Recombinant Tetraodon nigroviridis 40S ribosomal protein S3a (rps3a)

What is the genomic context of rps3a in Tetraodon nigroviridis compared to other vertebrates?

Tetraodon nigroviridis possesses one of the smallest known vertebrate genomes, making it an excellent model for studying genome architecture and evolution. Unlike mammals, Tetraodon exhibits remarkable compartmentalization of genetic elements, with pseudogenes and transposable elements primarily localized to heterochromatic regions corresponding to the short arms of small subtelocentric chromosomes . This compartmentalization likely extends to ribosomal protein genes and their pseudogenes. The rps3a gene in Tetraodon would be expected to follow the pattern seen with other ribosomal protein genes, which typically have multiple processed pseudogenes dispersed throughout the genome, arising from either duplication events or retro-transcription of mRNAs .

How does Tetraodon nigroviridis rps3a compare structurally to human RPS3A?

While specific structural data for Tetraodon nigroviridis rps3a is not extensively characterized in the provided sources, we can draw parallels with the human ortholog. Human 40S ribosomal protein S3a is encoded by the RPS3A gene and belongs to the S3AE family of ribosomal proteins . As a component of the 40S ribosomal subunit, it participates in protein synthesis. Given the conserved nature of ribosomal proteins across species, Tetraodon nigroviridis rps3a likely maintains the core structural elements required for ribosome assembly and function, while potentially exhibiting species-specific adaptations that could affect extra-ribosomal functions.

What are the expected extra-ribosomal functions of rps3a in Tetraodon nigroviridis?

Based on research on ribosomal proteins in other organisms, rps3a in Tetraodon nigroviridis likely possesses functions beyond protein synthesis. The related ribosomal protein S3 has been demonstrated to participate in several extra-ribosomal processes including DNA repair, apoptosis, selective gene transcription, and host-pathogen interactions . These functions are often regulated through post-translational modifications such as phosphorylation, methylation, and neddylation . Given the evolutionary relationship between S3 and S3a, it is reasonable to hypothesize that rps3a may also participate in cellular processes beyond its canonical role in translation, potentially including immune response mechanisms considering Tetraodon's evolved resistance mechanisms to pathogens .

What expression systems are most suitable for producing recombinant Tetraodon nigroviridis rps3a?

For recombinant expression of Tetraodon nigroviridis rps3a, researchers should consider several expression systems with their respective advantages:

• E. coli expression system: Most commonly used due to rapid growth, high protein yields, and cost-effectiveness. Recommended strains include BL21(DE3) or Rosetta for potentially problematic codon usage. Optimal expression conditions typically involve induction with 0.5-1.0 mM IPTG at OD600 0.6-0.8, followed by incubation at 16-25°C for 16-18 hours to minimize inclusion body formation.

• Insect cell expression: If mammalian-like post-translational modifications are critical, baculovirus expression systems using Sf9 or Hi5 cells may provide better functional properties.

• Yeast expression systems: Pichia pastoris or Saccharomyces cerevisiae can offer a compromise between bacterial and mammalian systems, particularly if disulfide bond formation is necessary for proper folding.
  The choice depends on research objectives, required protein modifications, and downstream applications.

What purification strategy would yield the highest activity for recombinant Tetraodon nigroviridis rps3a?

A multi-step purification protocol optimized for recombinant Tetraodon nigroviridis rps3a typically includes:

• Affinity chromatography: Using His6, GST, or other fusion tags for initial capture. For His-tagged constructs, immobilized metal affinity chromatography (IMAC) with Ni-NTA resin is effective, with elution using an imidazole gradient (50-300 mM).

• Ion-exchange chromatography: As a secondary purification step, using cation or anion exchange depending on the protein's theoretical isoelectric point.

• Size exclusion chromatography: Final polishing step to separate monomeric protein from aggregates and remove remaining impurities.
  Buffer optimization is critical throughout the process. A typical buffer system would include:

• Lysis buffer: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM DTT, protease inhibitors

• Purification buffers: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Storage buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT
  Protein activity should be assessed at each purification stage using appropriate functional assays.

What crystallization conditions are recommended for structural studies of Tetraodon nigroviridis rps3a?

For crystallization of recombinant Tetraodon nigroviridis rps3a, researchers should consider:

• Initial screening: Commercial sparse matrix screens (Hampton Research, Molecular Dimensions) at protein concentrations of 5-15 mg/ml using vapor diffusion methods (hanging or sitting drop).

