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-GlcNAc2, Man11-GlcNAc2 and Man12-GlcNAc2,
but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and Kd 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.
Cite this paper
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.
 Kito, M., et al. (2002) Construction of engineered CHO strains for high-level production of recombinant proteins. Applied Microbiological Biotechnology, 60, 442-448.
 Barnes, L.M., Bentley, C.M. and Dickson, A.J. (2000) Advances in animal cell recombinant protein production: GS-NS0 expression system. Cy-totechnology, 32, 109123. doi:10.1023/A:1008170710003
 Farid, S.S. (2006) Established bioprocesses for producing antibodies as a basis for future planning. Advances in Biochemical Engineering/Biotechnology, 101, 1-42.
 Cregg, J.M., et al. (2000) Recombinant protein expression in Pichia pastoris. Molecular Biotechnology, 16, 2352. doi:10.1385/MB:16:1:23
 Romanos, M.A., Scorer, C.A. and Clare, J.J. (1992) Foreign gene expression in yeast: A review. Yeast, 8, 423488. doi:10.1002/yea.320080602
 Cregg, J.M., et al. (1985) Pichia pastoris as a host system for transformations. Molecular Cell Biology, 5, 33763385.
 Grinna, L.S. and Tschopp, J.F. (1989) Size distribution and general structural features of N-linked oli-gosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast, 5, 107-115. doi:10.1002/yea.320050206
 Goodrick, J.C., et al. (2001) High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system. Biotechnology and Bioengineering, 74, 492-497.
 Schilling, B.M., Goodrick, J.C. and Wan, N.C. (2001) Scale-up of a high cell density continuous culture with Pichia pastoris X-33 for the con-stitutive expression of rhchitinase. Biotechnology Progress, 17, 629-633.
 Hellwig, S., et al. (2001) Analysis of single-chain antibody production in Pichia pastoris using online methanol control in fed-batch and mixed-feed fermentations. Biotechnology and bioengineering, 74, 344-352.
 Carter, P., et al. (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proceedings of National Academy Science of the USA, 89, 4285-4289.
 Shibui, T. and Nagahari, K. (1992) Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions. Applied Microbiological Biotechnology, 37, 352-357. doi:10.1007/BF00210991
 Cabilly, S. (1989) Growth at sub-optimal temperatures allows the production of functional, anti-gen-binding Fab fragments in Escherichia coli. Gene, 85, 553-557.
 Dean, N. (1999) Asparagine-linked glycosylation in the yeast Golgi. Biochimica et Biophysica Acta, 1426, 309322. doi:10.1016/S0304-4165(98)00132-9
 Ballou, C.E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymology, 185, 440-470.
 Yip, C.L., et al. (1994) Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins. Proceedings of National Academy Science of the USA, 91, 27232727. doi:10.1073/pnas.91.7.2723
 Niwa, R., et al. (2004) Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Research, 64, 2127-2133. doi:10.1158/0008-5472.CAN-03-2068
 Yamane-Ohnuki, N., et al. (2004) Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnology and bioengineering, 87, 614-622.