TITLE:
High-level secretory expression, purification, and characterization of an anti-human Her II monoclonal antibody, trastuzumab, in the methylotrophic yeast Pichia pastoris
AUTHORS:
Tatsuro Shibui, Keisuke Bando, Satoru Misawa
KEYWORDS:
Pichia Pastoris; Antibody; Secretory Production; Glycocylation; Her II
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.4 No.5,
May
30,
2013
ABSTRACT: 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.