Recombinant Tropheryma whipplei 50S ribosomal protein L21 (rplU)

What is Tropheryma whipplei 50S ribosomal protein L21 (rplU)?

Tropheryma whipplei 50S ribosomal protein L21 (rplU) is a critical component of the large (50S) subunit of bacterial ribosomes in T. whipplei, the causative agent of Whipple's disease. The full-length protein consists of 104 amino acids and plays an essential role in the ribosomal machinery responsible for protein synthesis in this bacterium . This protein is significant for understanding the minimal metabolism and pathogenicity of T. whipplei, as the organism has a remarkably reduced genome of approximately 927,303 base pairs . In protein databases, rplU is registered with the UniProt accession number Q83G55, indicating its biochemical characterization .

What are the optimal storage conditions for recombinant T. whipplei rplU?

Storage conditions for recombinant T. whipplei rplU vary depending on the protein formulation and intended use timeframe. The product's stability is influenced by multiple factors including storage state, buffer composition, temperature, and the protein's intrinsic stability characteristics .

For long-term storage:

• Liquid formulations: Store at -20°C to -80°C with an expected shelf life of approximately 6 months

• Lyophilized formulations: Store at -20°C to -80°C with an extended shelf life of approximately 12 months

For working solutions:

• Store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly compromise protein integrity

To maximize stability during freezer storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) before storing at -20°C or -80°C . When preparing the protein for experimental use, create smaller volume aliquots to minimize the number of freeze-thaw cycles required for future experiments.

What is the recommended reconstitution protocol for lyophilized rplU?

The optimal reconstitution protocol for lyophilized T. whipplei rplU involves several critical steps to ensure maximum protein recovery and biological activity:

• First, briefly centrifuge the vial prior to opening to ensure all lyophilized material is at the bottom of the container and to prevent product loss .

• Reconstitute the protein using deionized sterile water to achieve a final concentration between 0.1-1.0 mg/mL .

• For preparations intended for long-term storage, add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) .

• Prepare multiple small-volume aliquots to minimize future freeze-thaw cycles.

• For extended storage, maintain aliquots at -20°C to -80°C where they remain stable for approximately 6 months .

Working aliquots can be maintained at 4°C for up to one week for ongoing experiments . Throughout the reconstitution process, handle the protein gently to maintain structural integrity and functional activity, avoiding vigorous vortexing or prolonged exposure to room temperature.

How is T. whipplei rplU expressed and purified for research applications?

For research applications, T. whipplei rplU is typically produced using mammalian cell expression systems as indicated in the product specifications . This expression approach is selected because:

• Mammalian expression systems can provide appropriate protein folding and potential post-translational modifications when needed.

• The full protein sequence spanning residues 1-104 is expressed to maintain complete structural and functional characteristics .

• Standard protein purification protocols yield a product with confirmed purity exceeding 85% as verified by SDS-PAGE analysis .

The expression of T. whipplei proteins presents unique challenges, as the bacterium itself was historically difficult to cultivate, only being successfully grown in human fibroblasts in 2000 . This explains why heterologous expression systems are necessary for producing recombinant proteins from this organism.

For researchers developing their own expression systems, considerations should include:

• Codon optimization for the selected expression host

• Addition of purification tags (noting that "Tag type will be determined during the manufacturing process" )

• Implementation of appropriate purification techniques such as affinity chromatography

• Quality control verification using SDS-PAGE, mass spectrometry, or other analytical methods

What is the genomic context of the rplU gene in T. whipplei?

The rplU gene in T. whipplei exists within a highly condensed genome that exemplifies extreme genomic reduction. T. whipplei possesses a circular genome of approximately 927,303 base pairs (Twist strain) or 925,938 bp (strain TW08/27), making it one of the smallest genomes within the Actinobacteria phylum .

This reduced genome encodes approximately 808 predicted protein-coding genes in the Twist strain . While the specific genomic neighborhood of rplU is not detailed in the available search results, in most bacteria, ribosomal protein genes typically occur in conserved operons. Given T. whipplei's status as an organism with a minimal genome, it likely maintains essential gene arrangements, particularly those involved in fundamental processes like protein synthesis.

