Modified proteinase inhibitors

Abstract

Modified proteinase inhibitors having enhanced inhibition efficacy when compared to the naturally occurring counterparts. Also provided are polynucleotide molecules encoding the modified proteinase inhibitors, vectors and other recombinant nucleic acid constructs, transgenic cells, and transgenic organisms that comprise the polynucleotide molecules. Further disclosed are methods for producing the modified proteinase inhibitors, and methods and compositions for increasing the resistance of a cell or an organism to proteolytic damages, particularly the damages caused by a pest or a pathogen.

We claim:  
     
         1 . An isolated polynucleotide molecule comprising a sequence that encodes a polypeptide sequence, wherein said polypeptide sequence is encoded by CYT1-ORYSA-D86 of FIG. 1, and has increased proteinase inhibiting activity when compared to a naturally-occurring cystatin.  
     
     
         2 . An isolated polynucleotide molecule according to  claim 1 , wherein said polypeptide sequence comprises a sequence encoded by the nucleotide sequence of FIG. 2.  
     
     
         3 . An isolated polynucleotide molecule according to  claim 2 , wherein said polypeptide sequence is encoded by the nucleotide sequence of FIG. 2.  
     
     
         4 . An isolated polynucleotide molecule according to  claim 2 , wherein said polynucleotide comprises the nucleotide sequence of FIG. 2.  
     
     
         5 . An isolated polynucleotide molecule encoding a modified cystatin which has increased proteinase inhibiting activity when compared to its naturally-occurring counterpart, and which comprises at least a deletion of an amino acid residue corresponding to aspartic acid at position 86 of oryzacystatin.  
     
     
         6 . A vector comprising the polynucleotide of  claim 1  operatively linked to a regulator sequence.  
     
     
         7 . A vector according to  claim 6 , wherein the vector is an expression vector and the regulatory sequence is a promoter.  
     
     
         8 . A cell transformed with a vector of  claim 7 .  
     
     
         9 . A cell of  claim 8 , wherein the cell is a plant cell.  
     
     
         10 . A transgenic plant regerated from a cell of  claim 9 .  
     
     
         11 . A method of producing a proteinase inhibitor, the method comprising cultivating a cell of  claim 8  under conditions suitable for expression of said expression vector, and isolating the proteinase inhibitor expressed.  
     
     
         12 . An isolated polypeptide encoded by the polynucleotide of  claim 1 .  
     
     
         13 . A composition comprising an isolated polypeptide of  claim 12  and a suitable carrier.  
     
     
         14 . A modified cystatin which has increased proteinase inhibiting activity when compared to its naturally-occurring counterpart, and comprises at least a deletion of an amino acid residue corresponding to aspartic acid at position 86 of oryzacystatin I.  
     
     
         15 . A hybrid proteinase inhibitor comprises at least a partial sequence of an animal cystatin and at least a partial sequence of a piant cystatin linked together, wherein the hybrid proteinase inhibitor has an increased proteinase inhibitor activity when compared to a naturally occurring counterpart of said plant cystatin.  
     
     
         16 . A hybrid proteinase inhibitor according to  claim 15 , wherein said animal cystatin is CEWC.  
     
     
         17 . A hybrid proteinase inhibitor according to  claim 15 , wherein said plant cystatin is oryzacystatin I.  
     
     
         18 . A hybrid proteinase inhibitor according to  claim 15 , which comprises oc-nterm-1-15qvlsg40-63-cterm-97-116.  
     
     
         19 . A method for conferring to a cell resistance to proteolytic damages, said method comprising modifying the cell such that it expresses a polypeptide of  claim 12 .  
     
     
         20 . A method according to  claim 19 , wherein the cell is a plant cell.  
     
     
         21 . A method according to  claim 19 , wherein the polypeptide is encoded by a polynucleotide molecule which is operatively linked to a regulatory sequence.  
     
     
         22 . A method according to  claim 21 , wherein the regulatory sequence is a tissue-specific or a developmental stage-specific promoter.  
     
     
         23 . A method for increasing resistance of a plant to a pest, said method comprising providing a plant cell according to the method of  claim 19 , and regenerating said plant cell into a transgenic plant.  
     
     
         24 . A method according to  claim 23 , wherein the plant is a monocot.  
     
     
         25 . A method according to  claim 23 , wherein the plant is a dicot.  
     
     
         26 . A method according to  claim 23 , wherein the pest is a nematode.  
     
     
         27 . A method for reducing proteolytic damages in an organism, said method comprising administering an effective amount of the composition of  claim 13  to an organism in need thereof.  
     
     
         28 . A method according to  claim 27 , wherein said organism is a plant.