Recombinant Maconellicoccus hirsutus 60S ribosomal protein L17 (RpL17)
Description
Definition and Context
Recombinant Maconellicoccus hirsutus 60S ribosomal protein L17 (RpL17) refers to a bioengineered version of the ribosomal protein L17 derived from the pink hibiscus mealybug (Maconellicoccus hirsutus). Ribosomal proteins like RpL17 are essential components of the large ribosomal subunit (60S) in eukaryotes, playing critical roles in protein synthesis, translation regulation, and potentially other cellular processes . While RpL17 has been extensively studied in mammals (e.g., humans and mice) as a vascular smooth muscle cell growth inhibitor , its characterization in insects like M. hirsutus remains limited.
Functional Insights
In mammals, RpL17 inhibits vascular smooth muscle cell proliferation, acting as a tumor suppressor analog . In M. hirsutus, RpL17’s role could theoretically extend to regulating growth, reproduction, or stress responses, given the insect’s high reproductive potential and polyphagous nature . Horizontal gene transfer (HGT) events in mealybugs, as documented in transcriptome analyses, suggest that some genes involved in metabolism and detoxification have been acquired from endosymbionts . While RpL17 itself is not explicitly linked to HGT in M. hirsutus, its ribosomal function may interact with such pathways.
Mammalian RpL17 as a Model
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Growth Inhibition: In mice, RpL17 knockdown accelerates vascular cell proliferation, highlighting its role in suppressing intima-media thickening .
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Therapeutic Potential: Targeting RpL17 could modulate cardiovascular diseases in humans .
Implications for M. hirsutus Control
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Pest Management: If RpL17 exhibits similar growth-inhibitory effects in M. hirsutus, recombinant versions could be engineered to disrupt mealybug development, offering a novel biocontrol strategy .
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HGT and Detoxification: While RpL17 itself is not an HGT-acquired gene, its interaction with endosymbiont-derived pathways (e.g., amino acid metabolism) could influence pest resilience .
Challenges and Knowledge Gaps
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Lack of Direct Data: No studies explicitly characterize M. hirsutus RpL17’s structure, expression, or function.
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Bioengineering Limitations: Recombinant production of insect ribosomal proteins requires optimization of heterologous expression systems (e.g., bacterial or yeast hosts) to achieve proper folding and functionality.
Product Specs
Q&A
FAQs for Researchers on Recombinant Maconellicoccus hirsutus 60S Ribosomal Protein L17 (RpL17)
What expression systems are optimal for producing recombinant M. hirsutus RpL17 with high yield and stability?
To achieve high-yield recombinant RpL17 production:
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Prokaryotic systems (e.g., E. coli BL21) are cost-effective for large-scale production but may lack post-translational modifications. Tagging with His- or GST-facilitates purification ( ).
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Eukaryotic systems (e.g., insect cells or yeast) are preferable if native folding or modifications are critical. For example, baculovirus systems enable proper folding of ribosomal proteins.
| System | Yield | Post-Translational Modifications | Complexity |
|---|---|---|---|
| E. coli | High | None | Low |
| Insect Cells | Moderate | Yes | High |
| Yeast | Moderate | Limited | Medium |
Validation: Use SDS-PAGE and Western blotting with species-cross-reactive antibodies (e.g., anti-RpL17 validated in insects; ).
How can affinity chromatography methods balance purity and structural integrity during RpL17 purification?
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Immobilized Metal Affinity Chromatography (IMAC): Effective for His-tagged RpL17 but may require optimization of imidazole gradients to prevent aggregation.
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Size-Exclusion Chromatography (SEC): Post-IMAC SEC refines purity and assesses oligomeric state.
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Tag Removal: Use TEV protease for cleaving affinity tags if structural studies are planned.
Critical Step: Monitor protein stability via circular dichroism (CD) spectroscopy after each purification stage.
What in vitro assays validate the biological activity of recombinant RpL17?
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Ribosome Reconstitution Assays: Incorporate recombinant RpL17 into in vitro-assembled ribosomal subunits and assess translational activity using luciferase reporters.
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Binding Kinetics: Surface plasmon resonance (SPR) to measure interactions with rRNA or partner proteins ( ).
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Cell-Free Systems: Test RpL17’s ability to restore ribosome function in RpL17-depleted lysates (e.g., siRNA-treated insect cell extracts; ).
How can structural discrepancies between computational models and cryo-EM data for RpL17 be resolved?
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Integrative Modeling: Combine cryo-EM density maps (sub-3 Å resolution) with molecular dynamics simulations to refine flexible regions.
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Cross-Linking Mass Spectrometry (CLMS): Identify proximal amino acids to validate spatial arrangements.
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Mutagenesis Screens: Introduce point mutations at disputed regions (e.g., rRNA-binding domains) and assess ribosome assembly defects ( ).
What strategies address contradictions in RpL17’s role as a growth inhibitor versus ribosome biogenesis factor?
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Context-Specific Knockdowns: Use tissue-specific RNAi in insect models to dissect RpL17’s dual roles (e.g., fat body vs. ovarian cells; ).
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Ribosome Profiling: Compare translational efficiency in RpL17-deficient versus wild-type cells to identify differentially translated mRNAs.
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Single-Cell RNA-Seq: Resolve heterogeneity in RpL17 expression and correlate with cell cycle stages ( ).
Key Finding: In mice, RpL17 depletion increased proliferating vascular cells 8-fold, suggesting growth-inhibitory roles ( ).
Does recombinant RpL17 influence ribosome heterogeneity in M. hirsutus?
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rRNA Variant Analysis: Use northern blotting or long-read sequencing to detect alternative 5.8S rRNA forms (e.g., 5.8S<sub>C</sub> in RpL17-deficient mice; ).
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Polysome Profiling: Compare ribosomal subunit ratios (40S:60S) and polysome abundance in RpL17-modified strains.
| Condition | 60S Subunits | 5.8S Variants | Polysome Integrity |
|---|---|---|---|
| Wild-Type | Normal | 5.8S<sub>L</sub> | Intact |
| RpL17-Depleted | Reduced | 5.8S<sub>C</sub> | Disrupted |
Implication: Altered 5.8S rRNA may affect interactions with translocon components ( ).
How can researchers assess RpL17’s role in horizontal gene transfer (HGT) adaptation in M. hirsutus?
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Comparative Genomics: Screen M. hirsutus genomes for RpL17 orthologs in bacteria or fungi, leveraging known HGT clusters ( ).
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Functional Complementation: Express bacterial-origin RpL17 variants in insect cells and test ribosome assembly efficiency.
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CRISPR-Cas9 Knock-In: Replace endogenous RpL17 with HGT-acquired variants and quantify fitness metrics (e.g., symbiont dependence; ).