Several noteworthy features of T. whipplei's genome that may influence the context of rplU include:

• Genomic plasticity: Comparison of two T. whipplei genome sequences (Twist and TW08/27) revealed a large chromosomal inversion , indicating a degree of genomic rearrangement that could potentially affect gene organization.

• Repetitive elements: The genome contains numerous repeats, particularly associated with cell surface proteins , which can mediate genome rearrangements.

• Essential gene retention: Despite extensive genome reduction, T. whipplei has maintained genes critical for fundamental cellular functions, including ribosomal proteins like rplU.

How does the structure of T. whipplei rplU compare to other bacterial ribosomal L21 proteins?

While specific structural information for T. whipplei rplU is not provided in the available data, comparative analysis of its 104-amino acid sequence can yield important insights:

• Sequence conservation analysis of T. whipplei rplU against L21 proteins from other bacteria would likely reveal highly conserved regions that correspond to functionally critical domains. These conserved elements typically include RNA-binding motifs and regions involved in interactions with other ribosomal components.

• Ribosomal proteins generally maintain conserved structural elements due to their essential roles in ribosome assembly and function. The L21 protein typically features a globular domain that interacts with ribosomal RNA and neighboring proteins in the assembled ribosome.

• Given T. whipplei's reduced genome and adaptation to a host-dependent lifestyle , its rplU may exhibit unique adaptations compared to homologs from free-living bacteria. These could include specific amino acid substitutions that optimize function within the constraints of a minimal metabolism.

• The sequence "MFAVVKASGF QRLVEVGSVI SIDPTNVDSG GFVHFPVLLL VDGAEVVSDP DKLRLARVSA KFLRKLRGPK VRIHKFKNKT GYHKRQGHRQ GVYVFSVTSI ERGD" can be analyzed for characteristic RNA-binding motifs common to L21 proteins.

For researchers interested in structural comparisons, methods such as homology modeling, circular dichroism spectroscopy, or X-ray crystallography would provide valuable insights into the three-dimensional structure of T. whipplei rplU and how it compares to other bacterial L21 proteins.

What are the typical applications of recombinant T. whipplei rplU in studying Whipple's disease?

Recombinant T. whipplei rplU has several valuable applications in Whipple's disease research, particularly given the historical challenges in cultivating this pathogen:

• Diagnostic tool development: As a protein specific to T. whipplei, rplU can serve as a basis for developing serological assays to complement existing PCR-based diagnostic methods . Protein-based diagnostics could offer different sensitivity and specificity profiles compared to nucleic acid testing.

• Immunological research: The recombinant protein enables detailed investigation of host immune responses to T. whipplei, helping to elucidate why some individuals develop clinical Whipple's disease while others might carry the bacterium asymptomatically . This is particularly relevant given that T. whipplei DNA has been detected in individuals without clinical evidence of Whipple's disease .

• Structure-function analysis: Studying rplU provides insights into T. whipplei's adaptation to its environment, especially considering its reduced genome and unique metabolic constraints. This may reveal how essential cellular machinery has evolved in the context of genome reduction.

• Antimicrobial target investigation: The essential nature of ribosomal proteins makes them potential targets for antimicrobial development. Given that T. whipplei has a "mutation in DNA gyrase predicting a resistance to quinolone antibiotics" , alternative targets like rplU could be clinically relevant.

• Basic biology research: Work with rplU contributes to understanding the fundamental biology of T. whipplei, which remains incompletely characterized despite advances in molecular techniques .

These applications have particular value given that T. whipplei was cultured reproducibly only in 2000 , making recombinant proteins essential tools for studying this challenging organism.

How can I optimize experimental conditions for in vitro studies using T. whipplei rplU?

Successful in vitro studies using T. whipplei rplU require careful optimization of experimental conditions to ensure reliable and reproducible results:

• Protein stability considerations:

  • Buffer selection: Use physiological buffers (typically phosphate or Tris-based at pH 7.2-7.5) that maintain protein structure and function.

