Recombinant Hypophthalmichthys nobilis 60S ribosomal protein L15 (rpl15)

How conserved is RPL15 across fish species compared to other vertebrates?

RPL15 is highly conserved during eukaryotic evolution, making it valuable for comparative studies. Sequence analysis of RPL15 across multiple species shows remarkable conservation, particularly within the functional domains . When studying Hypophthalmichthys nobilis RPL15, researchers should perform multiple sequence alignments with other teleost fishes to identify species-specific variations. For methodology, use MUSCLE or ClustalW alignment tools followed by phylogenetic analysis with maximum likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) approaches to construct evolutionary trees . These approaches have successfully demonstrated RPL15's conservation across fifteen fish species from five orders under Teleostei.

What are the primary structural characteristics of RPL15 in Hypophthalmichthys nobilis?

The RPL15 protein belongs to the L15E family of ribosomal proteins and is a component of the 60S ribosomal subunit . In structural analysis, researchers should focus on the conserved domains that interact with rRNA and other ribosomal proteins. Methods for structural characterization include X-ray crystallography or cryo-EM to determine the three-dimensional structure. Comparative modeling using known structures from other species as templates can also provide insights into the protein's conformation. The predicted structure should be validated using tools like PROCHECK and VERIFY3D to ensure stereochemical quality.

What expression systems are most efficient for producing recombinant Hypophthalmichthys nobilis RPL15?

For optimal expression of recombinant RPL15, a bacterial expression system using E. coli BL21(DE3) is generally recommended for initial studies. Design the expression construct with a codon-optimized sequence for E. coli and include a His-tag or GST-tag for purification. For experimental protocol:

• Clone the RPL15 coding sequence into pET-28a(+) vector

• Transform into BL21(DE3) cells

• Induce expression with 0.5 mM IPTG at OD600 of 0.6-0.8

• Grow at 18°C overnight to minimize inclusion body formation

• Lyse cells using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors

For studies requiring post-translational modifications, consider eukaryotic expression systems such as yeast (Pichia pastoris) or insect cells (Sf9) using baculovirus expression vectors.

What purification strategy yields the highest purity and activity of recombinant RPL15?

A multi-step purification approach is recommended:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged protein)

• Ion-exchange chromatography (typically Q-Sepharose)

• Size-exclusion chromatography for final polishing

Table 1: Recommended Purification Buffers for RPL15

Monitor purification efficiency using SDS-PAGE and Western blotting with anti-RPL15 antibodies. Verify the functional integrity through ribosome binding assays or in vitro translation systems.

How can I assess the role of RPL15 in ribosome assembly in Hypophthalmichthys nobilis?

To analyze RPL15's role in ribosome assembly, use a combination of in vitro and in vivo approaches:

• Sucrose gradient ultracentrifugation: Isolate ribosomes from fish tissues or cell lines expressing recombinant RPL15 and analyze subunit profiles. Depletion of RPL15 typically results in decreased pre-60S ribosomal subunits and increased pre-40S subunits .

• Co-immunoprecipitation: Identify RPL15-interacting proteins within the ribosomal complex. Use antibodies against tagged RPL15 followed by mass spectrometry analysis.

• Fluorescence microscopy: Express fluorescently tagged RPL15 to visualize its localization during ribosome biogenesis. Co-staining with nucleolar markers like nucleolin or fibrillarin can provide insights into its role in nucleolar structure maintenance .

In functional studies, compare wild-type RPL15 with mutant variants to identify critical residues for ribosome assembly. Research has shown that RPL15 depletion affects nucleolar morphology, resulting in expanded nucleoli within the nucleus .

What methods are best for studying the impact of RPL15 on rRNA processing?

To investigate RPL15's role in rRNA processing:

• Northern blot analysis: Extract total RNA from control and RPL15-depleted samples. Design probes targeting specific rRNA precursors to detect processing defects.

• Pulse-chase experiments: Label nascent rRNA with 32P or 3H-uridine, then chase with non-labeled media. Analyze the processing kinetics of different rRNA species by gel electrophoresis and autoradiography.

• RNA-seq: Perform high-throughput sequencing to quantitatively assess the abundance of different rRNA intermediates.

• CRISPR-Cas9 mediated knockout/knockdown: Generate cell lines with reduced or absent RPL15 expression to study the resulting rRNA processing defects.

Previous research indicates that RPL15 participates in rRNA processing at the ITS1 site, and its depletion alters rRNA precursors required for both 60S and 40S ribosome biogenesis .

How effective is RPL15 as a phylogenetic marker for studying teleost fish relationships?

