Enhancing human glycoprotein hormones production in CHO cells using heterologous beta-chain signal peptides

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Abstract

We studied the influence of heterologous signal peptides in the β-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When replacing the natural signal peptides of the β-chains with the heterologous signal peptide of human serum albumin, cell productivity was increased by 2–2.5 times for human luteinizing hormone, human chorionic gonadotropin, human thyroid-stimulating hormone, but not for human follicle-stimulating hormone. No significant increase in cell productivity was observed for human azurocidin signal peptide and human glycoprotein hormone α-chain signal peptide. The used approach allows quick assessing the effect of heterologous signal peptides on the biosynthesis of heterodimeric proteins of various classes.

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About the authors

M. V. Sinegubova

The Federal State Institution “Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences”

Author for correspondence.
Email: mvsineg@gmail.com
Russian Federation, Moscow

D. E. Kolesov

The Federal State Institution “Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences”

Email: mvsineg@gmail.com
Russian Federation, Moscow

L. K. Dayanova

The Federal State Institution “Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences”

Email: mvsineg@gmail.com
Russian Federation, Moscow

I. I. Vorobiev

The Federal State Institution “Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences”

Email: mvsineg@gmail.com
Russian Federation, Moscow

N. A. Orlova

The Federal State Institution “Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences”

Email: mvsineg@gmail.com
Russian Federation, Moscow

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2. Fig. 1. A map of tricistrone expression plasmids encoding chains of glycoprotein hormones and sequences of signaling peptides. (a) Plasmid map, designations: pUC origin – the area of the beginning of replication of the pUC plasmid; bla – the open reading frame of beta-lactamase; EBVTR – the site of the terminal repeat of the human Epstein-Barr virus; CHO EEF1A1 UFR and CHO EEF1A1 DFR – areas flanking the EEF1A1 gene of the Chinese hamster contain a promoter, intron, terminator and polyadenylation signal of the EEF1A1 gene; TATA – TATA-box; PTS – the presumed point of transcription initiation; IRES – the natural internal binding site of wild-type EMCV ribosomes; IRES att – attenuated internal ribosome binding site; *SP – a variable signaling peptide of the β-chain; β – OPC β-chains of the corresponding hormone; aSP – signaling peptide α-chains of glycoprotein hormones; α – OPC α-chains; DHFR – ORC mouse dihydrofolate reductase. (a) Sequences of signaling peptides, the N-region is highlighted in purple font according to the algorithm

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3. Fig. 2. Titer of glycoprotein hormones secreted by transiently transfected cell culture CHO, when varying the signaling peptides of their β-chains. (a – d) The concentration of the heterodimeric form of hormones (according to ELISA data), normalized to the level of the green fluorescent protein eGFP in the cell lysate. (d – z) Titer of the secreted α-chain with a native signaling peptide (ELISA assessment), normalized to the titer of the heterodimeric form of the hormone. The average result of three independent biological repeats is presented, two repeats in ELISA for each sample, the values for the control peptide NSP are taken as 100%, * – p<0.05, single-factor analysis of variance, the Tukey criterion. Abbreviations: FSH – follicle stimulating hormone; LH – luteinizing hormone; hCG – human chorionic gonadotropin; TSH – thyroid stimulating hormone; NSP – native signaling peptide of the β-chain of the corresponding hormone; CHSA – human serum al- bumin; Azu – azurocidin; aSP — signaling peptide of the α-chain of glycoproteins

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