Glucuronyl C5-Epimerase, DNA encoding the same and uses thereof

Abstract

The invention is directed to a novel purified mouse C5-epimerase, fragments thereof, nucleic acids encoding the same and the recombinant production thereof. The invention is also directed to fragments of such epimerase, especially N-terminal fragments that are useful in fusion protein constructs to enhance the activity of recombinantly-produced heterologous epimerase enzymes.

1 . An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide, the amino acid sequence of which is at least 95% identical to a reference amino acid sequence selected from the group consisting of:
 (a) amino acids 1 to 118 of  FIG. 3 ;   (b) amino acids 1 to 119 of  FIG. 3 ;   (c) amino acids 1 to 120 of  FIG. 3 ;   (d) amino acids 1 to 121 of  FIG. 3 ;   (e) amino acids 119 to 618 of  FIG. 3 ;   (f) amino acids 120 to 618 of  FIG. 3 ;   (g) amino acids 121 to 618 of  FIG. 3 ;   (h) amino acids 122 to 618 of  FIG. 3 ;   (i) amino acids 34 to 147 of  FIG. 3 ;   (j) amino acids 35 to 154 of  FIG. 3 ;   (k) amino acids 34 to 154 of  FIG. 3 ;   (l) amino acids 1 to 154 of  FIG. 3 ;   (m) amino acids 155-618 of  FIG. 3  and   (n) amino acids 1-618 of  FIG. 3 .   
     
     
         2 . The polynucleotide of  claim 1  which is DNA. 
     
     
         3 . The polynucleotide of  claim 1  which is RNA. 
     
     
         4 . The polynucleotide of  claim 1 , further comprising a heterologous polynucleotide. 
     
     
         5 . The polynucleotide of  claim 4 , wherein said heterologous polynucleotide encodes a heterologous polypeptide. 
     
     
         6 . The polynucleotide of  claim 5 , wherein said heterologous polynucleotide is positioned at the 3′ of said nucleotide sequence. 
     
     
         7 . A vector comprising the polynucleotide of  claim 1 . 
     
     
         8 . The vector of  claim 7 , wherein said polynucleotide is operably linked to a heterologous regulatory polynucleotide. 
     
     
         9 . A host cell comprising the polynucleotide of  claim 1 . 
     
     
         10 . The host cell of  claim 9 , wherein said isolated polynucleotide is operably linked to a heterologous regulatory polynucleotide. 
     
     
         11 . A method of producing a protein that comprises culturing the host cell of  claim 10  under conditions such that said protein is expressed, and recovering said protein. 
     
     
         12 . An isolated C5-epimerase polypeptide, the amino acid sequence of which is at least 95% identical to a sequence selected from the group consisting of:
 (a) amino acids 1 to 118 of  FIG. 3 ;   (b) amino acids 1 to 119 of  FIG. 3 ;   (c) amino acids 1 to 120 of  FIG. 3 ;   (d) amino acids 1 to 121 of  FIG. 3 ;   (e) amino acids 119 to 618 of  FIG. 3 ;   (f) amino acids 120 to 618 of  FIG. 3 ;   (g) amino acids 121 to 618 of  FIG. 3 ;   (h) amino acids 122 to 618 of  FIG. 3 ;   (i) amino acids 34 to 147 of  FIG. 3 ;   (j) amino acids 35 to 154 of  FIG. 3 ;   (k) amino acids 34 to 154 of  FIG. 3 ;   (l) amino acids 1 to 154 of  FIG. 3 ;   (m) amino acids 155-618 of  FIG. 3 ; and   (n) amino acids 1-618 of  FIG. 3 .   
     
     
         13 . The isolated polypeptide of  claim 12 , which is produced or contained in a recombinant host cell. 
     
     
         14 . The isolated polypeptide of  claim 13 , wherein said recombinant host cell is an insect cell. 
     
     
         15 . A method of increasing the activity of a C5-epimerase, said method comprising:
 (a) providing a first polynucleotide comprising a nucleotide sequence encoding a polypeptide, the amino acid sequence of which is at least 80% identical to a reference amino acid sequence selected from the group consisting of amino acids 35 to 154 of  FIG. 3  and amino acids 34 to 154 of  FIG. 3 ;   (b) attaching said first polynucleotide of (a) to a second polynucleotide encoding a C5-epimerase; and   (c) expressing the fusion polynucleotide.   
     
     
         16 . The method of  claim 15 , wherein said first polynucleotide comprises a nucleotide sequence encoding a polypeptide, the amino acid sequence of which is amino acids 35 to 154 of  FIG. 3 . 
     
     
         17 . The method of  claim 15 , wherein said first polynucleotide comprises a nucleotide sequence encoding a polypeptide, the amino acid sequence of which is amino acids 34 to 154 of  FIG. 3 . 
     
     
         18 . The method of  claim 15 , wherein said second polynucleotide encoding a C5-epimerase encodes bovine C5-epimerase.