Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins
Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome-as...
Gespeichert in:
| Hauptverfasser: | , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
2021
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| In: |
Archives of biological sciences
Year: 2021, Jahrgang: 73, Heft: 1, Pages: 47-55 |
| ISSN: | 1821-4339 |
| DOI: | 10.2298/ABS20120557J |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.2298/ABS20120557J Verlag, lizenzpflichtig, Volltext: http://www.doiserbia.nb.rs/Article.aspx?ID=0354-46642000057J 
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| Verfasserangaben: | Bogdan Jovanovic, Lisa Schubert, Fabian Poetz, and Georg Stoecklin |
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| 245 | 1 | 0 | |a Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins |c Bogdan Jovanovic, Lisa Schubert, Fabian Poetz, and Georg Stoecklin |
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| 520 | |a Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome-associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions. | ||
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