Recombinant Cricetulus griseus 40S ribosomal protein S2 (RPS2)
Description
Introduction to Recombinant C. griseus 40S Ribosomal Protein S2
RPS2 is a conserved ribosomal protein integral to the 40S subunit, facilitating mRNA decoding and translational fidelity . The recombinant form, produced via biotechnological methods in Chinese hamster-derived cell lines, retains structural and functional properties of the native protein. Its study is pivotal for understanding ribosome assembly, translational regulation, and biopharmaceutical applications .
Biotechnological Production
Recombinant RPS2 is synthesized in engineered Chinese hamster cell lines, such as CHL-YN, which exhibit enhanced growth rates and protein yields compared to traditional CHO-K1 cells :
Table 1: Growth and productivity metrics of CHL-YN vs. CHO-K1 cells3
| Parameter | CHL-YN | CHO-K1 |
|---|---|---|
| Doubling time (hours) | 10.74 ± 0.20 | 21.29 ± 0.34 |
| IgG1 production (mg/L) | 7.13 ± 0.29 | 6.05 ± 0.73 |
| Specific growth rate (h⁻¹) | 0.0645 ± 0.0012 | 0.0326 ± 0.0005 |
CHL-YN cells achieve higher RPS2 expression (RPKM = 4,134.01) under serum-free conditions, enabling scalable recombinant protein production .
Functional Insights
-
Ribosome Assembly: RPS2 stabilizes 18S rRNA during 40S subunit maturation .
-
Translational Fidelity: Loss of RPS2 paralogs in yeast increases stop codon readthrough by 40–60%, underscoring its role in translational accuracy .
-
MDM2-p53 Pathway: RPS2 interacts with MDM2, modulating p53 activity under ribosomal stress .
Table 2: RPS2 expression in CHL-YN cells under varying conditions3
| Condition | RPS2 RPKM | Rank (Top Proteins) |
|---|---|---|
| EX-CELL CD (FBS −, day 3) | 4,134.01 | 2nd |
| IMDM (FBS +, day 2) | 1,576.86 | 4th |
Elevated RPS2 levels correlate with enhanced ribosomal biogenesis and IgG production in serum-free cultures .
Applications in Biopharmaceuticals
-
High-Yield Protein Production: CHL-YN cells expressing recombinant RPS2 achieve 18% faster IgG1 production than CHO-K1 .
-
Glycosylation Consistency: N-glycan profiles of IgG1 from CHL-YN and CHO-K1 are identical, ensuring therapeutic efficacy .
Challenges and Future Directions
Product Specs
Q&A
What is the functional significance of RPS2 in ribosome biogenesis?
RPS2 plays a critical role in ribosome assembly and maturation. Studies in fission yeast have demonstrated that RPS2 is essential for cell viability, with genetic depletion causing complete inhibition of 40S ribosomal subunit production . The pattern of pre-rRNA processing upon depletion of RPS2 reveals a reduction of 27SA2 pre-rRNAs and concomitant production of 21S rRNA precursors, indicating RPS2's role in efficient cleavage at site A2 within the 32S pre-rRNA .
Research methodological approach:
-
Use RNA pulse-chase assays to track pre-rRNA accumulation and processing kinetics in RPS2-depleted cells
-
Employ northern blot analysis with probes specific to different pre-rRNA regions
-
Conduct subcellular fractionation and gradient centrifugation to analyze pre-ribosomal particles
How conserved is RPS2 across species, and what implications does this have for research?
RPS2 demonstrates significant sequence and functional conservation across eukaryotes. Comparative analysis reveals high levels of amino acid identity in functional domains involved in rRNA binding and 40S subunit assembly.
| Species | Amino Acid Identity to C. griseus RPS2 (%) | Key Conserved Domains |
|---|---|---|
| Homo sapiens | ~98% | RNA-binding, 40S assembly |
| Mus musculus | ~97% | RNA-binding, 40S assembly |
| Saccharomyces cerevisiae | ~68% | RNA-binding, A2 cleavage site recognition |
| Schizosaccharomyces pombe | ~70% | Nuclear export, pre-40S assembly |
Methodological considerations:
-
Perform multiple sequence alignments using CLUSTAL or MUSCLE algorithms
-
Identify functionally significant domains through structure prediction software
-
Validate conserved regions through site-directed mutagenesis
What expression systems are most effective for recombinant Cricetulus griseus RPS2 production?