  • Temperature management: Conduct experiments at temperatures that preserve protein integrity, generally between 4°C and 37°C depending on the specific assay requirements.

  • Stabilizing additives: Consider incorporating glycerol (5-50%) or reducing agents if the protein contains cysteine residues that might form disulfide bonds.

• Functional assay design:

  • For RNA binding studies: Utilize purified ribosomal RNA or synthetic RNA oligonucleotides corresponding to known L21 binding sites.

  • For ribosome assembly investigations: Design reconstitution experiments incorporating other ribosomal components.

  • For structural studies: Optimize conditions specific to X-ray crystallography, NMR, or cryo-EM methodologies.

• Essential controls:

  • Include appropriate positive controls such as functionally characterized ribosomal proteins.

  • Implement negative controls including denatured protein preparations or buffer-only samples.

  • Consider using L21 proteins from related bacteria for comparative analyses.

• Analytical considerations:

  • Verify protein integrity before experiments using SDS-PAGE or mass spectrometry.

  • Accurately determine protein concentration using absorbance at 280 nm or colorimetric assays.

  • Perform experiments at multiple protein concentrations to establish dose-dependent effects.

• Product-specific recommendations:

  • For reconstituted protein, maintain concentration within 0.1-1.0 mg/mL .

  • For short-term studies, maintain working aliquots at 4°C for maximum of one week .

  • Minimize freeze-thaw cycles by preparing appropriately sized single-use aliquots .

Systematic optimization of these parameters enables development of robust in vitro assays to investigate the biochemical and structural properties of T. whipplei rplU.

What are the challenges in studying T. whipplei rplU function due to its reduced genome?

The study of T. whipplei rplU function presents several unique challenges related to the organism's reduced genome and specialized lifestyle:

• Minimal metabolic network: T. whipplei has a condensed genome of approximately 927,303 bp with notable deficiencies in amino acid metabolism pathways . This reduced metabolic capacity means that rplU functions within a highly streamlined cellular machinery, potentially with adaptations that may not be readily replicated in standard experimental systems.

• Host adaptation specializations: As an obligate intracellular pathogen, T. whipplei may have evolved specialized features in its ribosomal proteins. The organism demonstrates "a reduced capacity for energy metabolism" , suggesting potential adaptations in translation efficiency that could influence rplU function.

• Limited comparative models: As the only known reduced genome species within the Actinobacteria phylum , there are fewer closely related organisms available for meaningful comparative functional studies of rplU.

• Cultivation constraints: T. whipplei "resisted reproducible culture until grown in human fibroblasts in 2000" , making direct functional studies in the native organism technically challenging.

• Genomic plasticity considerations: The presence of "a large chromosomal inversion" between different strains suggests genomic rearrangements that could potentially affect gene expression regulation, including that of rplU.

• Complex host-pathogen interactions: The bacterium exhibits mechanisms for evading host defenses through genome rearrangements, which complicates understanding protein function in the context of infection dynamics.

To address these challenges, researchers might employ heterologous expression systems, comparative genomics across available T. whipplei strains, in vitro reconstitution approaches for ribosomal assembly, and computational modeling to complement experimental findings.

How can I design experiments to explore the role of rplU in T. whipplei pathogenesis?

Investigating the potential role of rplU in T. whipplei pathogenesis requires creative experimental approaches that address the challenges of working with this fastidious organism:

• Infection model systems:

  • Cell culture: Utilize human fibroblasts or macrophages (known to support T. whipplei growth ) to study the effects of targeting rplU expression or function.

  • RNA interference: Develop siRNAs targeting rplU mRNA to assess effects on bacterial survival in cell culture models.

  • Ex vivo systems: Consider intestinal tissue explants to better represent the physiological environment of infection.

• Molecular approach strategies:

  • Antisense oligonucleotides: Design molecules that specifically bind to rplU mRNA to inhibit translation and assess effects on bacterial viability.

  • Ribosome profiling: Analyze ribosome occupancy and translation efficiency patterns in T. whipplei under different conditions.

