Transformant, and method for producing transferrin
View Patent ↗To provide a method for producing transferrin by secretion with high productivity using a transformant of Schizosaccharomyces pombe , and a transformant suitable for the method. A transformant using Schizosaccharomyces pombe as a host, comprising a transferrin gene and a secretion signal peptide gene expressing transferrin to which a secretion signal peptide which functions in the host binds, located in an upstream region of the transferrin gene, and having gas2 gene endogenous to the host deleted or inactivated; and a method for producing transferrin, which comprises culturing the transformant in a liquid medium and obtaining transferrin from the liquid medium.
1. A schizosaccharomyces pombe host cell comprising a nucleic acid comprising a nucleotide sequence encoding and capable of expressing a secretion signal peptide and a transferrin protein, wherein the secretion signal peptide is fused to the N-terminus of the transferrin protein, wherein a gas2 gene, endogenous to the host cell, is deleted or inactivated.
2. The host cell according to claim 1 , wherein the transferrin protein is a mammalian transferrin protein.
3. The host cell according to claim 2 , wherein the mammalian transferrin protein is a human transferrin protein.
4. The host cell according to claim 2 , wherein the mammalian transferrin protein is a mutant mammalian transferrin protein in which an aspartic residue capable of having an N-linked glycosylation has been deleted or substituted with another amino acid residue.
5. The host cell according to claim 4 , wherein the mutant mammalian transferrin protein is a mutant human transferrin protein in which the aspartic acid residue capable of having an N-linked glycosylation has been deleted or substituted with another amino acid residue.
6. The host cell according to claim 1 , wherein at least one protease gene endogenous to the host cell is deleted or inactivated.
7. The host cell according to claim 6 , wherein the at least one protease gene is a gene selected from the group consisting of a metalloproteinase gene, a serine protease gene, a cysteine protease gene and an aspartic protease gene.
8. The host cell according to claim 6 , wherein the at least one protease gene is a gene selected from the group consisting of psp3gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene.
9. The host cell according to claim 1 , wherein the nucleotide sequence encodes and is capable of expressing a secretory carrier protein comprising the secretion signal peptide, which is fused to the N-terminus of the transferrin protein.
10. The host cell according to claim 9 , wherein the secretion signal peptide is selected from the group consisting of an S. pombe PDI1, a gene encoding an ab-domain portion of human PDI1, and a gene encoding an x-domain portion of S. pombe PDI1.
11. The host cell according to claim 1 , wherein the secretion signal peptide is fused to the N-terminus of the transferrin protein via a polypeptide linker.
12. A method for producing transferrin, which comprises culturing the host cell of claim 1 in a liquid medium and obtaining transferrin from the liquid medium.
13. The method according to claim 12 , wherein the liquid medium has a pH of from 5.5 to 6.5.
14. The method according to claim 12 , wherein the liquid medium comprises adenine.
15. The method according to claim 12 , wherein the host cell is cultured until the cell-density (OD 660 ) reaches 100 or higher, and then transferrin is obtained from the liquid medium.
16. A method for producing the S. pombe host cell of claim 1 , comprising introducing the nucleic acid comprising the nucleotide sequence encoding and capable of expressing the secretion signal peptide and the transferrin protein, wherein the secretion signal peptide is fused to the N-terminus of the transferrin protein, into an S. pombe host cell, and deleting or inactivating the gas2 gene endogenous to the host cell.