A rapid, simple, and economical method for the isolation of ribosomes and translational machinery for structural and functional studies

Abstract

Ribosomes are RNA-protein complexes essential for protein synthesis and quality control. Traditional methods for ribosome isolation are labor-intensive, expensive, and require a substantial amount of biological material. In contrast, our method, RNA affinity purification using poly-lysine (RAPPL), provides a rapid, simple, and cost-effective alternative applicable to various species and types of starting material (cell lysates, whole cells, organs, or whole organisms). It is also compatible with traditional isolation techniques. Here, we describe the use of RAPPL for rapid isolation, functional screening, and structural analysis of ribosomes and associated factors. We also demonstrate the application of RAPPL in investigating ribosome-associated resistance mechanisms in uropathogenic Escherichia coli samples and generating a 2.7-Å cryoEM ribosome structure from Cryptococcus neoformans. By significantly reducing the amount of the starting biological material and the time required for isolation, RAPPL has the potential to facilitate the study of ribosomal function, interactions, and antibiotic resistance and provide a versatile platform for academic, clinical, and industrial research.

Fig. 1. The RAPPL method.

A Schematic describing the advantages of RAPPL over conventional methods. Created in BioRender. Pavlovic Djuranovic, S. (2025) https:// BioRender.com/0uym2rp. B 2% agarose gel of RAPPLE-purified RNA samples from HEK-293 (HEK), human dermal fibroblasts (HDF), and HeLa human cell cultures. RNA isolated from commercial HeLa cell lysate (Con; Thermo Fisher HeLa IVT kit) and purified yeast tRNAs (tRNA, Ambion) were loaded as controls. NEB 100 bp and 1 Kb base pair markers (M1 and M2, respectively) are used to estimate the size of isolated RNAs. C Western blot analysis of HEK-293 lines uL4-HA and uS13-Flag tagged by CRISPR/Cas9 throughout the RAPPL purification process – lysate (Lys), flow-through (FT), wash (W), and elution (E1 and E2). RAPPL is selective for ribosome-associated factors, showing that the HA-tagged ribosomal protein uL4, Flag-tagged uS13, as well as the untagged uS9, are in the elution fractions. Translation factors are also enriched and purified by RAPPL, as seen by the visualization of eIF3A and eIF4A1 proteins by specific antibodies in elution fractions. Presence of GAPDH, as a control for loading, is detected in lysate and flow-through. Molecular markers indicate the size of detected proteins. Source data are provided as Source data. D (top) Plot of the log₁₀ of the average intensity-based absolute quantification (iBAQ) for each protein in the input, flow-through (FT), and bead-bound (IP) fractions. Crossbars represent the mean of 3 biological replicates for each group (HEK all proteins n = 3104, HEK ribosomal proteins n = 80).

Fig. 2. RAPPL can enrich and purify ribosomes from limited biological material.

A TEM visualization of RAPPL eluates from PureExpress® ribosomes, following a 10-fold sequential dilution scheme from 13.3 μM to 1.3 nM. The scale bar represents 500 nm. B Western blot using αHA antibody on RAPPL eluates of HEK-293 cells in which uL4 was HA-tagged by CRISPR/Cas9, whereby the starting material was diluted to 1 × 10⁶, 5 × 10⁵, 2.5 × 10⁵, 1 ×10⁵, 5 × 10⁴, 2.5 × 10⁴, 1 × 10⁴, 5 × 10³, 1 × 10³, and 0.1 × 10³ cells prior to lysis. C Western blot using αFlag antibody on RAPPL eluates of HEK-293 cells in which uS4 was Flag-tagged by CRISPR/Cas9, where the starting material was diluted to 1 × 10⁶, 5 × 10⁵, 1 × 10⁵, 5 × 10⁴, 2.5 × 10⁴, 1 × 10⁴, 5 × 10³ and 1 × 10³ cells prior to lysis. D Western blot using αHA antibody on RAPPL eluates of P. falciparum NF54 cells in which PfRACK was C-terminally tagged with mNeonGreen-HA, whereby the starting material was diluted to 5 × 10⁷, 1 × 10⁷, 5 × 10⁶, and 1 × 10⁶ cells prior to lysis. E Western blot using αuS11 antibody on RAPPL eluates of P. falciparum NF54 cells with HA-tagged RACK1, where the starting material was diluted to 5 × 10⁷, 1 × 10⁷, 5 × 10⁶, and 1 × 10⁶ cells prior to lysis. Molecular markers indicate the size of detected proteins. Source data are provided as Source Data.

