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

封面

如何引用文章

全文:

开放存取 开放存取
受限制的访问 ##reader.subscriptionAccessGranted##
受限制的访问 订阅存取

详细

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.

全文:

受限制的访问

作者简介

M. Sinegubova

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

编辑信件的主要联系方式.
Email: mvsineg@gmail.com
俄罗斯联邦, Moscow

D. Kolesov

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

Email: mvsineg@gmail.com
俄罗斯联邦, Moscow

L. Dayanova

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

Email: mvsineg@gmail.com
俄罗斯联邦, Moscow

I. Vorobiev

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

Email: mvsineg@gmail.com
俄罗斯联邦, Moscow

N. Orlova

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

Email: mvsineg@gmail.com
俄罗斯联邦, Moscow

参考

  1. Jackson R. C., Blobel G. Post-translational processing of full-length presecretory proteins with canine pancreatic signal peptidase // Ann. N. Y. Acad. Sci. 1980. V. 343. P. 391–404.
  2. Weintraub B. D., Stannard B. S., Linnekin D., Marshall M. Relationship of glycosylation to de novo thyroid-stimulating hormone biosynthesis and secretion by mouse pituitary tumor cells // J. Biol. Chem. 1980. V. 255. P. 5715–5723.
  3. Bernard M. P., Lin W., Kholodovych V., Moyle W. R. Human lutropin (hLH) and choriogonadotropin (CG) are assembled by different pathways: a model of hLH assembly // J. Biol. Chem. 2014. V. 289. P. 14360–14369.
  4. Orlova N. A., Kovnir S. V., Khodak Y. A., Polzikov M. A., Nikitina V. A., Skryabin K. G., Vorobiev I. I. High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors // PLoS One. 2019. V. 14. e0219434.
  5. Zhang L., Leng Q., Mixson A. J. Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo // J. Gene Med. 2005. V. 7. P. 354–365.
  6. Kapp K., Schrempf S., Lemberg M. K., Dobberstein B. Post-Targeting Functions of Signal Peptides. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience (2000–2013).
  7. Knappskog S., Ravneberg H., Gjerdrum C., Tröße C., Stern B., Pryme I. F. The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide // J. Biotechnol. 2007. V. 128. P. 705–715.
  8. Tan N. S., Ho B., Ding J. L. Engineering a novel secretion signal for cross-host recombinant protein expression // Protein Eng. Des. Sel. 2002. V. 15. P. 337–345.
  9. Kober L., Zehe C., Bode J. Optimized signal peptides for the development of high expressing CHO cell lines // Biotechnol. Bioeng. 2013. V. 110. P. 1164–1173.
  10. Yu X., Conyne M., Lake M. R., Walter K. A., Min J. In silico high throughput mutagenesis and screening of signal peptides to mitigate N-terminal heterogeneity of recombinant monoclonal antibodies // MAbs. 2022. V. 14.
  11. Park J. H., Lee H. M., Jin E. J., Lee E. J., Kang Y. J., Kim S., Yoo S. S., Lee G. M., Kim Y. G. Development of an in vitro screening system for synthetic signal peptide in mammalian cell-based protein production // Appl. Microbiol. Biotechnol. 2022. V. 106. P. 3571–3582,
  12. Haryadi R., Ho S., Kok Y. J., Pu H. X., Zheng L., Pereira N. A., Li B., Bi X., Goh L. T., Yang Y., et al. Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells // PLoS One 2015. V. 10. e0116878.
  13. Orlova N. A., Kovnir S. V., Hodak J. A., Vorobiev I. I., Gabibov A. G., Skryabin K. G. Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells // BMC Biotechnol. 2014. V. 14. P. 56.
  14. Sinegubova M., Orlova N., Vorobiev I. Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins // Peer J. 2023. V. 11. e16287.
  15. Orlova N. A., Kovnir S. V., Khodak Y. A., Polzikov M. A., Vorobiev I. I. Recombinant human luteinizing hormone for the treatment of infertility: the generation of producer cell lines // Obstet. Gynecol. Reprod. 2017. V. 11. P. 33–42.
  16. Teufel F., Almagro Armenteros J. J., Johansen A. R., Gíslason M. H., Pihl S. I., Tsirigos K. D., Winther O., Brunak S., von Heijne G., Nielsen H. SignalP 6.0 predicts all five types of signal peptides using protein language models // Nat. Biotechnol. 2022. V. 40. P. 1023.

补充文件

附件文件
动作
1. JATS XML
下载
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

下载 (392KB)
索引源数据
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

下载 (467KB)
索引源数据

版权所有 © Russian Academy of Sciences, 2024