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Applied and Environmental Microbiology, January 2008, p. 446-453, Vol. 74, No. 2
0099-2240/08/$08.00+0     doi:10.1128/AEM.02106-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Kousuke Kuroda,^1,8 Kazuo Kobayashi,¹ Yoshinori Kitagawa,¹ Taishiro Nakagawa,¹ Haruhiko Tsumura,² Toshihiro Komeda,³ Daisuke Shinmi,⁴ Eiji Mori,⁴ Kazuhiro Motoki,⁴ Kazumi Fuju,⁴ Teruyuki Sakai,⁴ Koichi Nonaka,⁵ Takeshi Suzuki,⁵ Kimihisa Ichikawa,⁶ Yasunori Chiba,⁷ and Yoshifumi Jigami^7,8*

Kirin Pharma Co., Ltd., CMC R & D Laboratories, Gunma, Japan,¹ Kirin Pharma Co., Ltd., Production Planning, Tokyo, Japan,² Kirin Holdings Co., Ltd., Central Laboratories for Frontier Technology, Gunma, Japan,³ Kirin Pharma Co., Ltd., Discovery Research Laboratories, Gunma, Japan,⁴ Daiichi Sankyo Co., Ltd., Process Technology Research Laboratories, Fukushima, Japan,⁵ Daiichi Sankyo Co., Ltd., Advanced Technology Research Laboratories, Tokyo, Japan,⁶ National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan,⁷ Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan⁸

Received 14 September 2007/ Accepted 13 November 2007

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

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* Corresponding author. Mailing address: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Phone: 81-29-861-6160. Fax: 81-29-861-6161. E-mail: jigami.yoshi{at}aist.go.jp

Published ahead of print on 26 November 2007.

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Applied and Environmental Microbiology, January 2008, p. 446-453, Vol. 74, No. 2
0099-2240/08/$08.00+0     doi:10.1128/AEM.02106-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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