The method of  claim 1 , wherein said recombinant mite Group 1 protein is selected from the group consisting of a pro-form of said recombinant mite Group 1 protein and a mature form of said recombinant mite Group 1 protein. 
     
     
         14 . The method of  claim 1 , wherein said recombinant mite Group 1 protein is secreted by said methyltrophic yeast microorganism. 
     
     
         15 . The method of  claim 1 , wherein said recombinant mite Group 1 protein binds to IgE of at least about 70% of serum samples comprising IgE that selectively bind to a native mite Group 1 protein. 
     
     
         16 .- 40 . (canceled) 
     
     
         41 . A methyltrophic yeast microorganism comprising a nucleic acid molecule encoding a mite Group 1 protein fragment thereof wherein said nucleic acid molecule consists of a nucleic acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:81, SEQ ID NO:86, and SEQ ID NO:91 or a fragment thereof, said recombinant mite Group 1 protein or fragment is selected from the group consisting of:  Dermatophagoides farinae, Dermatophagoides pteronyssinus , and  Euroglyphus maynei  recombinant mite Group 1 proteins (Der f 1, Der p 1 and Eur m 1) or a fragment thereof and has a function selected from the group consisting of: selectively binding IgE and causing proliferation of a T cell that proliferates in response to a native mite Group 1 protein selected from the group consisting of Der f 1, Der p 1 and Eur m 1, said nucleic acid molecule being operatively linked to a transcription control sequence and wherein said nucleic acid molecule is optionally fused to a signal sequence. 
     
     
         42 . (canceled) 
     
     
         43 . The methyltrophic yeast microorganism of  claim 41 , wherein said nucleic acid molecule consists of a nucleic acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:81, SEQ ID NO:86, and SEQ ID NO:91. 
     
     
         44 . (canceled) 
     
     
         45 . The methyltrophic yeast microorganism of  claim 41 , wherein said recombinant mite Group 1 protein is selected from the group consisting of:
 (a) a recombinant mite Group 1 protein consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35. SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, and SEQ ID NO:92; and   (b) a recombinant mite Group 1 protein that is a fragment of any of said proteins of (a), wherein said fragment has a function selected from the group consisting of selectively binding IgE and causing proliferation of a T cell that proliferates in response to a native mite Group 1 protein.   
     
     
         46 . The methyltrophic yeast microorganism of  claim 41 , wherein said methyltrophic yeast microorganism is of a genus selected from the group consisting of a  Pichia  microorganism, a  Hansenula  microorganism, a  Torulopsis  microorganism, and a  Candida  microorganism. 
     
     
         47 . The methyltrophic yeast microorganism of  claim 46 , wherein said  Pichia  microorganism is  P. pastoris.    
     
     
         48 . The methyltrophic yeast microorganism of  claim 47 , wherein said nucleic acid molecule encodes a recombinant mite Group 1 protein fused to a  S. cerevisiae  alpha factor signal sequence. 
     
     
         49 .- 56 . (canceled) 
     
     
         57 . A method to produce a recombinant protein comprising a recombinant mite Group 1 protein or a fragment thereof, comprising the steps of:
 (a) culturing a methyltrophic yeast microorganism transformed with a nucleic acid molecule encoding said recombinant mite Group 1 protein or fragment; and   (b) recovering said recombinant mite Group 1 protein or fragment from said methyltrophic yeast microorganism;   
       wherein
 said recombinant mite Group 1 protein or fragment is selected from the group consisting of:  Dermatophagoides farinae, Dermatophagoides pteronyssinus , and  Euroglyphus maynei  recombinant mite Group 1 proteins (Der f 1, Derp 1 and Eur m 1) or a fragment thereof and has a function selected from the group consisting of: selectively binding IgE and causing proliferation of a T cell that proliferates in response to a native mite Group 1 protein selected from the group consisting of Der f 1, Der p 1 and Eur m 1 and 
 said nucleic acid molecule hybridizes under stringent conditions with a nucleic acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:81, SEQ ID NO:86, and SEQ ID NO:91 or a fragment thereof and wherein said nucleic acid molecule is optionally fused to a signal sequence. 
 
     
     
         58 . A method to produce a recombinant protein comprising a recombinant mite Group 1 protein or a fragment thereof, comprising the steps of:
 (a) culturing a methyltrophic yeast microorganism transformed with a nucleic acid molecule encoding said recombinant mite Group 1 protein or fragment; and   (b) recovering said recombinant mite Group 1 protein or fragment from said methyltrophic yeast microorganism;   
       wherein
 said recombinant mite Group 1 protein or fragment is selected from the group consisting of:  Dermatophagoides farinae, Dermatophagoides pteronyssinus , and  Euroglyphus maynei  recombinant mite Group 1 proteins (Der f 1, Der p 1 and Eur m 1) or a fragment thereof and has a function selected from the group consisting of: selectively binding IgE and causing proliferation of a T cell that proliferates in response to a native mite Group 1 protein selected from the group consisting of Der f 1, Der p 1 and Eur m 1 and 
 said nucleic acid molecule having a sequence identity of at least 95% with a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:81, SEQ ID NO:86, and SEQ ID NO:91 or a fragment thereof and wherein said nucleic acid molecule is optionally fused to a signal sequence.