Recombinant Spodoptera frugiperda 60S ribosomal protein L41 (RpL41)

What is the structure and function of 60S ribosomal protein L41 in S. frugiperda?

The 60S ribosomal protein L41 in S. frugiperda, similar to its homologs in other species, is a small protein component of the large 60S ribosomal subunit involved in protein synthesis. While the specific structure in S. frugiperda has not been fully characterized, comparative analysis with human RPL41 suggests it likely contains approximately 25 amino acids with a characteristic alpha helix (positions 3-14) and a turn (positions 15-18). The protein is likely rich in basic amino acids, particularly arginine and lysine, consistent with its role in ribosome functioning .

RpL41 belongs to the L41E family of ribosomal proteins and shares sequence similarity with the yeast ribosomal protein YL41. In the cellular context, it functions as part of the ribosomal machinery that catalyzes protein synthesis. Ribosomes consist of small 40S and large 60S subunits, which together comprise 4 RNA species and approximately 80 structurally distinct proteins forming the complete translation apparatus .

How does S. frugiperda RpL41 compare to homologous proteins in other species?

Comparative analysis reveals high conservation of L41 proteins across species. The human RPL41 gene, for instance, shows 99.5% homology in exonic regions when compared with corresponding genes in yeast, though the human gene contains introns not observed in the yeast counterpart. This high degree of conservation suggests strong evolutionary pressure to maintain the sequence and function of this protein .

The table below summarizes key comparative features of L41 proteins across select species:

While the complete sequence and structure of S. frugiperda RpL41 have not been fully elucidated, its predicted features are based on the high conservation observed in L41 proteins across species .

What expression systems are suitable for recombinant production of S. frugiperda RpL41?

For recombinant production of S. frugiperda RpL41, the baculovirus expression vector system (BEVS) using Spodoptera frugiperda cells (Sf9 or Sf21) is particularly appropriate, offering a homologous expression environment. This system has been well-established for producing various recombinant proteins, vaccines, and potential gene therapy vectors .

The general methodology involves:

• Gene design and synthesis based on the known or predicted amino acid sequence of S. frugiperda RpL41

• Construction of an expression vector containing the RpL41 gene

• Introduction of the vector into baculovirus

• Infection of S. frugiperda cells with the recombinant baculovirus

• Expression, isolation, and purification of the recombinant protein

How can I optimize the design of experiments (DoE) for recombinant S. frugiperda RpL41 expression?

Optimizing recombinant S. frugiperda RpL41 expression requires a systematic DoE approach rather than the inefficient one-factor-at-a-time method. The multifactorial nature of protein expression makes DoE particularly valuable for identifying optimal conditions and understanding the complex interactions between experimental variables .

For RpL41 expression in S. frugiperda cells, a methodological DoE approach should include:

• Factor Identification: Determine key variables affecting expression, including:

  • Multiplicity of infection (MOI)

  • Cell density at time of infection

  • Post-infection harvest time

  • Medium composition

  • Temperature

  • pH

• Experimental Design Selection: Choose an appropriate design based on the number of factors:

  • For initial screening: Fractional factorial designs to identify significant factors

  • For optimization: Response surface methodology (RSM) to determine optimal levels

• Analysis of Factor Interactions: Use statistical software to analyze how factors interact and affect:

  • Protein yield

  • Protein solubility

  • Functional activity

  • Purity

A typical experimental matrix for initial screening might look like this:

Analysis of results would identify the most significant factors affecting RpL41 expression, which would then be further optimized using response surface methodology. This approach significantly reduces experimental time and resources while providing robust statistical analysis of the results .

What proteomics approaches can be applied to study S. frugiperda RpL41 during cellular growth and viral infection?

Stable isotope labeling by amino acids in cell culture (SILAC) represents a powerful approach for quantitative proteomics analysis of S. frugiperda RpL41 during cellular growth and baculovirus infection. This methodology allows for precise comparison of protein expression levels across different conditions .

