Author(s)

Tatsuro Shibui, Keisuke Bando, Satoru Misawa

API Corporation, Bio and Chemical Process Discovery Laboratory, 1000 Kamoshida-Cho, Aoba-Ku, Yokohama, Japan.
Food Biotechnology Laboratory, School of Food Sciences, Nippon Veterinary and Life Science University, 2-17-5 Sakai, Musashi- no-Shi, Tokyo, Japan.
Mitsubishi Chemical Group Science and Technology Research Center, 1000 Kamoshida-Cho, Aoba-Ku, Yokohama, Japan.

DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. These fragments were inserted into a site between the AOX 1-promoter and -terminator in pPICZ A to be expressed by P. pastoris. The expression vector was linearized, and introduced into P. pastoris GS115 by electroporation. After the checking of several transformants with PCR to ensure a precise insertion, one was selected and cultured to examine antibody production. The level of production reached 10 mg/L in a flask with medium containing 1% methanol. The heavy chain and light chain of the product were assembled to form a hetero tetramer, as detected by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal amino acid sequencing revealed that the signal peptides of both chains were well processed. The mobility of the product in SDS-PAGE after treatment with Peptide N-Glycosidase F indicated the heavy chain to be N-glycosylated. Further analysis of the N-glycans with a mass spectrometer revealed a mixture of Man₉-GlcNAc₂, Man₁₀-GlcNAc_2, Man₁₁-GlcNAc₂ and Man₁₂-GlcNAc₂, but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and K_d value for the antigen, HER II, and reactivity to a HER2-overexpressing breast cancer cell-line, SK-BR-3, to be almost the same as for the clinically used trastuzumab produced by CHO.

Pichia Pastoris; Antibody; Secretory Production; Glycocylation; Her II

Shibui, T. , Bando, K. and Misawa, S. (2013) High-level secretory expression, purification, and characterization of an anti-human Her II monoclonal antibody, trastuzumab, in the methylotrophic yeast Pichia pastoris. Advances in Bioscience and Biotechnology, 4, 640-646. doi: 10.4236/abb.2013.45084.