Antifreeze proteins from basidiomycetes

Abstract

The present invention provides antifreeze proteins produced by a basidiomycete. The antifreeze protein has a high antifreeze activity such as a thermal hysteresis activity or an ice-recrystallization inhibition activity.

What is claimed is:  
     
         1 . An isolated antifreeze protein produced by a basidiomycete.  
     
     
         2 . The isolated antifreeze protein as defined in  claim 1 , wherein said basidiomycete is a member of the order Aphyllophorales.  
     
     
         3 . The isolated antifreeze protein as defined in  claim 2 , wherein said basidiomycete is a member of the family Ramariaceae.  
     
     
         4 . The isolated antifreeze protein as defined in  claim 1 , wherein said basidiomycete is a member of the order Agaricales.  
     
     
         5 . The isolated antifreeze protein as defined in  claim 4 , wherein said basidiomycete is a member of the family Coprinaceae or Tricholomataceae.  
     
     
         6 . The isolated antifreeze protein as defined in any one of  claims 1  to  5 , which depresses the freezing point of an aqueous solution.  
     
     
         7 . The isolated antifreeze protein as defined in  claim 1 , wherein said protein comprises an N-terminal amino acid sequence selected from the group consisting of: 
 Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Leu-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 1),    Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 2),    Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 3),    Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 4),    Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-Lys-Ala (SEQ ID NO: 5),    Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Thr-Tyr-Ala-Ile-Leu-Thr-Lys-Ala (SEQ ID NO: 6),    Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-Lys-Thr (SEQ ID NO: 7), and    an N-terminal amino acid sequence substantially homologous to any one of SEQ ID NOS: 1 to 7.    
     
     
         8 . An isolated polypeptide comprising an N-terminal amino acid sequence selected from the group consisting of: 
 Ala-Gly-Pro-Ser-Ala-Val-Ala-Gly-Leu-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 1),    Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Val-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 2),    Ala-Gly-Pro-Thr-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 3),    Ala-Gly-Pro-Ser-Ala-Val-Pro-Leu-Gly-Thr-Ala-Gly-Asn-Tyr-Ala-Ile-Leu-Ala-Ser-Thr (SEQ ID NO: 4),    Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-Lys-Ala (SEQ ID NO: 5),    Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Thr-Tyr-Ala-Ile-Leu-Thr-Lys-Ala (SEQ ID NO: 6),    Ala-Gly-Pro-Thr-Ala-Val-Asn-Leu-Gly-Thr-Ala-Lys-Asn-Tyr-Ala-Ile-Leu-Thr-Lys-Thr (SEQ ID NO: 7), and    an N-terminal amino acid sequence substantially homologous to any one of SEQ ID NOS: 1 to 7,    wherein said polypeptide has a molecular mass of 15 to 30 kDa, and depresses the freezing point of an aqueous solution.    
     
     
         9 . An isolated polypeptide comprising a polypeptide selected from the group consisting of an amino acid sequence shown in any one of SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, and an amino acid sequence having one or more amino acid deletions, substitutions or additions relative to an amino acid sequence shown in any one of SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, 
 wherein said polypeptide depresses the freezing point of an aqueous solution.    
     
     
         10 . An isolated polynucleotide encoding a polypeptide selected from the group consisting of an amino acid sequence shown in any one of SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, and an amino acid sequence having one or more amino acid deletions, substitutions or additions relative to an amino acid sequence shown in any one of SEQ ID NOS: 9, 11, 13, 15, 17, 19 and 21, 
 wherein said polypeptide depresses the freezing point of an aqueous solution.    
     
     
         11 . An isolated polynucleotide comprising a polynucleotide selected from the group consisting of a polynucleotide shown in any one of SEQ ID NOS: 8, 10, 12, 14, 16, 18 and 20, and a polynucleotide which hybridizes under stringent conditions with a polynucleotide complementary to a polynucleotide consisting of all or a part of a base sequence shown in any one of SEQ ID NOS: 8, 10, 12, 14, 16, 18 and 20, 
 wherein said polynucleotide encodes a protein which depresses the freezing point of an aqueous solution.    
     
     
         12 . A recombinant vector comprising a polynucleotide as defined in  claim 10  or  11 .  
     
     
         13 . A transformant comprising the recombinant vector as defined in  claim 12 .  
     
     
         14 . A method of preparing an antifreeze protein, comprising culturing the transformant as defined in  claim 13  under conditions such that said transformant produces said antifreeze protein, and collecting the antifreeze protein produced from the resulting culture.  
     
     
         15 . An antifreezing composition comprising the protein as defined in  claim 1  or the polypeptide as defined in  claim 9 , and a carrier or diluent.  
     
     
         16 . An antibody which reacts specifically with the protein as defined in  claim 1  or the polypeptide of  claim 9 .  
     
     
         17 . A protein-antibody complex, said complex comprising a protein bound by an antibody that specifically recognizes and binds an epitope of said protein, wherein said antibody is an antibody as defined in  claim 16 , and wherein said complex depresses the freezing point of an aqueous solution.  
     
     
         18 . A method of preparing an antifreeze protein, comprising culturing a basidiomycete that produces an antifreeze protein under low temperature and conditions such that said basidiomycete produces said antifreeze protein, and collecting the produced antifreeze produced from the resulting culture.