For functional studies of recombinant C. griseus RPS2, selection of an appropriate expression system is critical. Based on established protocols for ribosomal proteins:
| Expression System | Advantages | Limitations | Optimal Applications |
|---|---|---|---|
| E. coli | High yield, economical, rapid | Lacks PTMs, potential folding issues | Structural studies, antibody production |
| Insect cells | Better folding, some PTMs | Higher cost, longer production time | Functional assays, protein-protein interaction studies |
| Mammalian cells | Native PTMs, proper folding | Highest cost, complex protocols | In vivo functional studies, complex formation analysis |
Methodological recommendations:
-
For E. coli expression: Use BL21(DE3) strain with pET vector systems containing N-terminal His-tag
-
For mammalian expression: Consider using CHO cells themselves with inducible promoters
-
Optimize codon usage for the expression host
-
Purify using a combination of affinity chromatography and size exclusion methods
How does RPS2 contribute to nuclear export of pre-40S particles?
Analysis of steady-state RNA levels reveals that pre-40S particles are produced in RPS2-depleted cells but are retained in the nucleolus . This suggests a critical role for RPS2 in monitoring pre-40S export competence.
Recommended experimental approach:
-
Employ fluorescent in situ hybridization (FISH) with probes targeting pre-rRNA to visualize nuclear retention
-
Use immunofluorescence to co-localize pre-40S particles with nucleolar markers
-
Perform immunoprecipitation of RPS2 followed by mass spectrometry to identify interaction partners involved in nuclear export
-
Develop conditional RPS2 mutants to identify specific domains involved in export
What role does RPS2 play in cellular transformation and cancer models?
Research indicates that RPS2 may have oncogenic potential when aberrantly expressed. Studies show that RPS2 is overexpressed in malignant prostate cancer cell lines and archived tumor specimens . This suggests RPS2 may promote cancer and represent a therapeutic target.
Experimental strategies:
-
Use siRNA or CRISPR-Cas9 to knockdown RPS2 in cancer cell lines and measure effects on proliferation and apoptosis
-
Evaluate RPS2 expression levels across normal, benign, and malignant cell lines using RT-PCR and Western blotting
-
Develop "ribozyme-like" oligonucleotides (similar to DNAZYM-1P described for human RPS2) to target C. griseus RPS2
-
Establish xenograft models to assess in vivo effects of RPS2 knockdown on tumor growth
Comparative RPS2 expression in cell lines (based on human prostate model):
| Cell Type | Relative RPS2 Expression | Phenotype |
|---|---|---|
| Normal epithelial | Low | Non-tumorigenic |
| Benign hyperplasia | Low to moderate | Non-tumorigenic |
| Early carcinoma | Moderate to high | Moderately tumorigenic |
| Metastatic carcinoma | High | Highly tumorigenic |
What methods are effective for studying RPS2 post-translational modifications in C. griseus?
Post-translational modifications (PTMs) of ribosomal proteins regulate their function, localization, and participation in extra-ribosomal activities. For C. griseus RPS2:
Recommended methodological workflow:
-
Immunoprecipitate RPS2 from CHO cells under different conditions
-
Perform mass spectrometry analysis with particular focus on:
-
Phosphorylation sites (common regulatory mechanism)
-
Ubiquitination and SUMOylation (affecting stability and localization)
-
Methylation and acetylation (regulatory modifications)
-
-
Generate site-specific antibodies against identified PTMs
-
Validate functional significance through site-directed mutagenesis
How can evolutionary analysis of RPS2 inform functional studies?
