Recombinant Silurus asotus 60S ribosomal protein L15 (rpl15)

What is the typical sequence length and molecular weight of RPL15 proteins across species?

RPL15 proteins typically contain between 144-204 amino acids depending on the species. For instance, the human RPL15 contains 204 amino acids with a molecular weight of approximately 24.14 kDa and a theoretical pI of 12.11 . The Pseudomonas aeruginosa RPL15 is shorter at 144 amino acids . While specific data for Silurus asotus RPL15 is not directly presented in the literature, fish species like Danio rerio show approximately 79.02% nucleotide sequence homology with other vertebrate RPL15 sequences . Researchers should expect similar molecular characteristics for Silurus asotus, with potential species-specific variations in exact length and post-translational modifications.

What are the key structural domains and functional motifs identified in RPL15 proteins?

Topology analysis of RPL15 proteins reveals several conserved functional sites including:

• Two cAMP- and cGMP-dependent protein kinase phosphorylation sites

• Four N-myristoylation sites

• Two Protein kinase C phosphorylation sites

• Two Casein kinase II phosphorylation sites

• Two Amidation sites

• One Ribosomal protein L15e signature

These motifs are highly conserved across vertebrate species, suggesting their functional importance. When designing experiments with recombinant Silurus asotus RPL15, researchers should verify the presence of these motifs using tools like PROSITE or similar motif prediction software to ensure the recombinant protein maintains native functionality.

What expression systems are most effective for producing functional recombinant RPL15?

Multiple expression systems have been successfully used for RPL15 production, with varying advantages depending on research needs:

For Silurus asotus RPL15, E. coli or yeast expression systems would likely provide a good balance of yield and functionality, with E. coli BL21 being particularly effective as demonstrated with other species' RPL15 proteins .

What purification strategies yield the highest purity and activity for recombinant RPL15?

A multi-step purification process is recommended for recombinant RPL15:

• Initial capture using affinity chromatography: His-tag purification is common and effective for recombinant RPL15 proteins .

• Secondary purification: Ion exchange chromatography is recommended due to RPL15's high theoretical pI (12.11).

• Polishing step: Size exclusion chromatography to remove aggregates and ensure homogeneity.

Purities of >90% are typically achievable and sufficient for most research applications . When working with Silurus asotus RPL15, researchers should consider maintaining reducing conditions throughout purification to prevent potential disulfide bond formation affecting protein structure.

How can I effectively study RPL15 subcellular localization patterns and do these vary between species?

RPL15 displays a distinctive localization pattern that can be studied using these approaches:

• Immunofluorescence microscopy: RPL15 has been shown to colocalize strongly with nucleolin (R value 0.86±0.05) and ribosomal protein RPL11, while showing partial colocalization with RPS6 (R value 0.6±0.10) .

• Subcellular fractionation: Biochemical separation followed by immunoblotting reveals that RPL15 is present in both cytoplasmic and nuclear fractions, with a higher percentage in nuclear fractions compared to other ribosomal proteins like RPL11 .

• Co-localization studies: RPL15 colocalizes with nucleolin, fibrillarin, and UBF in nucleoli, consistent with its role in ribosome assembly .

For species-specific variations in Silurus asotus RPL15, research should employ both endogenous protein detection and exogenous expression of tagged constructs (GFP-RPL15) to compare localization patterns with known models. Differential localization could indicate species-specific functional adaptations of this ribosomal protein.

What methods are most reliable for studying RPL15's role in ribosome biogenesis?

To investigate RPL15's function in ribosome biogenesis, researchers should consider:

• siRNA-mediated depletion: Specific siRNAs targeting RPL15 have been successfully used to study its function. Monitoring nucleolar morphology through nucleolin staining before and after depletion can reveal RPL15's impact on nucleolar structure and function .

• Quantitative nucleolar analysis: Image processing algorithms that measure nucleolar area relative to nuclear area have been developed to quantitatively assess nucleolar morphology defects resulting from RPL15 depletion .

• Polysome profiling: To assess the impact of RPL15 on ribosome assembly and translation.

• Pre-rRNA processing analysis: Northern blotting to examine how RPL15 affects specific steps in rRNA maturation.

When applying these techniques to Silurus asotus RPL15, researchers should design species-specific siRNAs based on the cloned sequence to ensure effective knockdown.

How does RPL15 expression correlate with cancer progression and what mechanisms are involved?

RPL15 has shown significant correlation with cancer progression, particularly in colon cancer:

• Expression patterns: RPL15 is overexpressed in colon cancer cells and tissues, with expression levels closely associated with colon carcinogenesis .

• Functional impact: Depletion of RPL15 causes different cellular responses in:

  • Cancer cells: Induces apoptosis in colon cancer cells

  • Non-transformed cells: Causes ribosomal stress leading to G1-G1/S cell cycle arrest

• Potential mechanisms: RPL15 depletion likely triggers ribosomal stress response pathways differently in cancer versus normal cells.

