Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones
| Autor: | Kimihisa Ichikawa, Rie Ito, Masayuki Yabuta, Minako Ono, Yasunori Chiba, Satoshi Baba, Takeshi Suzuki, Koichi Nonaka |
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| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine Glycosylation medicine.drug_class Bioengineering Biology Monoclonal antibody Endoplasmic Reticulum 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology medicine Animals Secretion Protein disulfide-isomerase Endoplasmic reticulum Antibodies Monoclonal Molecular biology Yeast 030104 developmental biology chemistry Biochemistry Saccharomycetales biology.protein Protein folding Antibody Protein Processing Post-Translational Biotechnology Molecular Chaperones |
| Zdroj: | Journal of bioscience and bioengineering. 124(2) |
| ISSN: | 1347-4421 |
| Popis: | A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta. |
| Databáze: | OpenAIRE |
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