The methodological workflow would include:

• SILAC Labeling:

  • Culture S. frugiperda cells in media containing either "light" (natural) or "heavy" (isotopically labeled) amino acids

  • Infect one population with baculovirus while maintaining the other as control

  • Harvest cells at multiple time points post-infection

• Sample Processing:

  • Extract and quantify total protein from each sample

  • Enzymatically digest proteins into peptides

  • Combine "heavy" and "light" samples for comparative analysis

• Mass Spectrometry Analysis:

  • Perform LC-MS/MS to identify and quantify peptides

  • The mass difference between "heavy" and "light" peptides allows for precise relative quantification

  • Identify RpL41 and associated proteins through database searching

• Bioinformatics Analysis:

  • Compile a search database with protein annotations from various insect species to maximize proteome coverage

  • Analyze differential expression patterns of RpL41 during viral infection

  • Examine potential interaction partners and post-translational modifications

This approach has successfully identified differentially expressed proteins related to energy metabolism, endoplasmic reticulum function, and oxidative stress during baculovirus infection in S. frugiperda cells, and could reveal how RpL41 expression and function changes during viral infection .

What purification strategies are most effective for recombinant S. frugiperda RpL41?

Purifying recombinant S. frugiperda RpL41 presents unique challenges due to its small size (approximately 25 amino acids) and basic nature. An effective purification strategy must account for these characteristics while maximizing yield and maintaining protein functionality.

The recommended methodological approach includes:

• Initial Extraction and Clarification:

  • Harvest cells 48-72 hours post-infection (optimized based on DoE results)

  • Lyse cells using gentle detergents or mechanical disruption

  • Remove cellular debris by centrifugation (10,000-15,000 × g, 30 minutes)

  • Filter supernatant through 0.45 μm filters

• Affinity Chromatography (if using tagged constructs):

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC)

  • Load clarified lysate onto Ni-NTA or cobalt resin

  • Wash extensively with buffer containing low imidazole concentrations

  • Elute with buffer containing 250-500 mM imidazole

  • Consider on-column tag cleavage if desired

• Ion Exchange Chromatography:

  • Given the basic nature (high arginine/lysine content) of RpL41, cation exchange chromatography is particularly effective

  • Use strong cation exchangers like Sulfopropyl (SP) or Sulfoethyl (SE)

  • Employ a salt gradient (0-1 M NaCl) for elution

  • This step is highly effective for removing nucleic acid contamination

• Size Exclusion Chromatography:

  • Final polishing step to remove aggregates and ensure homogeneity

  • Use columns designed for small proteins (e.g., Superdex 75 or equivalent)

  • Analyze fractions by SDS-PAGE with appropriate visualization for small proteins

• Quality Control:

  • Assess purity by SDS-PAGE (using tricine gels optimized for small proteins)

  • Confirm identity by mass spectrometry

  • Evaluate folding using circular dichroism spectroscopy

  • Test functionality through ribosome binding assays

This multi-step approach addresses the challenges of purifying small, basic proteins like RpL41 while maintaining their native structure and function for subsequent studies .

How can I assess the functional integrity of purified recombinant S. frugiperda RpL41?

Assessing the functional integrity of purified recombinant S. frugiperda RpL41 requires a combination of structural and functional assays tailored to its role in ribosome function and potential extraribosomal activities.

The comprehensive methodological approach includes:

• Structural Integrity Assessment:

  • Circular Dichroism (CD) Spectroscopy: Evaluate secondary structure elements (alpha helix, turn)

  • Mass Spectrometry: Confirm exact molecular weight and detect any post-translational modifications

  • Dynamic Light Scattering: Assess homogeneity and detect potential aggregation

  • NMR Spectroscopy: For detailed structural analysis of this small protein

• Ribosomal Incorporation Assays:

  • In vitro ribosome reconstitution using purified ribosomal components

  • Sucrose gradient centrifugation to verify RpL41 association with 60S subunits or 80S ribosomes

  • Cryo-electron microscopy to visualize RpL41 positioning within reconstituted ribosomes

• Translation Functionality Tests:

  • In vitro translation assays using:

    • RpL41-depleted ribosomes

    • RpL41-depleted ribosomes supplemented with recombinant RpL41

  • Measure translation efficiency, fidelity, and kinetics

  • Analyze translation products by gel electrophoresis and autoradiography

• Binding Partner Interaction Studies:

  • Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) to measure binding kinetics with:

    • rRNA components

    • Other ribosomal proteins

    • Potential regulatory proteins (e.g., kinases like CKII)

  • Co-sedimentation assays with purified components

• Functional Complementation:

  • Rescue experiments in RpL41-depleted S. frugiperda cell extracts

  • Measure restoration of translation activity

  • Compare activity to wild-type cell extracts

Results from these assays can be presented in a functionality assessment matrix:

This comprehensive approach provides multiple lines of evidence for the functional integrity of the purified recombinant protein .

