Production of recombinant selenoprotein mutants with enhanced catalytic activity

Abstract

The present invention generally relates to the production of industrially relevant quantities of selenoprotein enzymes in eukaryotic cell cultures. More specifically, the present invention generally relates to the production of such enzymes wherein one or more catalytic cysteine or serine residues are mutagenically replaced by selenocysteine.

1 - 8 . (canceled) 
     
     
         9 . A mutant papain enzyme, comprising selenocysteine. 
     
     
         10 . The mutant papain enzyme of  claim 9 , wherein cysteine-25 in the active site is replaced by selenocysteine. 
     
     
         11 . The mutant papain enzyme of  claim 10 , which exhibits enhanced catalytic activity compared to that of papain containing cysteine-25 in the active site. 
     
     
         12 . A method of making a mutant papain containing selenocysteine at amino acid position 25 located in the active site, in a eukaryotic algal host cell, comprising:
 a) introducing into said eukaryotic algal host cell an expression cassette comprising, operably linked for expression in said eukaryotic algal host cell in the following order:
 i) a tightly regulated gene promoter active in a eukaryotic alga; 
 ii) a nucleic acid sequence encoding papain, wherein UGA replaces the codon encoding cysteine at amino acid position 25, located in the active site of said papain; and 
 iii) a 3′ untranslated eukaryotic selenocysteine insertion sequence; 
   b) selecting transformed cells;   c) culturing selected cells in a medium that promotes expression of said mutant selenocysteine-containing papain; and   d) recovering said mutant selenocysteine-containing papain from said eukaryotic algal host cell or said medium when said papain is secreted.   
     
     
         13 . The method of  claim 12 , wherein said tightly regulated gene promoter is the Fea1 promoter. 
     
     
         14 . The method of  claim 12 , wherein said nucleic acid sequence encoding papain comprises a nucleic acid sequence encoding a FLAG tag. 
     
     
         15 . The method of  claim 12 , wherein said expression cassette further comprises a selection sequence. 
     
     
         16 . The method of  claim 15 , wherein said selection sequence is an antibiotic or auxotrophic selection sequence. 
     
     
         17 . The method of  claim 12 , wherein said expression cassette further comprises a signal sequence that causes secretion of said papain from said eukaryotic alga. 
     
     
         18 . The method of  claim 17 , wherein said signal sequence is a 5′ perplasmic targeting sequence. 
     
     
         19 . The method of  claim 18 , wherein said signal sequence is that of the Fea1 gene, which causes said papain to accumulate between the cell membrane and cell wall. 
     
     
         20 . The method of  claim 12 , wherein said expression cassette further comprises an immuno-tagging sequence located downstream from said signal sequence, at the N-terminal end of said papain. 
     
     
         21 . The method of  claim 20 , wherein said immuno-tagging sequence is a FLAG epitope. 
     
     
         22 . The method of  claim 12 , wherein said expression cassette further comprises a sequence coding for an RNAi that down-regulates expression of a gene product that interferes with expression of said papain. 
     
     
         23 . The method of  claim 22 , wherein said sequence coding for an RNAi that down-regulates expression of a gene product that interferes with expression of said papain is a copy of at least a portion of a nucleic acid sequence encoding a translated wild-type gene in reverse orientation. 
     
     
         24 . The method of  claim 23 , wherein said gene product that interferes with expression of said papain is wild-type Fea1 protein. 
     
     
         25 . The method of  claim 12 , wherein said 3′ untranslated eukaryotic selenocysteine insertion sequence is  Chlamydomonas  selenocysteine insertion sequence SeIKI. 
     
     
         26 . The method of  claim 25 , wherein said SeIKI sequence is part of a SeIKI terminator sequence. 
     
     
         27 . The method of  claim 12 , wherein said expression cassette is contained in an expression vector. 
     
     
         28 . The method of  claim 27 , wherein said expression vector is a plasmid. 
     
     
         29 . The method of  claim 28 , wherein said plasmid is pUC18 or pUC19. 
     
     
         30 . The method of  claim 12 , wherein said expression cassette is introduced into said eukaryotic algal host cell via a virus, electroporation, transfection, or the glass bead method. 
     
     
         31 . The method of  claim 12 , further comprising introducing into said eukaryotic algal host cell a cassette that reduces expression of periplasmic-targeted proteins to facilitate purification of said selenocysteine-containing papain. 
     
     
         32 . The method of  claim 12 , further comprising separately introducing into said eukaryotic algal host cell a selection sequence by co-transforming said eukaryotic algal host cell with an additional plasmid comprising said selection sequence. 
     
     
         33 . The method of  claim 32 , wherein said selection sequence is an antibiotic or auxotrophic selection sequence. 
     
     
         34 . The method of  claim 12 , wherein said medium contains 100 μg/L sodium selenite. 
     
     
         35 . The method of  claim 12 , wherein expression of expression of said mutant selenocysteine-containing papain is induced by elevated carbon dioxide, low iron, or high cadmium levels. 
     
     
         36 . The method of  claim 12 , wherein said eukaryotic algal host cell is  Chlamydomonas.    
     
     
         37 . A method of making a mutant protein containing selenocysteine in a eukaryotic algal host cell, comprising:
 a) introducing into said eukaryotic algal host cell an expression cassette comprising, operably linked for expression in said eukaryotic algal host cell in the following order:
 i) a tightly regulated gene promoter active in a eukaryotic alga; 
 ii) a nucleic acid sequence encoding said protein, wherein UGA replaces a codon encoding cysteine at a desired amino acid position in said protein; and 
 iii) a 3′ untranslated eukaryotic selenocysteine insertion sequence; 
   b) selecting transformed cells;   c) culturing selected cells in a medium that promotes expression of said mutant selenocysteine-containing protein; and   d) recovering said mutant selenocysteine-containing protein from said eukaryotic algal host cell or said medium when said protein is secreted.