Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb;102(3):1215-1228.
doi: 10.1007/s00253-017-8684-8. Epub 2017 Dec 15.

Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination

P M Sant'Ana  1 , J E Oliveira  1 , E R Lima  1 , C R J Soares  1 , C N Peroni  1 , P Bartolini  1 , Maria Teresa C P Ribela  2
Affiliations

Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination

P M Sant'Ana et al. Appl Microbiol Biotechnol. 2018 Feb.

Abstract

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and β-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Keywords: Circulatory half-life; HEK293 cells; In vivo bioactivity; MALDI-TOF-MS; N-glycan composition; Thyroid-stimulating hormone.

PubMed Disclaimer

References

    1. Böhn E, Seyfried BK, Dockal M, Graninger M, Hasslacher M, Neurath M, Konetschny C, Matthiessen P, Mitterer A, Scheiflinger F (2015) Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells. BMC Biotechnol 15(1):87–10. https://doi.org/10.1186/s12896-015-0205-1 - DOI
    1. Boime I, Ben-Menahem D (1999) Glycoprotein hormone structure-function and analog design. Recent Prog Horm Res 54:271–288 - PubMed
    1. Brooks SA (2006) Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins. Expert Rev Proteomics 3(3):345–349. https://doi.org/10.1586/14789450.3.3.345 - DOI - PubMed
    1. Butler M, Spearman M (2014) The choice of mammalian cell host and possibilities for glycosylation engineering. Curr Opin Biotechnol 30:107–112. https://doi.org/10.1016/j.copbio.2014.06.010 - DOI - PubMed
    1. Capone MVN, Suzuki MF, Oliveira JE, Damiani R, Soares CRJ, Bartolini P (2015) N-glycoprofiling analysis in a simple glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin. J Biotechnol 202:78–87. https://doi.org/10.1016/j.jbiotec.2014.11.034 - DOI - PubMed

LinkOut - more resources