RPL15 is an excellent phylogenetic marker for resolving teleostean relationships, particularly at higher taxonomic levels (interordinal relationships) . For phylogenetic studies:

• Sequence the complete open reading frame (ORF) of RPL15 from multiple fish species

• Perform multiple sequence alignment using MUSCLE or ClustalW

• Construct phylogenetic trees using NJ, MP, and ML methods with appropriate evolutionary models

• Use bootstrap analysis (1000 replicates) to evaluate the robustness of tree topology

Table 2: Comparative Analysis of RPL15 as a Phylogenetic Marker

When analyzing Hypophthalmichthys nobilis RPL15 in phylogenetic studies, include Anguilla japonica as an outgroup, as this approach has produced phylogenetic trees largely congruent with morphology-based classifications .

How do post-translational modifications of RPL15 vary across fish species?

Study post-translational modifications (PTMs) using:

• Mass spectrometry: Perform LC-MS/MS analysis of purified RPL15 to identify phosphorylation, methylation, acetylation, and ubiquitination sites.

• Western blotting: Use modification-specific antibodies (anti-phospho, anti-acetyl) to detect PTMs.

• 2D gel electrophoresis: Separate protein isoforms based on charge and mass to identify modified variants.

• Comparative analysis: Compare PTM patterns across different fish species to identify conserved modification sites.

Create a comprehensive map of RPL15 modifications and correlate with functional differences across species. This approach can reveal evolutionary adaptations in ribosomal function specific to different aquatic environments.

How can I investigate the role of RPL15 in cell proliferation and apoptosis in fish cell lines?

To study RPL15's impact on cell proliferation and apoptosis:

• siRNA-mediated knockdown: Design specific siRNAs targeting Hypophthalmichthys nobilis RPL15. Transfect fish cell lines and assess:

  • Cell proliferation using MTT assay at multiple time points

  • BrdU incorporation to measure DNA synthesis

  • Cell cycle analysis by flow cytometry

  • Apoptosis using Annexin V/PI staining and caspase activation assays

• Overexpression studies: Express recombinant RPL15 in fish cell lines and measure effects on proliferation and survival.

Research in human cells has shown that RPL15 depletion results in different outcomes depending on cell type: G1-G1/S cell cycle arrest in non-transformed epithelial cells versus apoptosis in cancer cells . Similar differential responses might be observed in fish normal versus transformed cell lines.

What is the relationship between RPL15 and ribosomal stress response in fish models?

To investigate RPL15's role in ribosomal stress:

• Stress induction: Treat fish cells with ribosomal stressors (actinomycin D, 5-FU) and analyze RPL15 expression and localization.

• Protein-protein interaction analysis: Identify stress-specific interactions using co-immunoprecipitation or proximity ligation assays, focusing on p53 pathway components.

• Gene expression analysis: Measure expression of stress response genes (p53, p21) after RPL15 depletion using qRT-PCR and Western blotting.

Human studies show that RPL15 depletion induces ribosome stress leading to p53 and p21 accumulation . Comparative analysis between fish and mammalian models can reveal conserved stress response mechanisms.

How can I resolve solubility issues when expressing recombinant RPL15?

Ribosomal proteins often face solubility challenges during recombinant expression. Address these issues with:

• Optimization of expression conditions:

  • Reduce induction temperature to 16-18°C

  • Decrease IPTG concentration to 0.1-0.2 mM

  • Use rich media like Terrific Broth

  • Co-express with chaperones (GroEL/ES, DnaK/DnaJ)

• Solubility tags:

  • Fusion with solubility enhancers (MBP, SUMO, Thioredoxin)

  • Include cleavage sites for tag removal

• Buffer optimization during purification:

  • Include stabilizing agents (glycerol 10-15%, arginine 50-100 mM)

  • Optimize ionic strength (300-500 mM NaCl)

  • Test different pH ranges (7.0-8.5)

If inclusion bodies form despite optimization, develop a refolding protocol using gradual dialysis from denaturing conditions (8M urea) to native buffer.

What are the best approaches for studying RPL15 interactions with rRNA and other ribosomal proteins?

To characterize RPL15 interactions within the ribosomal complex:

• RNA immunoprecipitation (RIP): Pull down RPL15 and analyze bound RNAs by RT-PCR or sequencing.

• UV crosslinking: Identify direct RNA-protein contact sites using UV-induced crosslinking followed by immunoprecipitation and sequencing (CLIP-seq).

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map protein-protein and protein-RNA interaction surfaces by measuring solvent accessibility changes.

• Structural analysis: Use cryo-EM to visualize RPL15 within the assembled ribosome structure.

• Mutagenesis: Generate point mutations in conserved residues and assess their impact on interactions and function.

For troubleshooting non-specific interactions, increase stringency in washing steps and include competitors like heparin or yeast tRNA to reduce background.