Conus protein disulfide isomerase

Abstract

Protein disulfide isomerase is a major component of Conus venom ducts. The invention relates to a protein disulfide isomerase (PDI) from Conus snails, a nucleic acid sequence encoding the Conus protein disulfide isomerase, and to methods for folding disulfide-rich peptides using a protein disulfide isomerase. Oxidative folding of conotoxin precursors, catalyzed by a PDI, was more efficient and decreased the number and concentration of transiently accumulated folding species. The PDI-assisted oxidative folding of conotoxins was also influenced by the propeptide relative to the mature peptide.

What is claimed is:  
     
         1 . An isolated nucleic acid comprising: 
 a nucleic acid encoding a protein disulfide isomerase protein selected from the group consisting of:    a) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:2;    b) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:4;    c) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:6;    d) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:8;    e) a protein disulfide isomerase having at least 57% identity to a protein disulfide isomerase selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, having protein disulfide isomerase activity; and    f) a fragment of said protein disulfide isomerase of (a), (b), (c), (d) or (e), wherein said fragment encodes a protein having protein disulfide isomerase activity.    
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein said isolated nucleic acid is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.  
     
     
         3 . The isolated nucleic acid of  claim 1 , further comprising vector sequences.  
     
     
         4 . The isolated nucleic acid of  claim 3 , wherein said vector is an expression vector.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein said isolated nucleic acid is from a member of the genus  Conus.    
     
     
         6 . A host cell comprising a cell containing the vector of  claim 3 .  
     
     
         7 . A host cell comprising a cell containing the vector of  claim 4 .  
     
     
         8 . The host cells of  claim 7 , further comprising an expression vector encoding a disulfide-rich peptide, wherein said expression vector encoding a disulfide-rich peptide and said expression vector encoding a protein disulfide isomerase comprise one or more nucleic acid molecules.  
     
     
         9 . A method for producing a protein disulfide isomerase comprising: 
 introducing into a host cell a nucleic acid encoding protein disulfide isomerase selected from the group consisting of:    a) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:2;    b) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:4;    c) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:6;    d) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:8;    e) a protein disulfide isomerase having at least 57% identity to a protein disulfide isomerase selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, having protein disulfide isomerase activity; and    f) a fragment of said protein disulfide isomerase of (a), (b), (c), (d) or (e), wherein said fragment encodes a protein having protein disulfide isomerase activity;    expressing said protein disulfide isomerase; and    isolating said protein disulfide isomerase.    
     
     
         10 . The method according to  claim 9 , wherein said cell is selected from the group consisting of an insect cell, HIGH FIVE™, Sf9, Sf21,  Drosophila  Schneider2, a mammalian cell, COS 1, NIH 3T3, HeLa, 293, CHO, U266, a plant cells, Baculovirus,  Saccharomyces, Schizosaccharomyces, Aspergillus, E. coli,  and  Bacillus.    
     
     
         11 . A method for producing a correctly-folded disulfide-rich peptide comprising: 
 introducing a nucleic acid encoding a protein disulfide isomerase and a nucleic acid encoding a disulfide-rich peptide into a host cell, wherein said nucleic acid encoding a protein disulfide isomerase and a disulfide-rich peptide comprise one or more nucleic acid molecules;    expressing a protein disulfide isomerase peptide and a disulfide-rich peptide, wherein said disulfide-rich peptide is a conotoxin; and    isolating a correctly-folded disulfide-rich peptide.    
     
     
         12 . The method according to  claim 11 , wherein said host cell is selected from the group consisting of an insect cell, HIGH FIVE™, Sf9, Sf21,  Drosophila  Schneider2, a mammalian cell, COS 1, NIH 3T3, HeLa, 293, CHO, U266, a plant cells, Baculovirus,  Saccharomyces, Schizosaccharomyces, Aspergillus, E. coli,  and  Bacillus.    
     
     
         13 . The method according to  claim 11 , wherein said protein disulfide isomerase peptide is from a member of the genus  Conus.    
     
     
         14 . A method for producing a correctly-folded disulfide-rich peptide comprising: 
 introducing into a host cell a nucleic acid encoding a protein disulfide isomerase selected from the group consisting of:    a) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:2;    b) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:4;    c) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:6;    d) a protein disulfide isomerase comprising an amino acid sequence as set forth in SEQ ID NO:8;    e) a protein disulfide isomerase having at least 57% identity to a protein disulfide isomerase selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, having protein disulfide isomerase activity; and    f) a fragment of said protein disulfide isomerase of (a), (b), (c), (d) or (e), wherein said fragment encodes a protein having protein disulfide isomerase activity    introducing a nucleic acid encoding a disulfide-rich peptide, wherein said nucleic acid encoding a protein disulfide isomerase and said nucleic acid encoding a disulfide-rich peptide comprise one or more nucleic acid molecules;    expressing a protein disulfide isomerase peptide and a disulfide-rich peptide; and    isolating a correctly-folded disulfide-rich peptide.    
     
     
         15 . The method according to  claim 14 , wherein said cell is selected from the group consisting of an insect cell, HIGH FIVE™, Sf9, Sf21,  Drosophila  Schneider2, a mammalian cell, COS 1, NIH 3T3, HeLa, 293, CHO, U266, a plant cells, Baculovirus,  Saccharomyces, Schizosaccharomyces, Aspergillus, E. coli,  and  Bacillus.    
     
     
         16 . The method according to  claim 14 , wherein said disulfide-rich peptide is a conotoxin.  
     
     
         17 . An isolated protein disulfide isomerase polypeptide comprising a protein disulfide isomerase selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a protein having at least 57% identity to said protein disulfide isomerase and a fragment thereof, wherein said protein disulfide isomerase or fragment thereof has protein disulfide isomerase activity.  
     
     
         18 . A method for producing a correctly-folded disulfide-rich peptide comprising: 
 combining a disulfide-rich peptide and a protein disulfide isomerase, wherein said protein disulfide isomerase is selected from the group consisting of a protein disulfide isomerase as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a protein having at least 57% identity to said protein disulfide isomerase and a fragment thereof, wherein said protein disulfide isomerase or fragment thereof has protein disulfide isomerase activity; and    isolating said correctly-folded disulfide-rich peptide.    
     
     
         19 . The method according to  claim 18 , wherein said disulfide-rich peptide is a conotoxin polypeptide.  
     
     
         20 . The method according to  claim 18 , further comprising: 
 adding a cell extract.    
     
     
         21 . The method according to  claim 20 , wherein said cell extract comprises a translation system.  
     
     
         22 . The method according to  claim 21 , wherein said cell extract further comprising a transcription system.  
     
     
         23 . A method for producing a correctly-folded disulfide-rich peptide comprising: 
 combining a disulfide-rich conotoxin peptide and a protein disulfide isomerase or fragment thereof, wherein said fragment has protein disulfide isomerase activity; and    isolating said correctly-folded disulfide-rich peptide.    
     
     
         24 . The method according to  claim 23 , further comprising: 
 adding a cell extract.    
     
     
         25 . The method according to  claim 24 , wherein said cell extract comprises a translation system.  
     
     
         26 . The method according to  claim 25 , wherein said cell extract further comprising a transcription system.  
     
     
         27 . The method according to  claim 23 , wherein said protein disulfide isomerase is from a member of the genus  Conus.