Evolutionary analysis can provide insights into functional domains and selection pressures. Studies on other RPS genes have shown diverse evolutionary patterns that inform function .
Analytical approach:
-
Collect RPS2 sequences across multiple species
-
Calculate nucleotide diversity (π) and average number of segregating sites (θ)
-
Apply Tajima's D statistic to test for selection
-
Generate a haplotype tree to visualize evolutionary relationships
When applied to resistance genes like RPS2 in Arabidopsis, such analysis revealed high level of diversity with π = 13.444 and θ = 13.246 , suggesting possible selection for diversity in pathogen interaction contexts. Similar approaches could reveal whether C. griseus RPS2 has undergone selection for specialized functions in this species.
What CRISPR-Cas9 strategies are most effective for studying essential genes like RPS2?
Since complete knockout of RPS2 is likely lethal (as observed in yeast) , specialized CRISPR approaches are required:
| Strategy | Methodology | Applications | Considerations |
|---|---|---|---|
| Inducible knockdown | Tet-regulated sgRNA or shRNA | Temporal control of RPS2 depletion | Expression leakage may be problematic |
| Conditional knockout | loxP-flanked exons with inducible Cre | Complete but controlled gene deletion | Requires engineering cell lines with loxP sites |
| Degron tagging | CRISPR knock-in of AID or SMASh tag | Rapid protein degradation upon inducer addition | May affect protein function even without inducer |
| Domain-specific edits | Precise editing of functional domains | Structure-function studies | Requires detailed knowledge of protein domains |
Implementation protocol:
-
Design multiple sgRNAs targeting non-essential domains or regulatory regions
-
Screen for optimal targeting efficiency using T7 endonuclease assay
-
For essential function studies, employ homology-directed repair to introduce conditional elements
-
Validate edited clones via sequencing and functional assays
How can C. griseus RPS2 be leveraged in CHO cell engineering for biopharmaceutical production?
CHO cells are the predominant system for biopharmaceutical protein production. Understanding and potentially modifying RPS2 could enhance their productivity:
Research strategies:
-
Create CHO cell lines with controlled RPS2 expression to optimize ribosome biogenesis and protein synthesis
-
Investigate effects of RPS2 variants on stress response and protein production under bioreactor conditions
-
Develop RPS2-based selection markers for CHO cell line development
What are the methodological considerations for studying RPS2 interactions with pre-rRNA and other proteins?
To characterize RPS2's role in ribosome assembly, several techniques can be employed:
| Technique | Application | Advantages | Technical Considerations |
|---|---|---|---|
| RNA immunoprecipitation (RIP) | Identify direct RNA binding sites | Preserves native interactions | Requires high-quality antibodies |
| Cross-linking and immunoprecipitation (CLIP) | Map precise RNA contact sites | Nucleotide resolution | Complex protocol with potential artifacts |
| Proximity labeling (BioID/TurboID) | Identify protein interaction network | Captures transient interactions | Fusion protein may alter function |
| Cryo-EM of pre-ribosomes | Structural roles in assembly | Direct visualization | Technically challenging, heterogeneous samples |
Methodological workflow:
-
Generate tagged RPS2 constructs (ensuring tags don't interfere with function)
-
Validate expression and incorporation into pre-ribosomes
-
Perform interaction studies under various cellular conditions
-
Validate key interactions through multiple complementary techniques
How does RPS2 contribute to the nucleolar stress response pathway?
Nucleolar stress activates p53-dependent and independent pathways. Research suggests ribosomal proteins like RPS2 may play regulatory roles in these pathways.
Experimental approach:
-
Induce nucleolar stress using actinomycin D, 5-fluorouracil, or nutrient deprivation
-
Monitor RPS2 localization via immunofluorescence microscopy
-
Analyze RPS2 interaction with MDM2 and p53 via co-immunoprecipitation
-
Assess effects of RPS2 depletion on nucleolar stress response using RNA-seq
