Nucleic acids encoding recombinant soluble endoproteases

Abstract

The present invention relates to the expression and secretion in Saccharomyces cerevisiae of readily purifiable soluble variants of the Kex1 endopeptidase of Kluyveromyces lactis and the purification and use thereof for the in vitro processing of recombinant proteins usable in industrial applications. The soluble Kex1 endoproteases described here are free from the transmembrane domain of the native enzyme; the deletion of the transmembrane domain is achieved by removing at least 57 amino acid residues from the C-terminal.

1 - 18 . (canceled) 
     
     
         19 . A DNA fragment coding for a recombinant soluble Kex1 endoprotease consisting of the amino acid sequence set forth as amino acid residues 103 through 600 of SEQ ID NO: 3. 
     
     
         20 . A DNA fragment coding for a recombinant soluble Kex1 endoprotease consisting of the amino acid sequence set forth as amino acid residues 103 through 611 of SEQ ID NO: 3. 
     
     
         21 . The DNA fragment of  claim 19 , wherein the DNA fragment comprises nucleic acids 307 to 1800 of SEQ ID NO:1. 
     
     
         22 . The DNA fragment of  claim 20 , wherein the DNA fragment comprises nucleic acids 307 to 1833 of SEQ ID NO:1. 
     
     
         23 . A plasmid expression vector comprising the DNA fragment according to  claim 19 . 
     
     
         24 . A plasmid expression vector comprising the DNA fragment according to  claim 20 . 
     
     
         25 . A plasmid expression vector comprising the DNA fragment according to  claim 21 . 
     
     
         26 . A plasmid expression vector comprising the DNA fragment according to  claim 22 . 
     
     
         27 . Host cells transformed by the expression vector according to  claim 23 . 
     
     
         28 . Host cells transformed by the expression vector according to  claim 24 . 
     
     
         29 . Host cells transformed by the expression vector according to  claim 25 . 
     
     
         30 . Host cells transformed by the expression vector according to  claim 26 . 
     
     
         31 . The host cells according to  claim 27 , wherein the cells are yeast cells. 
     
     
         32 . The host cells according to  claim 28 , wherein the cells are yeast cells. 
     
     
         33 . The host cells according to  claim 29 , wherein the cells are yeast cells. 
     
     
         34 . The host cells according to  claim 30 , wherein the cells are yeast cells. 
     
     
         35 . The host cells according to  claim 31 , wherein the cells are  Saccharomyces cerevisiae  yeast cells. 
     
     
         36 . The host cells according to  claim 32 , wherein the cells are  Saccharomyces cerevisiae  yeast cells. 
     
     
         37 . The host cells according to  claim 33 , wherein the cells are  Saccharomyces cerevisiae  yeast cells. 
     
     
         38 . The host cells according to  claim 34 , wherein the cells are  Saccharomyces cerevisiae  yeast cells.