  • Protein interaction studies: Identify potential binding partners of rplU using techniques such as co-immunoprecipitation or yeast two-hybrid screening.

• Structural and functional investigations:

  • Protein-protein interaction mapping: Use recombinant rplU to identify interactions with host proteins that might contribute to pathogenesis.

  • Subcellular localization: Develop antibodies against rplU to track its localization during different stages of infection.

  • Translation efficiency studies: Assess the impact of rplU on protein synthesis rates using reconstituted translation systems.

• Comparative genomics and transcriptomics:

  • Compare rplU sequences across different T. whipplei strains, particularly those with documented variations in virulence.

  • Analyze rplU expression levels during different stages of infection to identify potential regulatory patterns.

  • Investigate potential regulatory mechanisms controlling rplU expression in response to environmental cues.

• Potential therapeutic targeting:

  • Given T. whipplei's "mutation in DNA gyrase predicting a resistance to quinolone antibiotics" , investigate whether rplU might serve as an alternative drug target.

  • Screen for compounds that specifically inhibit T. whipplei rplU function without affecting human ribosomal proteins.

These experimental strategies can provide insights into whether rplU plays roles beyond protein synthesis in T. whipplei pathogenesis, potentially contributing to our understanding of Whipple's disease pathophysiology.

What methods can be used to study potential interactions between T. whipplei rplU and the host immune system?

Investigating potential interactions between T. whipplei rplU and the host immune system requires multidisciplinary approaches combining immunological, molecular, and cellular techniques:

• Serological and immunological assays:

  • ELISA development: Detect and quantify antibodies against rplU in serum samples from Whipple's disease patients compared to appropriate controls.

  • T-cell response analysis: Assess whether rplU can stimulate T-cell proliferation in peripheral blood mononuclear cells from patients and healthy individuals.

  • Cytokine profiling: Measure cytokine production patterns (particularly IL-1β, TNF-α, IL-6, and IL-10) following recombinant rplU stimulation of immune cells.

• Molecular interaction studies:

  • Co-immunoprecipitation: Identify host immune proteins that directly interact with rplU during infection.

  • Surface plasmon resonance: Quantitatively characterize binding kinetics between rplU and candidate host receptors.

  • Protein-protein interaction screening: Discover novel interactions between rplU and human immune proteins using techniques such as yeast two-hybrid screening.

• Cellular response assays:

  • Macrophage activation analysis: Determine if rplU affects macrophage polarization states (M1/M2) or phagocytic capacity.

  • Dendritic cell functional studies: Evaluate if rplU influences dendritic cell maturation, antigen presentation, or cytokine production.

  • Reporter assays: Determine if rplU activates immune signaling pathways such as NF-κB or inflammasome formation.

• Advanced microscopy approaches:

  • Confocal microscopy: Visualize the localization of rplU during infection using fluorescently labeled antibodies.

  • Proximity ligation assays: Detect protein-protein interactions in situ with high specificity.

  • Live-cell imaging: Track the dynamics of host-pathogen interactions involving rplU in real time.

• Bioinformatic prediction tools:

  • Epitope mapping: Identify potential B-cell and T-cell epitopes within the rplU sequence that might be recognized by the immune system.

  • Molecular docking simulations: Predict potential interactions between rplU and host immune receptors.

These methodologies can help determine whether rplU contributes to T. whipplei's documented ability to evade host defenses , potentially explaining why some individuals develop clinical disease while others remain asymptomatic despite exposure to the bacterium.

How can I use genomic data to compare rplU sequences across different T. whipplei strains?

Comparative analysis of rplU sequences across T. whipplei strains requires systematic bioinformatic approaches that can reveal evolutionary patterns and functional implications:

• Sequence acquisition and alignment methodology:

  • Obtain rplU sequences from public databases (NCBI, UniProt) for available T. whipplei strains, including the well-characterized Twist strain and TW08/27 .

  • Align sequences using multiple sequence alignment algorithms such as MUSCLE, CLUSTAL, or T-Coffee.

  • Visualize and analyze alignments using tools like Jalview or AliView to identify conserved regions and variation hotspots.