What are common challenges in expressing recombinant S. frugiperda RpL41 and how can they be addressed?

Expressing recombinant S. frugiperda RpL41 presents several challenges due to its small size, basic nature, and potential cytotoxicity when overexpressed. Here are common issues researchers encounter and methodological approaches to address them:

• Low Expression Levels:

  • Cause: Suboptimal codon usage, mRNA instability, or toxicity to host cells

  • Solution:

    • Optimize codon usage for expression host

    • Use stronger promoters (e.g., polyhedrin for baculovirus)

    • Consider inducible expression systems

    • Reduce expression temperature to 27°C (for Sf9 cells)

    • Test different MOI values (0.1-10) and harvest times

• Protein Degradation:

  • Cause: Small proteins are often more susceptible to proteolytic degradation

  • Solution:

    • Add protease inhibitors during extraction

    • Use fusion partners (e.g., GST, MBP) to increase size and stability

    • Optimize harvest time to collect protein before degradation occurs

    • Consider co-expression with chaperones

• Poor Solubility:

  • Cause: Basic proteins may aggregate due to interactions with nucleic acids

  • Solution:

    • Extract under high salt conditions (500 mM NaCl)

    • Include nucleases in extraction buffer

    • Test different detergents and solubilizing agents

    • Consider extraction under denaturing conditions followed by refolding

• Difficulty in Detection:

  • Cause: Small size makes visualization on standard SDS-PAGE difficult

  • Solution:

    • Use tricine-SDS-PAGE optimized for small proteins

    • Include larger tags for easier detection

    • Employ Western blotting with specific antibodies

    • Consider silver staining or fluorescent dyes with higher sensitivity

• Disruption of Host Cell Translation:

  • Cause: Overexpression of ribosomal proteins may interfere with host translation

  • Solution:

    • Use tightly controlled inducible systems

    • Optimize expression to balance yield and host cell viability

    • Monitor cell viability during expression

The table below summarizes experimental adjustments for common issues:

By systematically addressing these challenges through optimization of expression conditions and purification protocols, researchers can significantly improve the yield and quality of recombinant S. frugiperda RpL41 .

How can I investigate the role of S. frugiperda RpL41 in response to baculovirus infection?

Investigating the role of S. frugiperda RpL41 during baculovirus infection requires a multifaceted approach to monitor changes in expression, localization, and function. The baculovirus-host interaction provides a unique model to study how ribosomal proteins may be regulated or repurposed during viral infection.

A comprehensive methodological framework includes:

• Expression Profiling:

  • Quantitative Proteomics: Apply SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) to compare RpL41 levels in infected versus uninfected cells at multiple timepoints

  • RT-qPCR: Monitor RpL41 mRNA levels throughout the infection cycle

  • Western Blotting: Track protein expression with RpL41-specific antibodies

  • Ribosome Profiling: Assess translational efficiency of RpL41 mRNA during infection

• Localization Studies:

  • Immunofluorescence Microscopy: Visualize potential redistribution of RpL41 during infection

  • Subcellular Fractionation: Compare RpL41 distribution in cytoplasmic, nuclear, and membrane fractions

  • Polysome Profiling: Determine association of RpL41 with active ribosomes versus free subunits

• Functional Analysis:

  • RNA Interference: Knockdown RpL41 and assess effects on viral replication

  • CRISPR/Cas9 Gene Editing: Generate RpL41 mutants to identify critical functional residues

  • Overexpression Studies: Examine consequences of RpL41 overexpression on viral replication

• Interaction Networks:

  • Immunoprecipitation-Mass Spectrometry: Identify proteins interacting with RpL41 during infection

  • Cross-linking Studies: Capture dynamic interactions with viral components

  • Yeast Two-Hybrid Screening: Screen for interactions with viral proteins

• Viral Replication Assessment:

  • Plaque Assays: Quantify viral titers under various RpL41 manipulation conditions

  • qPCR: Measure viral DNA replication

  • Reporter Assays: Monitor viral gene expression using reporter constructs

This comprehensive approach allows researchers to determine whether RpL41 plays a passive role as part of the cellular translation machinery that is hijacked by the virus, or whether it has a more active role in the viral replication cycle or host defense. Recent studies have shown that some ribosomal proteins have non-canonical functions during stress or infection, making this an important area of investigation .

