Proteases and methods for producing them

Abstract

A secreted mature polypeptide derived from an S2A or S1E protease which after maturation has protease activity, which polypeptide when expressed and before maturation comprises a heterologous pro-region.

1 - 21 . (canceled)  
     
     
         22 . A secreted mature polypeptide which after maturation has protease activity, which polypeptide when expressed and before maturation comprises a heterologous pro-region, and which polypeptide: 
 (a) comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of the mature part of the polypeptide shown in SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO: 43; or SEQ ID NO: 45;    (b) comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of the mature part of the polypeptide encoded by the polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 25; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; or SEQ ID NO: 44;    (c) comprises a mature part which is a variant of the mature part of the polypeptide having the amino acid sequence of SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO: 43; or SEQ ID NO: 45, the segment comprising a substitution, deletion, extension, and/or insertion of one or more amino acids;    (d) is a fragment of (a), (b) or (c).    
     
     
         23 . The polypeptide according to  claim 22 , wherein the heterologous pro-region is derived from a protease.  
     
     
         24 . The polypeptide according to  claim 22 , wherein the heterologous pro-region is derived from an S2A or S1E protease.  
     
     
         25 . The polypeptide according to  claim 22 , wherein the heterologous pro-region is 70% identical to the pro-region shown in SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53.  
     
     
         26 . The polypeptide according to  claim 22  which comprises at least three non-polar or uncharged polar amino acids within the last four amino acids of the C-terminus of the polypeptide  
     
     
         27 . The polypeptide according to  claim 22 , wherein said polypeptide comprises one or more added amino acids, and where said one more added amino acids are non-polar or uncharged.  
     
     
         28 . The polypeptide according to  claim 27 , wherein the one or more added amino acid(s) are selected from the group consisting of Q, S, V, A, or P.  
     
     
         29 . The polypeptide according to  claim 27 , wherein the one or more added amino acids are selected from the group consisting of: QSHVQSAP, QSAP, QP, TL, TT, QL, TP, LP, TI, IQ, QP, PI, LT, TQ, IT, QQ, and PQ.  
     
     
         30 . The polypeptide according to  claim 22  which when expressed and before maturation comprises a heterologous secretion signal-peptide which is cleaved from the polypeptide when the polypeptide is secreted.  
     
     
         31 . The polypeptide according to  claim 30 , wherein the heterologous secretion signal peptide comprises an amino acid sequence having a sequence identity of at least 70% with the amino acid sequence encoded by polynucleotides 1-81 of SEQ ID NO: 2, or SEQ ID NO: 44.  
     
     
         32 . An isolated polynucleotide encoding a polypeptide as defined in  claim 22 .  
     
     
         33 . A recombinant expression vector comprising a polynucleotide as defined in  claim 32 .  
     
     
         34 . A recombinant host cell comprising a polynucleotide as defined in  claim 32 .  
     
     
         35 . The recombinant host cell according to  claim 34  which is a  Bacillus  cell.  
     
     
         36 . A transgenic plant or plant part, comprising a polynucleotide as defined in  claim 32 .  
     
     
         37 . A transgenic, non-human animal comprising a polynucleotide as defined in  claim 32 .  
     
     
         38 . A method for producing a polypeptide, the method comprising: (a) cultivating a recombinant host cell as defined in  claim 34 , to produce a supernatant comprising the polypeptide, and (b) recovering the polypeptide.  
     
     
         39 . The polypeptide of  claim 22 , wherein the polypeptide: 
 (a) comprises an amino acid sequence which is at least 80% identical to the amino acid sequence of the mature part of the polypeptide shown in SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO: 43; or SEQ ID NO: 45; or    (b) comprises an amino acid sequence which is at least 80% identical to the amino acid sequence of the mature part of the polypeptide encoded by the polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 25; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; or SEQ ID NO: 44.    
     
     
         40 . The polypeptide of  claim 22 , wherein the polypeptide: 
 (a) comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of the mature part of the polypeptide shown in SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO: 43; or SEQ ID NO: 45; or    (b) comprises an amino acid sequence which is at least 9% identical to the amino acid sequence of the mature part of the polypeptide encoded by the polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 25; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; or SEQ ID NO: 44.    
     
     
         41 . The polypeptide of  claim 22 , wherein the polypeptide: 
 (a) comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of the mature part of the polypeptide shown in SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO: 43; or SEQ ID NO: 45; or    (b) comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of the mature part of the polypeptide encoded by the polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 25; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; or SEQ ID NO: 44.