Mutant Proteinase with Reduced Self-Cleavage Activity and Method of Purification

Abstract

The present invention provides a mutant 27 kDa NIa proteinase having reduced self-cleavage activity relative to the self-cleavage activity of its wild-type proteinase. The mutant has the same substrate cleavage activity as the wild-type proteinase but is more stable than the wild-type proteinase. The present invention also provides a method of obtaining large quantities of active 27 kDa NIa proteinase for use as a tool for purification of other proteins.

1 . An isolated mutant proteinase having a molecular weight of about 27 kDa and reduced self-cleavage activity relative to the self-cleavage activity of its wild-type proteinase. 
     
     
         2 . The mutant proteinase of  claim 1 , wherein the mutant proteinase comprises an amino acid sequence in which the residue corresponding to Ser 219 of the 27 kDa NIa proteinase is replaced with another residue. 
     
     
         3 . The mutant proteinase of  claim 2 , wherein the residue replacing Ser 219 of 27 kDa NIa proteinase is Asn. 
     
     
         4 . A composition comprising the mutant proteinase of  claim 1  and a carrier. 
     
     
         5 . A fusion protein comprising the mutant proteinase of  claim 1  fused to a heterologous polypeptide. 
     
     
         6 . The fusion protein of  claim 5 , wherein the heterologous polypeptide consists of six histidines. 
     
     
         7 . An isolated nucleic acid molecule comprising a sequence encoding a mutant proteinase having a molecular weight of about 27 kDa and reduced self-cleavage activity relative to the self-cleavage activity of its wild-type proteinase. 
     
     
         8 . An isolated nucleic acid molecule of  claim 7 , wherein the nucleic acid molecule encodes an amino acid sequence in which the residue corresponding to Ser 219 of the 27 kDa NIa proteinase is replaced with another amino acid. 
     
     
         9 . An isolated nucleic acid of  claim 8 , wherein the nucleic acid comprises a sequence that encodes an amino acid sequence in which the residue corresponding to Ser 219 of the 27 kDa NIa proteinase in Asn. 
     
     
         10 . A vector comprising the nucleic acid of  claim 7 . 
     
     
         11 . An expression vector comprising the nucleic acid of  claim 7 . 
     
     
         12 . A host cell comprising the expression vector of  claim 11 . 
     
     
         13 . A method of producing a proteinase having a molecular weight of about 27 kDa and reduced self-cleavage activity relative to the self-cleavage activity of its wild-type proteinase activity comprising cultivating the host cell of  claim 12  under conditions that allow expression of the proteinase. 
     
     
         14 . a nucleic acid encoding a fusion protein comprising a proteinase, having a molecular weight of about 27 kDA and reduced self-cleavage activity relative to the self-cleavage activity of its wild-type proteinase, fused to a heterologous protein. 
     
     
         15 . The nucleic acid of  claim 14 , wherein the heterologous protein consists of six histidines. 
     
     
         16 . A method of purifying a polypeptide that forms inclusion bodies in a cell comprising the following steps:
 a) obtaining cells expressing the polypeptide;   b) lysing the cells;   c) pelleting inclusion bodies by centrifugation of the lysed cells;   d) centrifuging the inclusion bodies;   e) solubilizing the inclusion bodies in buffer containing solubilization   f) centrifuging the solubilized inclusion bodies to obtain a supernatant containing the polypeptide;   g) loading the supernatant over a single denaturing column;   h) collecting the proteinase; and   i) renaturing the collected polypeptide.   
     
     
         17 . The method of  claim 16 , wherein the polypeptide has a molecular weight of about 27 kDa and reduced self-cleavage activity relative to its wild-type polypeptide. 
     
     
         18 . The method of  claim 16 , wherein the polypeptide is selected from the group consisting of tobacco etch virus (TEV) 27 kDa NIa proteinase and mutant TPSN 27 kDa NIa proteinase. 
     
     
         19 . The method of  claim 16 , wherein in step (g) the denaturing column contains Ni-NTA. 
     
     
         20 . A method of using the proteinase of  claim 1 , comprising incubating the proteinase with a protein for a sufficient amount of time to allow cleavage of the protein.