Please use this identifier to cite or link to this item:
https://biore.bio.bg.ac.rs/handle/123456789/5177
Title: | Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins | Authors: | Jovanović, Bogdan Schubert, Lisa Poetz, Fabian Stoecklin, Georg |
Keywords: | RPS9;Ribosome-associated proteins;Ribosome purification;Streptavidin-binding peptide;Affinity purification | Issue Date: | 30-Dec-2020 | Rank: | M23 | Publisher: | Serbian Biological Society | Citation: | Jovanovic B, Schubert L, Poetz F, Stoecklin G. Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins. Arch Biol Sci Belgrade 2021;73(1):47-55. doi: 10.2298/ABS20120557J. | Journal: | Archives of Biological Sciences | Abstract: | 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. |
URI: | https://biore.bio.bg.ac.rs/handle/123456789/5177 | ISSN: | 0354-4664 1821-4339 |
DOI: | 10.2298/ABS20120557J |
Appears in Collections: | Journal Article |
Show full item record
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.