Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing α2,6-sialyltransferase
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2009
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Protein Expression and Purification
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A CHO cell line, previously genetically modified by the introduction of rat α2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of α2,3- and 39% of α2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 μM methotrexate, presented a secretion level of ∼2 μg hTSH/106 cells/day, useful for product purification and characterization.
The relative molecular masses (Mr) of the heterodimer and of the α- and β-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.6-fold more potent than native hTSH (p < 0.001).
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DAMIANI, RENATA; OLIVEIRA, JOAO E.; VORAUER-UHL, KAROLA; PERONI, CIBELE N.; VIANNA, ELIZABETH G.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P. Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing α2,6-sialyltransferase. Protein Expression and Purification, v. 67, n. 1, p. 7-14, 2009. DOI: 10.1016/j.pep.2009.04.005. Disponível em: http://repositorio.ipen.br/handle/123456789/4850. Acesso em: 19 Mar 2025.
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