• Optimization parameters:

  • pH range: 6.0-8.0

  • Precipitants: PEG variations (PEG 3350, PEG 4000) at 10-25%

  • Salt additives: MgCl₂ (5-20 mM), NaCl (50-200 mM)

  • Temperature: 4°C and 18°C trials in parallel

  • Additives: Glycerol (5-10%), reducing agents (2-5 mM DTT or TCEP)

• Co-crystallization: If studying functional interactions, consider co-crystallization with ribosomal RNA fragments or potential binding partners.

• Post-crystallization treatments: Dehydration, annealing, or additive soaking may improve diffraction quality.
  Cryo-protection typically employs glycerol, ethylene glycol, or low molecular weight PEGs at 25-30% concentration prior to flash-cooling in liquid nitrogen.

How can I assess the RNA-binding properties of recombinant Tetraodon nigroviridis rps3a?

RNA-binding characteristics of recombinant Tetraodon nigroviridis rps3a can be evaluated through multiple complementary approaches:

• Electrophoretic Mobility Shift Assay (EMSA): Using labeled RNA fragments (typically 25-100 nucleotides) derived from 18S rRNA or synthetic oligonucleotides with varying concentrations of purified rps3a (1-500 nM). Run on 6-8% native polyacrylamide gels in TBE buffer at 4°C.

• Fluorescence Polarization: Using fluorescently-labeled RNA oligonucleotides to determine binding kinetics and affinity constants. Typical Kd values for ribosomal protein-RNA interactions range from 10⁻⁸-10⁻⁶ M.

• Surface Plasmon Resonance (SPR): Immobilizing either the protein or RNA on a sensor chip to measure real-time binding kinetics.

• Isothermal Titration Calorimetry (ITC): For thermodynamic characterization of the binding interaction.

• UV Crosslinking: To identify specific amino acid residues involved in RNA binding.
  Data analysis should include determination of binding constants, stoichiometry, and binding specificity compared to other ribosomal proteins.

How is rps3a expression regulated in Tetraodon nigroviridis tissues?

The expression regulation of rps3a in Tetraodon nigroviridis likely follows patterns similar to other ribosomal proteins, with tissue-specific and developmental variations. To investigate this:

• Quantitative PCR methodology: Design primers specific to Tetraodon nigroviridis rps3a, avoiding cross-reactivity with pseudogenes. Reference genes for normalization should include beta-actin, GAPDH, and at least one tissue-specific stable reference.

• Tissue panel analysis: Examine expression across multiple tissues including liver, muscle, brain, gonad, gill, and kidney. Typical results would likely show higher expression in metabolically active and proliferating tissues.

• Developmental profiling: Quantify expression at different developmental stages from embryo to adult.

• Promoter analysis: The rps3a promoter likely contains typical ribosomal protein gene elements including a polypyrimidine tract, binding sites for transcription factors such as YY1, and potentially TOP (terminal oligopyrimidine) sequences characteristic of translational regulation.
  Based on comparative studies, expression might be altered during immune responses, as the related ribosomal protein S3 has been implicated in immune signaling pathways .

What post-translational modifications regulate Tetraodon nigroviridis rps3a function?

Post-translational modifications of Tetraodon nigroviridis rps3a likely play crucial roles in its functional regulation. Based on studies of related ribosomal proteins, important modifications may include:

• Phosphorylation: Based on the related ribosomal protein S3, phosphorylation at threonine residues (similar to the T221 identified in RPS3) may be mediated by protein kinases such as PKCδ . Phosphorylation sites can be identified using:

  • Mass spectrometry phosphoproteomic analysis

  • Site-directed mutagenesis of predicted phosphorylation sites

  • Phospho-specific antibodies for Western blotting

• Ubiquitination: Likely regulates protein turnover and potentially extra-ribosomal functions.

• Acetylation: May influence nuclear localization or protein-protein interactions.
  A comprehensive post-translational modification map would require:

  • Immunoprecipitation of native rps3a from Tetraodon tissues

  • High-resolution mass spectrometry analysis

  • Comparison of modification patterns under different physiological conditions
    These modifications could regulate rps3a's participation in ribosome assembly, RNA binding affinity, and potential involvement in signaling pathways.

How has rps3a evolved in Tetraodon nigroviridis compared to other vertebrates?

Evolutionary analysis of rps3a in Tetraodon nigroviridis reveals important insights about selection pressures and functional constraints:

How can differences between Tetraodon nigroviridis rps3a and mammalian homologs inform structural studies?

