High-level secretory expression, purification, and characterization of an anti-human Her II monoclonal antibody, trastuzumab, in the methylotrophic yeast Pichia pastoris

Tatsuro Shibui, Keisuke Bando, Satoru Misawa
2013 Advances in Bioscience and Biotechnology  
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
more » ... f 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 Man 9 -GlcNAc 2 , Man 10 -GlcNAc 2, Man 11 -GlcNAc 2 and Man 12 -GlcNAc 2 , 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.
doi:10.4236/abb.2013.45084 fatcat:f2ylg5jfmvgs7pi47oypyngzq4