Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase.
A CHO cell line, previously genetically modified by the introduction of rat alpha2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of alpha2,3- and 39% of alpha2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 microM methotrexate, presented a secretion level of approximately 2 microg 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).
Damiani
Renata
R
Biotechnology Department, IPEN-CNEN, Av. Professor Lineu Prestes, 2242, Cidade Universitária, 05508-900 São Paulo, Brazil.
Oliveira
João Ezequiel
JE
Vorauer-Uhl
Karola
K
Peroni
Cibele Nunes
CN
Vianna
Elizabeth Gimbo
EG
Bartolini
Paolo
P
Ribela
Maria Teresa C P
MT
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D037102:Lectins; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D013972:Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D037102:Lectins; D021122:Protein Subunits; D011994:Recombinant Proteins; D013972:Thyrotropin; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase