Recombinant Cenarchaeum symbiosum Protein pelota homolog (pelA)

What is Cenarchaeum symbiosum and why is its pelota homolog significant?

Cenarchaeum symbiosum is a marine archaeon belonging to the Thaumarchaeota phylum, closely related to Nitrosopumilus as indicated in taxonomic studies . It was first isolated as a symbiont of marine sponges and represents one of the first thoroughly studied mesophilic archaea. The pelota homolog (pelA) is particularly significant in understanding translation quality control mechanisms in archaea and potentially provides evolutionary insights into similar mechanisms in eukaryotes.

The protein is preserved in a Tris-based buffer with 50% glycerol and requires storage at -20°C, or -80°C for extended preservation . This recombinant protein is produced specifically for research applications and is not intended for human, veterinary, or therapeutic applications .

How does pelA contribute to cellular function in C. symbiosum?

Based on homology with pelota proteins in other organisms, pelA likely plays a critical role in mRNA surveillance and ribosome recycling pathways. These pathways are essential for maintaining translation fidelity by eliminating defective mRNAs and rescuing stalled ribosomes. Research approaches to investigate this function include:

• In vitro translation assays with purified components

• Ribosome binding studies using gradient centrifugation

• mRNA decay analysis in reconstituted systems

• Protein-protein interaction studies with predicted pathway components

What experimental approaches reveal pelA's role in archaeal translation quality control?

To investigate pelA's role in translation quality control, researchers should consider the following methodological approaches:

• Reconstituted in vitro translation systems incorporating purified recombinant pelA

• Site-directed mutagenesis of conserved residues to identify functional domains

• Cryo-EM structural studies of pelA-ribosome complexes

• Comparative analysis with eukaryotic pelota proteins using chimeric constructs

These approaches should be designed to test specific hypotheses about pelA's molecular function, particularly focusing on its interaction with mRNA surveillance machinery unique to archaeal systems.

How does archaeal pelA compare with eukaryotic pelota homologs?

While the search results don't provide direct comparative data, this research question requires methodological approaches including:

• Sequence alignment and phylogenetic analysis

• Structural superimposition of solved structures

• Functional complementation studies in heterologous systems

• Domain swapping experiments to identify functionally conserved regions

These methodologies allow researchers to address fundamental questions about the evolution of translation quality control mechanisms across domains of life.

What techniques can identify novel binding partners of pelA?

To identify previously unknown protein-protein interactions involving pelA, researchers should consider:

• Affinity purification coupled with mass spectrometry (AP-MS)

• Yeast two-hybrid screening adapted for archaeal proteins

• Protein microarray analysis

• Cross-linking mass spectrometry (XL-MS) to capture transient interactions

Each method offers distinct advantages for capturing different types of interactions, and researchers should consider employing multiple complementary approaches for comprehensive interaction mapping.

What expression systems optimize recombinant pelA production?

The optimal expression system for recombinant pelA production depends on research objectives:

For initial purification, researchers should verify that their recombinant pelA maintains proper folding and activity before proceeding to functional studies.

What buffer conditions optimize pelA stability for functional studies?

Based on the product information, pelA is supplied in a Tris-based buffer with 50% glycerol . For functional studies, researchers should consider:

• Maintaining protein in Tris or HEPES buffers (pH 7.5-8.0) to mimic physiological conditions

• Including stabilizing agents such as glycerol (10-20%) to prevent aggregation

• Adding reducing agents (DTT or β-mercaptoethanol) to maintain cysteine residues

• Incorporating divalent cations (Mg²⁺) that might be required for functional activity

Storage at -20°C is appropriate for short-term use, while -80°C is recommended for long-term storage to maintain protein integrity .

How can researchers validate recombinant pelA activity?

Functional validation of recombinant pelA should include:

• Ribosome binding assays to confirm interaction with translation machinery

• ATPase activity measurements if pelA exhibits predicted enzymatic function

• mRNA substrate binding studies using fluorescence anisotropy or electrophoretic mobility shift assays

• In vitro reconstitution of translation quality control using defined components

These assays provide complementary information about different aspects of pelA function and should be selected based on specific research questions.

What controls are essential for pelA functional studies?

Rigorous experimental design for pelA studies should include:

• Negative controls:

  • Buffer-only controls without pelA

  • Inactive mutant versions of pelA (with mutations in predicted functional domains)

  • Heterologous proteins of similar size and charge properties

• Positive controls:

  • Well-characterized pelota homologs from model organisms

  • Native (non-recombinant) pelA when available

  • Parallel experiments with established components of the quality control machinery

• Validation controls:

  • Multiple independent protein preparations

  • Concentration gradients to establish dose-dependence

  • Time-course experiments to capture kinetic parameters

How should researchers design experiments to investigate temperature-dependent activity of archaeal pelA?

C. symbiosum is a mesophilic archaeon, suggesting its proteins function optimally at moderate temperatures. To investigate temperature-dependent activity:

• Perform activity assays across a temperature range (4-60°C)

• Monitor protein stability using circular dichroism at different temperatures

• Compare activity profiles with pelota homologs from thermophilic and psychrophilic archaea

• Correlate temperature optima with the ecological niche of C. symbiosum

Data should be presented as temperature-activity profiles with appropriate statistical analysis to identify significant temperature effects.

How can researchers reconcile contradictory findings about pelA function?

When facing contradictory results regarding pelA function, researchers should:

• Systematically compare experimental conditions between studies

• Evaluate differences in protein preparation methods that might affect activity

• Consider species-specific differences if comparing pelA homologs

• Perform side-by-side experiments with standardized protocols

A comprehensive meta-analysis approach using the following framework helps resolve contradictions:

What statistical approaches are appropriate for pelA functional data analysis?

When analyzing functional data for recombinant pelA:

• Use appropriate replication (minimum n=3) for all experimental conditions

• Apply parametric tests (t-test, ANOVA) for normally distributed data

• Consider non-parametric alternatives when assumptions of normality are violated

• Report effect sizes alongside p-values to indicate biological significance

• Use multiple comparison corrections (e.g., Bonferroni) when testing numerous hypotheses

Experimental data should be presented with clear statistical annotations and appropriate graphical representations that accurately depict both central tendency and variability.

How can researchers address solubility issues with recombinant pelA?

Archaeal proteins often present solubility challenges when expressed in heterologous systems. Methodological approaches include:

• Optimize expression conditions:

  • Reduce induction temperature (16-20°C)

  • Use weaker promoters to slow expression rate

  • Co-express molecular chaperones

• Modify protein constructs:

  • Create truncated versions focusing on functional domains

  • Remove hydrophobic regions that may cause aggregation

  • Add solubility tags (MBP, SUMO, TRX)

• Adjust purification protocols:

  • Include stabilizing agents (glycerol, low concentrations of denaturants)

  • Use higher salt concentrations in buffers

  • Incorporate detergents for partially hydrophobic proteins

What strategies help resolve activity loss during purification and storage?

To maintain pelA activity throughout purification and storage:

• Minimize freeze-thaw cycles by aliquoting protein after initial purification

• Store in appropriate conditions (-20°C for short-term, -80°C for long-term storage)

• Include stabilizing agents in storage buffer (50% glycerol as indicated in product specifications)

• Add protease inhibitors to prevent degradation

• Consider protein-specific stabilizing factors identified through thermal shift assays