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.