Comparative analysis between Tetraodon nigroviridis rps3a and mammalian homologs provides valuable insights for structural biology approaches:

• Divergent regions: Areas of sequence divergence, while maintaining function, often indicate regions tolerant to modification. These can be targeted for:

  • Crystallization construct design by removing flexible regions

  • Surface entropy reduction mutations to enhance crystallizability

  • Design of species-specific antibodies or probes

• Conserved motifs: Highly conserved regions likely represent:

  • Critical RNA binding interfaces

  • Ribosome integration sites

  • Protein-protein interaction domains

• Thermal stability differences: Fish proteins often show adaptations for function at lower temperatures compared to mammalian homologs. This may manifest as:

  • Different amino acid composition in surface-exposed regions

  • Modified hydrophobic core packing

  • Altered salt bridge and hydrogen bonding networks
    A detailed structural bioinformatics analysis should include:

  • Multiple sequence alignment across diverse vertebrates

  • Homology modeling based on available ribosomal protein structures

  • Molecular dynamics simulations to identify stable structural elements

  • Prediction of intrinsically disordered regions that might differ between species
    These insights can guide experimental design for both structural studies and functional assays.

How can recombinant Tetraodon nigroviridis rps3a be used to study ribosome assembly?

Recombinant Tetraodon nigroviridis rps3a offers powerful approaches for investigating ribosome assembly mechanisms:

• In vitro reconstitution studies: Using purified rps3a with other recombinant ribosomal proteins and rRNA to study assembly hierarchies and kinetics. Methodological considerations include:

  • Order-of-addition experiments to determine assembly pathways

  • Temperature and ionic strength variations to assess stability

  • Time-resolved structural techniques (SAXS, cryo-EM) to capture assembly intermediates

• Fluorescence-based assays: Fluorescently labeled rps3a can track incorporation into pre-ribosomal particles in real-time through:

  • FRET pairs with other labeled ribosomal components

  • Fluorescence correlation spectroscopy to measure diffusion properties

  • Single-molecule tracking in reconstituted systems

• Atomic force microscopy: To visualize topological changes during assembly steps involving rps3a incorporation.

• Cross-linking coupled with mass spectrometry: To map interaction networks during assembly, identifying assembly factors that specifically recognize rps3a.
  Researchers should consider species-specific factors that might influence Tetraodon nigroviridis ribosome assembly compared to mammalian systems, potentially relating to its compact genome and unique evolutionary history .

What potential role might Tetraodon nigroviridis rps3a play in immune responses?

While direct evidence for immune functions of Tetraodon nigroviridis rps3a is not provided in the search results, we can form hypotheses based on related findings:

• Potential NF-κB signaling involvement: The related ribosomal protein S3 functions as a non-Rel subunit of NF-κB and is involved in regulating specific NF-κB target gene transcription . Research approaches to investigate whether rps3a has similar functions include:

  • Co-immunoprecipitation with NF-κB components

  • Chromatin immunoprecipitation to identify potential DNA binding sites

  • Reporter gene assays with NF-κB responsive elements

• Response to pathogen challenge: Tetraodon nigroviridis has evolved mechanisms to resist pathogen infection, particularly bacterial pathogens like Vibrio parahaemolyticus . Experimental approaches to investigate rps3a's role include:

  • Expression profiling following bacterial challenge

  • RNAi-mediated knockdown to assess impact on immune responses

  • Identification of potential interaction partners in immune signaling pathways

• Post-translational modifications during immune activation: Phosphorylation of rps3a could be modulated during immune responses, similar to how RPS3 phosphorylation is regulated by PKCδ in other contexts . Methods to investigate include:

  • Phosphoproteomics before and after immune stimulation

  • Mutagenesis of predicted phosphorylation sites followed by functional assays

  • Kinase inhibitor studies to identify regulatory enzymes
    A multi-omics approach combining transcriptomics, proteomics, and phosphoproteomics would provide a comprehensive view of rps3a's potential role in Tetraodon nigroviridis immune responses.

How can I overcome solubility issues when expressing recombinant Tetraodon nigroviridis rps3a?

Solubility challenges with recombinant Tetraodon nigroviridis rps3a can be addressed through systematic optimization:

• Expression condition optimization:

  • Reduce induction temperature (16-20°C)

  • Lower IPTG concentration (0.1-0.5 mM)

  • Use auto-induction media for gradual protein expression

  • Co-express with molecular chaperones (GroEL/GroES, trigger factor)

• Construct design strategies:

  • Remove putative aggregation-prone regions identified through bioinformatics

  • Create truncated constructs focusing on stable domains

  • Add solubility-enhancing fusion partners (MBP, SUMO, Trx)

  • Introduce surface mutations to increase hydrophilicity

• Buffer optimization during purification:

  • Screen various buffer systems (HEPES, Tris, phosphate) at pH range 6.5-8.0

  • Test additives: arginine (50-500 mM), proline (25-100 mM), glycerol (5-20%)

  • Include stabilizing co-factors or binding partners

  • Evaluate deterrents including reduced/non-reduced glutathione mixtures

• Refolding approaches if necessary:

  • Gradual dialysis from denaturant (8M to 0M urea/GuHCl)

  • On-column refolding using affinity chromatography

  • Pulsed dilution techniques to prevent aggregation
    Experimental design should include parallel optimization of multiple parameters followed by functional assays to ensure that soluble protein retains biological activity.

