Neublastin expression constructs
View Patent ↗A method of producing a secreted neublastin polypeptide using a heterologous signal sequence is disclosed. The secreted neublastin does not contain a neublastin pro sequence.
1. A method of making a secreted neublastin polypeptide, the method comprising:
providing a eukaryotic host cell transformed with a DNA comprising a nucleotide sequence that encodes a secreted neublastin polypeptide lacking a functional neublastin signal peptide and a neublastin pro-domain, wherein the neublastin polypeptide is operatively linked to a signal sequence selected from the group consisting of rat albumin signal sequence and human growth hormone signal sequence, and
culturing the eukaryotic cell under conditions so that the neublastin polypeptide is expressed and secreted.
2. The method of claim 1 , wherein the eukaryotic host cell is a mammalian cell.
3. The method of claim 2 , wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
4. The method of claim 1 , wherein the secreted neublastin polypeptide is selected from the group consisting of:
the 113 C-terminal amino acids of human neublastin;
the 112 C-terminal amino acids of human neublastin;
the 111 C-terminal amino acids of human neublastin;
the 110 C-terminal amino acids of human neublastin;
the 109 C-terminal amino acids of human neublastin;
the 108 C-terminal amino acids of human neublastin;
the 107 C-terminal amino acids of human neublastin;
the 106 C-terminal amino acids of human neublastin;
the 105 C-terminal amino acids of human neublastin;
the 104 C-terminal amino acids of human neublastin;
the 103 C-terminal amino acids of human neublastin;
the 102 C-terminal amino acids of human neublastin;
the 101 C-terminal amino acids of human neublastin;
the 100 C-terminal amino acids of human neublastin; and
the 99 C-terminal amino acids of human neublastin.
5. The method of claim 4 , wherein the secreted neublastin polypeptide is the 104 C-terminal amino acids of human neublastin.
6. The method of claim 1 , wherein the signal sequence is a rat albumin signal sequence.
7. The method of claim 1 , wherein the signal sequence is a human growth hormone signal sequence.
8. A nucleic acid comprising a nucleotide sequence that encodes a secreted neublastin polypeptide lacking a functional neublastin signal peptide and a neublastin pro-domain, wherein the neublastin polypeptide is operatively linked to a signal sequence selected from the group consisting of a rat albumin signal sequence, the modified albumin signal sequence MKWVTFLLFLLFISGEAFA (amino acids 1 to 19 of SEQ ID NO:26), and a human growth hormone signal sequence.
9. A host cell comprising the nucleic acid of claim 8 .
10. The host cell of claim 9 , wherein the host cell is a mammalian cell.
11. The host cell of claim 10 , wherein the mammalian cell is a CHO cell.
12. The nucleic acid of claim 8 , comprising:
the nucleotide sequence of SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:25; or
a nucleotide sequence encoding the polypeptide of SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:26.
13. The nucleic acid of claim 8 , wherein the nucleotide sequence has been optimized for expression in a mammalian host cell.
14. The nucleic acid of claim 8 , wherein the signal sequence is a rat albumin signal sequence.
15. The nucleic acid of claim 8 , wherein the signal sequence is the modified albumin signal sequence MKWVTFLLFLLFISGEAFA (amino acids 1 to 19 of SEQ ID NO:26).
16. The nucleic acid of claim 8 , wherein the signal sequence is a human growth hormone signal sequence.
17. The nucleic acid of claim 8 , wherein the secreted neublastin polypeptide is selected from the group consisting of:
the 113 C-terminal amino acids of human neublastin;
the 112 C-terminal amino acids of human neublastin;
the 111 C-terminal amino acids of human neublastin;
the 110 C-terminal amino acids of human neublastin;
the 109 C-terminal amino acids of human neublastin;
the 108 C-terminal amino acids of human neublastin;
the 107 C-terminal amino acids of human neublastin;
the 106 C-terminal amino acids of human neublastin;
the 105 C-terminal amino acids of human neublastin;
the 104 C-terminal amino acids of human neublastin;
the 103 C-terminal amino acids of human neublastin;
the 102 C-terminal amino acids of human neublastin;
the 101 C-terminal amino acids of human neublastin;
the 100 C-terminal amino acids of human neublastin; and
the 99 C-terminal amino acids of human neublastin.
18. The nucleic acid of claim 17 , wherein the secreted neublastin polypeptide is the 104 C-terminal amino acids of human neublastin.