Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing alpha 2,6-sialyltransferase

Autor(es)

Damiani, Renata ^[1] ; Oliveira, Joao Ezequiel ^[1] ; Vorauer-Uhl, Karola ^[2] ; Peroni, Cibele Nunes ^[1] ; Vianna, Elizabeth Gimbo ^[1] ; Bartolini, Paolo ^[1] ; Ribela, Maria Teresa C. P. ^[1]

Afiliação do(s) autor(es)

^[1] IPEN CNEN, Dept Biotechnol, BR-05508900 Sao Paulo - Brazil

^[2] Univ Nat Resources & Appl Life Sci, Dept Biotechnol, Vienna - Austria

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 67, n. 1, p. 7-14, SEP 2009.
• Citações Web of Science: 13

Resumo

A CHO cell line, previously genetically modified by the introduction of rat alpha 2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of alpha 2,3- and 39% of alpha 2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 mu M methotrexate, presented a secretion level of similar to 2 mu g hTSH/10(6) cells/day, useful for product purification and characterization. The relative molecular masses (M(r)) of the heterodimer and of the alpha- and beta-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 T(4) 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). (C) 2009 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 07/56094-2 - Sintese de tireotrofina humana (r-htsh) em celulas cho "humanizadas", capazes de introduzir a ligacao alfa-2,6 entre o acido sialico e a galactose.
• Beneficiário: Maria Teresa de Carvalho Pinto Ribela
• Modalidade de apoio: Auxílio à Pesquisa - Regular