• Phylogenetic analysis approaches:

  • Construct phylogenetic trees using Maximum Likelihood, Neighbor-Joining, or Bayesian inference methods.

  • Assess evolutionary relationships between T. whipplei strains based on rplU sequence patterns.

  • Compare the rplU phylogeny with whole-genome phylogenies to identify potential horizontal gene transfer events or gene-specific selection pressures.

• Variation pattern analysis:

  • Calculate nucleotide and amino acid diversity metrics (π, dN/dS ratios) to identify selection signatures.

  • Identify sites under positive or purifying selection using evolutionary models.

  • Correlate observed sequence variations with strain characteristics, such as clinical presentation or geographical origin.

• Structural implication assessment:

  • Map identified sequence variations onto predicted protein structures using homology modeling.

  • Determine whether variations occur in functional domains, RNA-binding regions, or protein-protein interaction sites.

  • Predict how sequence differences might affect protein function or stability.

• Regulatory element comparison:

  • Examine the promoter regions and potential regulatory elements controlling rplU expression across strains.

  • Identify potential differences in gene expression regulation that might influence pathogenicity.

  • Consider the genomic context of rplU, noting that T. whipplei exhibits "frequent genome rearrangements" .

• Contextual genomic analysis:

  • Place rplU variation within the broader context of genomic diversity among T. whipplei strains.

  • Consider the six different T. whipplei subtypes previously identified through 16S-23S rDNA spacer region analysis .

  • Evaluate whether rplU diversity correlates with the "unexpected degree of sequence variation" observed in surface proteins .

This systematic approach to comparing rplU sequences can provide insights into the evolution and adaptation of T. whipplei, potentially revealing correlations between genetic variation and clinical manifestations of Whipple's disease.

What role might rplU play in the unique metabolism of T. whipplei given its genome constraints?

The functional role of rplU in T. whipplei must be considered within the context of the organism's highly constrained genome and specialized metabolic adaptations:

• Translation efficiency optimization:

  • As a 50S ribosomal protein, rplU is fundamental to protein synthesis , which becomes particularly critical in an organism with a reduced genome encoding only 808 predicted proteins .

  • Given T. whipplei's documented "deficiencies in amino acid metabolisms" and "reduced capacity for energy metabolism" , efficient translation becomes essential for resource conservation.

  • The rplU protein may have evolved specific adaptations to optimize translation efficiency under these severe metabolic constraints.

• Nutritional dependency adaptations:

  • T. whipplei's genome lacks "key biosynthetic pathways" , indicating a reliance on host-derived resources.

  • The rplU protein might participate in specialized ribosomal functions that prioritize translation of proteins essential for nutrient acquisition from the host environment.

  • It could potentially contribute to translational regulation under fluctuating nutritional conditions encountered during different stages of infection.

• Stress response mechanisms:

  • The genome notably lacks "clear thioredoxin and thioredoxin reductase homologs" , suggesting altered redox homeostasis mechanisms.

  • The rplU protein might function within ribosomes that have adapted to maintain functionality under oxidative stress or other metabolic challenges.

  • It could potentially participate in stress-specific translation programs that help the bacterium survive hostile host environments.

• Host interaction considerations:

  • Beyond canonical translation functions, rplU might serve moonlighting functions in host interaction.

  • Some bacterial ribosomal proteins have been documented to function as virulence factors or immune modulators in other pathogens.

  • Such secondary functions could be particularly important for a pathogen with a minimal genome where proteins often serve multiple functions.

• Therapeutic targeting implications:

  • Understanding rplU's role in T. whipplei metabolism could identify vulnerabilities for antimicrobial development.

  • Given the organism's documented "mutation in DNA gyrase predicting a resistance to quinolone antibiotics" , alternative targets such as rplU might represent valuable therapeutic opportunities.

Research approaches to investigate these aspects could include comparative structural studies with L21 proteins from bacteria with different metabolic capacities, in vitro translation systems to assess function under varying conditions, and proteomic analyses to identify potential interaction partners specific to T. whipplei.