How can I analyze differential protein expression data to understand the role of RpL41 in S. frugiperda cells during viral infection?

Analyzing differential protein expression data to understand the role of RpL41 in S. frugiperda cells during viral infection requires sophisticated bioinformatics approaches and careful interpretation. The goal is to place RpL41 expression changes within the broader context of cellular response to infection.

• Data Preprocessing and Quality Control:

  • Normalize mass spectrometry data to account for technical variations

  • Apply appropriate transformations (e.g., log2) for statistical analysis

  • Filter out low-quality peptide identifications

  • Evaluate reproducibility across replicates

• Differential Expression Analysis:

  • Apply statistical tests (e.g., t-test, ANOVA) with appropriate multiple testing correction

  • Calculate fold changes in RpL41 expression between infected and control samples

  • Establish significance thresholds (typically p < 0.05 and fold change > 1.5)

  • Create volcano plots highlighting RpL41 and related proteins

• Temporal Analysis:

  • Plot RpL41 expression changes across infection time course

  • Identify proteins with similar or opposite expression patterns

  • Apply clustering algorithms to group proteins with similar temporal profiles

  • Correlate RpL41 expression with viral protein production phases

• Network Analysis:

  • Construct protein-protein interaction networks including RpL41

  • Identify modules of co-regulated proteins

  • Calculate network centrality measures to assess RpL41's importance

  • Map expression changes onto known ribosomal and translation-related pathways

• Functional Enrichment Analysis:

  • Perform Gene Ontology (GO) enrichment for biological processes, molecular functions, and cellular components

  • Conduct KEGG pathway analysis to identify affected pathways

  • Use reactome analysis for detailed pathway mapping

  • Analyze enrichment of proteins co-regulated with RpL41

A sample visualization of RpL41 temporal expression might look like:

What are the future research directions for S. frugiperda RpL41 in recombinant protein production and viral infection studies?

The study of S. frugiperda RpL41 presents several promising avenues for future research, both in fundamental understanding of ribosomal biology and in applications for recombinant protein production systems.

Key future research directions include:

• Structural and Functional Characterization:

  • Complete determination of S. frugiperda RpL41 structure using cryo-EM and X-ray crystallography

  • Investigation of potential extraribosomal functions, similar to those observed in mammalian systems

  • Detailed analysis of how RpL41 contributes to ribosome stability and translation efficiency

• Host-Virus Interactions:

  • Exploration of how baculovirus infection modulates RpL41 expression and function

  • Investigation of whether viral proteins directly interact with RpL41

  • Determination if RpL41 alterations contribute to the viral protein production phase

• Biotechnological Applications:

  • Development of RpL41-derived peptides as potential cell-penetrating delivery vehicles

  • Engineering of S. frugiperda cells with modified RpL41 to enhance recombinant protein production

  • Creation of biosensors based on RpL41 interactions for monitoring cellular stress

• Comparative Studies Across Species:

  • Examination of RpL41 conservation and divergence across insect species

  • Functional comparison between insect and mammalian L41 proteins

  • Investigation of species-specific interactions and regulatory mechanisms

• Therapeutic and Agricultural Applications:

  • Exploration of RpL41 as a potential target for specific insect control methods

  • Development of strategies to modulate RpL41 function for enhanced baculovirus-based bioprocesses

  • Investigation of potential antimicrobial peptides derived from RpL41 sequences

These research directions will contribute to both fundamental understanding of ribosomal biology in insect systems and practical applications in biotechnology and agriculture. As recombinant protein production in insect cells continues to be an important platform for vaccines, therapeutics, and research reagents, deeper understanding of S. frugiperda RpL41 function may lead to improved expression systems and process optimization .