For researchers interested in Silurus asotus RPL15 in cancer models, comparative studies between fish and mammalian RPL15 could provide insights into conserved oncogenic mechanisms across vertebrate evolution.

What techniques are most effective for studying the differential effects of RPL15 depletion between normal and cancer cells?

To study differential effects of RPL15 depletion, researchers should employ:

• Cell viability assays: MTT or ATP-based assays to quantify differential survival between normal and cancer cells following RPL15 depletion.

• Apoptosis detection methods:

  • Annexin V/PI staining followed by flow cytometry

  • TUNEL assay for DNA fragmentation

  • Western blot analysis of apoptotic markers (cleaved caspase-3, PARP)

• Cell cycle analysis: Propidium iodide staining and flow cytometry to detect G1-G1/S arrest in non-transformed cells versus apoptosis in cancer cells.

• Mechanistic pathway analysis: Western blotting for key stress-response proteins (p53, p21) to understand divergent responses.

When applying these techniques to Silurus asotus RPL15 research, fish cell lines provide an excellent model system for comparative oncology studies.

How conserved is RPL15 across species and what does this suggest about its functional importance?

RPL15 shows remarkable evolutionary conservation:

• Sequence homology analysis has revealed high conservation at the amino acid level. The deduced amino acid sequences show 100% homology across multiple mammalian species including Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, and Canis familiaris .

• Nucleotide sequence comparisons show varying degrees of homology:

  • 94.31% between giant panda and Homo sapiens

  • 89.92% between giant panda and Bos taurus

  • 91.54% between giant panda and Mus musculus

  • 91.22% between giant panda and Rattus norvegicus

  • 96.59% between giant panda and Canis familiaris

  • 79.02% between giant panda and Danio rerio (fish)

This high conservation suggests essential fundamental roles in ribosome function that have been maintained through evolutionary pressure. The slightly lower homology in fish (79.02% in Danio rerio) indicates that while core functionality is preserved, there may be species-specific adaptations in Silurus asotus RPL15 worth investigating.

What genomic organization patterns of RPL15 are observed across species and how might this impact expression studies?

The genomic organization of RPL15 shows defined patterns:

• Exon-intron structure: In giant panda, the RPL15 genomic sequence spans 1,835 bp, containing three exons and two introns .

• Regulatory elements: The promoter regions of RPL15 genes typically contain binding sites for transcription factors involved in growth and proliferation regulation.

• Species variations: While the protein-coding regions are highly conserved, the non-coding regions (introns, UTRs) show greater variability across species.

For Silurus asotus RPL15 expression studies, researchers should consider:

• Designing primers spanning exon-exon junctions to avoid genomic DNA amplification

• Accounting for potential differences in promoter elements when studying expression regulation

• Using appropriate reference genes from the same species when performing qPCR analyses

What are common challenges in producing soluble, correctly folded recombinant RPL15 and how can they be addressed?

Researchers frequently encounter these challenges when working with recombinant RPL15:

• Inclusion body formation: RPL15's high basicity (pI 12.11) can lead to aggregation in E. coli.

  • Solution: Express at lower temperatures (16-18°C) and use solubility-enhancing tags like SUMO or MBP.

• Incomplete post-translational modifications: E. coli lacks machinery for eukaryotic modifications.

  • Solution: For studies requiring authentic modifications, consider yeast or baculovirus expression systems .

• Nucleic acid contamination: RPL15's natural RNA binding properties may lead to co-purification with host RNAs.

  • Solution: Include high salt washes (0.5-1M NaCl) and RNase treatment during purification.

• Structural variations between species: Fish RPL15 may have unique properties compared to mammalian versions.

  • Solution: Optimize purification protocols specifically for Silurus asotus RPL15 rather than relying solely on mammalian protocols.

What control experiments are essential when studying RPL15 function to differentiate general ribosomal stress from RPL15-specific effects?

To distinguish RPL15-specific effects from general ribosomal stress:

• Parallel depletion studies: Compare effects of RPL15 depletion with depletion of other ribosomal proteins (e.g., RPL11, RPS6) to identify unique phenotypes .

• Rescue experiments: Complement RPL15 depletion with wild-type and mutant versions to identify functionally important domains.

• Timing analysis: Examine the temporal sequence of cellular events following RPL15 depletion compared to other ribosomal protein depletions.

• Pathway-specific inhibitors: Use inhibitors of p53, mTOR, or other stress pathways to determine which mechanisms mediate RPL15 depletion effects.

• Global translation assessment: Measure protein synthesis rates using puromycin incorporation or similar techniques to distinguish translation defects from other cellular responses.

These controls are particularly important when studying Silurus asotus RPL15 to establish whether any observed species-specific effects are truly unique to this protein or represent general ribosomal stress responses.