Fig. 3. RAPPL is a versatile method that works with single-cell organisms, tissues, and whole multicellular organisms.

A TEM visualization of RAPPL eluates purified from several single-cell organisms (E. coli, S. cerevisiae, T. gondii, P. falciparum, C. parvum). B TEM visualization of RAPPL eluates purified from mouse tissue sections of spleen and liver, as well as from whole organisms (D. rerio and C. elegans). C TEM visualization of compartment-specific ribosomes generated from RAPPL eluates of cytoplasmic, mitochondrial, and nuclear (ribosome biogenesis) fractions. The scale bar represents 100 or 500 nm. Created in BioRender. Pavlovic Djuranovic, S. (2025) https://BioRender.com/0uym2rp.

Fig. 4. RAPPL eluates can be used in downstream applications.

A HEK-293 cells were treated with cycloheximide, anisomycine, and harringtonine, with untreated cells used as a control. Ribosomes were purified using RAPPL in the presence of inhibitors, and the eluates were visualized via TEM. B HEK-293 lysates were fractionated using polysome profiling. Fractions corresponding to ribosome subunit and monosomes, light polysomes, and heavy polysomes were pooled, respectively. These pools were diluted 1:5 to ensure that sucrose did not interfere with binding. The diluted, pooled samples were subjected to RAPPL, and the eluates were visualized via TEM. C Schematic of arabinose-inducible reporter expressing a 2xHA affinity-tagged eGFP reporter separated by a TEV protease cleavage site (top). RAPPL was performed on bacterial lysates in the absence or presence of bacterial translation elongation inhibitor chloramphenicol (CHL), followed by αHA magnetic bead purification, again with and without CHL, and eluted with TEV protease. Eluates were visualized via TEM. Non-induced cells are used as controls for lack of protein production and subsequent non-specific binding to αHA beads. The scale bar represents 500 nm. Created in BioRender. Pavlovic Djuranovic, S. (2025) https://BioRender.com/0uym2rp.

Fig. 5. RAPPL-isolated ribosomes are translationally active and can be used for clinical applications.

A We used RAPPL to capture ribosomes from E. coli DH5α cells grown to exponential phase, eluting them in 30 μL of RAPPL elution buffer. Eluates were then used in the PURExpress® in vitro translation system instead of kit-provided ribosomes. A PCR product encoding for eGFP harboring the T7 promoter and a polyA tail were used in the reaction (see “Methods”). Reactions were incubated for 4 h. Ribosomes purified using RAPPL were active and able to translate mRNA. B The activity of RAPPL purified E. coli BL21 ribosomes in the in vitro PURExpress® assays was observed using a kinetics protocol that measured eGFP fluorescence (excitation, 488 ± 9 nm; emission, 507 ± 9 nm). The graph shows relative eGFP fluorescence with standard deviation of technical triplicates over 2.5 h, in reactions with and without the DNA template. Error bars represent the standard deviation of three technical replicates. C Plate bacterial growth assays were performed to demonstrate resistance of various strains to erythromycin (ERY), kanamycin (KAN), chloramphenicol (CHL), and clindamycin (CLI). The DH5α strain was used as the control strain. D eGFP reporter was synthesized using RAPPL-isolated ribosomes in the absence and presence of antibiotics (ERY, KAN, CHL, and CLI) that target E. coli ribosomes. For each strain, 4.5 μL of 1.5 µg / µL of RAPPL-isolated ribosomes was used in the standard 25 μL PURExpress® in vitro Δ ribosome translation reaction (see “Methods”). Western blot analysis was performed on samples collected after 4 h of incubation at 37 °C and visualized using an αGFP-specific antibody. Source data are provided as Source data.

Fig. 6. Structural determination of RAPPL-isolated materials can yield high-resolution CryoEM maps.

C. neoformans cells (~ 10⁸) in exponential phase were lysed, and the clarified lysate was used for RAPPL. The ribosomes were eluted in 30 μL of RAPPL elution buffer. The eluate was first screened using TEM. Subsequently, grids were prepared using 5 μL of eluate. Movies that were captured resulted in a global resolution of approximately 2.7 Å. Created in BioRender. Pavlovic Djuranovic, S. (2025) https:// BioRender.com/0uym2rp.