METHOD FOR PRODUCING A MUCIN-TYPE GLYCOPROTEIN
The present invention relates to a means for generating a mucin-type glycopeptide or glycoprotein on a large scale in yeast. Specifically, the invention relates to a method which comprises introducing into a yeast at least one selected from the group consisting of a gene encoding UDP-GalNAc synthetase, a gene encoding UDP-GalNAc transporter, and a gene encoding polypeptide:O-GalNAc transferase, and, if desired, a gene encoding a mucin-type glycopeptide; and producing a mucin-type glycoprotein having O-GalNAc by use of the yeast.
1 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising the step of introducing into a yeast at least one selected from the group consisting of a gene encoding UDP-GalNAc synthetase, a gene encoding UDP-GalNAc transporter, and a gene encoding polypeptide:O-GalNAc transferase.
2 . The method according to claim 1 , wherein the method comprises the steps of:
(1) introducing the gene encoding UDP-GalNAc synthetase; and
(2) introducing the gene encoding UDP-GalNAc transporter into the yeast.
3 . The method according to claim 1 , wherein the method comprises the steps of:
(1) introducing the gene encoding UDP-GalNAc synthetase; and
(2) introducing the gene encoding polypeptide:O-GalNAc transferase into the yeast.
4 . The method according to claim 1 , wherein the method comprises the steps of:
(1) introducing the gene encoding UDP-GalNAc transporter; and
(2) introducing the gene encoding polypeptide:O-GalNAc transferase into the yeast.
5 . The method according to claim 1 , wherein the method comprises the steps of:
(1) introducing the gene encoding UDP-GalNAc synthetase;
(2) introducing the gene encoding UDP-GalNAc transporter; and
(3) introducing the gene encoding polypeptide:O-GalNAc transferase into the yeast.
6 . The method according to claim 1 , wherein the polypeptide:O-GalNAc transferase comprises the transmembrane domain of a protein localized in the yeast Golgi apparatus.
7 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising the step of further introducing a gene encoding a mucin-type glycoprotein into the yeast obtained by the method according to claim 1 .
8 . The method according to claim 7 , wherein the gene encoding a mucin-type glycoprotein comprises a secretion signal sequence derived from yeast.
9 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising the step of further introducing a gene encoding core 1 β1-3 Gal transferase, core 2 β-1,6-GlcNAc transferase, or core 3 β-1,3-GlcNAc transferase into the yeast obtained by the method according to claim 1 .
10 . The method according to claim 9 , wherein the core 1 β1-3 Gal transferase comprises the transmembrane domain of a protein localized in the yeast Golgi apparatus.
11 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising further performing the steps of:
(1) introducing a gene encoding UDP-Gal synthetase into the yeast obtained by the production method according to claim 9 , when the UDP-GalNAc synthetase has no UDP-Gal synthetic activity; and/or
(2) introducing a gene encoding a UDP-Gal transporter into the yeast obtained by the production method according to claim 9 , when the UDP-GalNAc transporter has no UDP-Gal transport activity.
12 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising the step of further introducing a gene encoding a mucin-type glycoprotein into the yeast obtained by the method according to claim 9 .
13 . The method according to claim 12 , wherein the gene encoding a mucin-type glycoprotein comprises a secretion signal sequence derived from yeast.
14 . The method according to claim 1 , wherein the yeast is a methanol-utilizing yeast.
15 . The method according to claim 14 , wherein the methanol-utilizing yeast is Ogataea minuta.
16 . The method according to claim 1 , wherein the yeast is Saccharomyces cerevisiae.
17 . A transformed yeast obtained by the method according to claim 1 .
18 . A method for producing a mucin-type glycoprotein, comprising the steps of:
culturing the transformed yeast according to claim 17 ; and
collecting the mucin-type glycoprotein from the culture.
19 . The method according to claim 18 , further comprising the step of exposure to a compound inhibiting the activity of an O-mannosylation enzyme.
20 . A mucin-type glycoprotein obtained by the method according to claim 18 .
21 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising the step of further disrupting an enzyme having O-mannosilation activity in the yeast obtained by the method according to claim 1 .
22 . A method for producing a transformed yeast for generating a mucin-type glycoprotein having O-GalNAc, comprising the step of further introducing a gene encoding a mucin-type glycoprotein into the yeast obtained by the method according to claim 21 .
23 . The method according to claim 22 , wherein the gene encoding a mucin-type glycoprotein comprises a secretion signal sequence derived from yeast.
24 . A transformed yeast obtained by the method according to claim 21 .
25 . A method for producing a mucin-type glycoprotein, comprising the steps of:
culturing the transformed yeast according to claim 24 ; and
collecting the mucin-type glycoprotein from the culture.
26 . A mucin-type glycoprotein obtained by the method according to claim 25 .