Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

Antibody production by a protease-deficient strain of methylotrophic yeast, Ogataea minuta

  • Kousuke Kuroda1,
  • Yoshinori Kitagawa1,
  • Kazuo Kobayashi1,
  • Haruhiko Tsumura1,
  • Toshihiro Komeda2,
  • Yasunori Chiba3 and
  • Yoshihumi Jigami3
Microbial Cell Factories20065(Suppl 1):P64

https://doi.org/10.1186/1475-2859-5-S1-P64

Published: 10 October 2006

Background

At present the expression system of mammalian cells such as CHO has been adopted as the conventional method to produce antibody for pharmaceuticals. However a novel method of producing antibody has been sought after as a substitute for the costly production of antibody by mammalian cells. Therefore, we tried to construct a novel antibody production system by using methylotrophic yeast, O. minuta.

Results

When human antibody genes were introduced in the methylotrophic yeast O. minuta to produce an antibody, the heavy chain of the antibody was partially degraded (see Figure 1, lane 1). Peptide sequencing revealed that degradation occurred in the CH1 region (see Figure 2). In order to inhibit this degradation, the YPS1 gene coding Aspartic protease attached to the plasma membrane, a homologue of Saccharomyces cerevisiae, was cloned from O. minuta, and we constructed the Δyps1 strain. As a result, the Δyps1 strain repressed the partial degradation of the antibody (see Figure 1, lane2).
Figure 1

Culture supernatant from the yeast O. minuta was subjected to Western analysis with HRP conjugated anti-human Fc antibody. Lane1: heavy chain secreted by YPS1+ strain. Lane2: heavy chain secreted by Δyps1 strain.

Figure 2

Position of the partial degradation in the heavy chain on its CH1-hinge region produced by O. minuta YPS1 + strain. Arrows show the position of the degradation.

Conclusion

We constructed a protease-deficient strain and confirmed the secretion of full-length antibody by yeast. Improving the production of antibody should be a subject of further research.

Declarations

Acknowledgements

We thank N.Kawashima, A. Oonishi, Y. Yamamoto, M. Tezuka and K. Sato for their valuable assistance with this research. We are also grateful to Dr. M. Tsukahara for helpful suggestions.

Authors’ Affiliations

(1)
Kirin Brewery Co., Ltd., CMC R&D Lab
(2)
Kirin Brewery Co., Ltd., Central Lab for Frontier Technology
(3)
National Institute Advanced Industrial Science and Technology (AIST)

References

  1. Bourbonnais Y, Larouche C, Tremblay GM: Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional yapsin 1 (Yps1p) endoprotease. Protein Expr Purif. 2000, 20: 485-491. 10.1006/prep.2000.1338.View ArticleGoogle Scholar
  2. Wood CR, Boss MA, Kenten JH, Calvert JE, Roberts NA, Emtage JS: The synthesis and in vivo assembly of functional antibodies in yeast. Nature. 1985, 314: 446-449. 10.1038/314446a0.View ArticleGoogle Scholar

Copyright

© Kuroda et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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