What strategies can resolve non-specific DNA/RNA contamination during purification of recombinant Tetraodon nigroviridis rps3a?

Nucleic acid contamination is a common challenge when purifying RNA-binding proteins like rps3a. Effective strategies include:

• Preventive measures during cell lysis:

  • Include nucleases in lysis buffer (DNase I 10 μg/ml, RNase A 5 μg/ml)

  • Increase salt concentration (500-750 mM NaCl) to disrupt nucleic acid interactions

  • Add polyethyleneimine (0.05-0.1%) to precipitate nucleic acids before initial capture

• Chromatographic approaches:

  • Incorporate a heparin affinity step, which acts as a nucleic acid mimetic and can help separate protein-nucleic acid complexes

  • Use anion exchange chromatography with optimized salt gradients

  • Add a hydroxyapatite chromatography step which differentially binds proteins and nucleic acids

• Selective precipitation techniques:

  • Ammonium sulfate fractionation at concentrations that precipitate protein but leave nucleic acids in solution

  • Differential PEG precipitation

• Post-purification treatments:

  • High salt washes (1-2 M NaCl) of protein bound to affinity resin

  • Extended dialysis against buffers containing 10-20 mM EDTA to chelate divalent cations required for nucleic acid binding
    Monitor nucleic acid contamination by A260/A280 ratio measurements (target <0.7 for pure protein) and agarose gel electrophoresis of purified protein samples with sensitive nucleic acid stains.

What methodologies are recommended for identifying Tetraodon nigroviridis rps3a interaction partners?

To comprehensively map the interactome of Tetraodon nigroviridis rps3a, researchers should employ multiple complementary approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged rps3a (FLAG, HA, or His) in relevant cell lines or native tissues

  • Optimize crosslinking conditions (formaldehyde 0.1-1% or DSS/BS3 1-2 mM)

  • Perform stringent washes to reduce non-specific interactions

  • Analyze by LC-MS/MS with label-free quantification or SILAC for higher confidence

  • Compare against appropriate negative controls (tag-only, irrelevant protein)

• Proximity-dependent labeling:

  • Generate BioID or TurboID fusions to rps3a for in vivo biotinylation of proximal proteins

  • Express APEX2-rps3a fusions for spatial proteomics approaches

  • Optimize labeling conditions (biotin concentration, labeling time)

• Yeast two-hybrid screening:

  • Use both N- and C-terminal fusions as baits

  • Screen against Tetraodon-specific cDNA libraries

  • Validate interactions through co-immunoprecipitation

• Computational predictions:

  • Apply homology-based interaction prediction based on known interactions of mammalian RPS3A

  • Use co-expression network analysis from transcriptomic data

  • Employ structural modeling to predict potential interaction interfaces
    Based on studies with related proteins, potential interactors might include other ribosomal proteins, translation factors, and components of signaling pathways such as NF-κB or TRIP13-related proteins .

How can I investigate the role of Tetraodon nigroviridis rps3a in ribosome heterogeneity?

Ribosome heterogeneity represents an emerging area of research, and Tetraodon nigroviridis rps3a can be studied in this context through:

• Ribosome profiling and selective ribosome profiling:

  • Generate antibodies specific to Tetraodon nigroviridis rps3a

  • Immunoprecipitate ribosomes containing rps3a followed by sequencing of protected mRNA fragments

  • Compare translational profiles of rps3a-containing vs. rps3a-depleted ribosomes

• Cryo-electron microscopy approaches:

  • Isolate distinct ribosome populations using sucrose gradient fractionation

  • Perform structural analysis to identify conformational differences

  • Map rps3a positioning and potential isoform incorporation

• Quantitative proteomics of ribosome composition:

  • Use SILAC or TMT labeling to compare stoichiometry of ribosomal proteins

  • Analyze post-translational modifications specific to various ribosome populations

  • Identify tissue-specific or condition-specific variations in rps3a incorporation

• Genetic manipulation studies:

  • Generate conditional knockdown models of rps3a

  • Perform polysome profiling to assess impact on translation

  • Create mutant versions with altered post-translational modification sites

  • Assess tissue-specific phenotypes resulting from altered rps3a function
    This research could reveal specialized roles of rps3a-containing ribosomes in translating specific mRNA subsets, particularly those involved in stress responses or immune functions given the potential role of ribosomal proteins in these processes .