Recombinant Ostreococcus lucimarinus 40S ribosomal protein S3a (OSTLU_28528)

What is Ostreococcus lucimarinus 40S ribosomal protein S3a (OSTLU_28528)?

OSTLU_28528 is a recombinant protein corresponding to the 40S ribosomal protein S3a from Ostreococcus lucimarinus (strain CCE9901). It is part of the small ribosomal subunit and plays an essential role in protein synthesis. The full-length mature protein consists of 259 amino acids (residues 2-260) and has been successfully expressed in E. coli expression systems to produce the recombinant version with high purity (>85% as determined by SDS-PAGE) .

What database identifiers are associated with this protein?

The protein is associated with several key identifiers that can be used for further research and reference:

• UniProt Accession Number: A4SAD2

• Product Code (for commercially available recombinant): CSB-EP020444ODL

• Gene name: OSTLU_28528

What are the optimal storage conditions for OSTLU_28528 recombinant protein?

For optimal stability of OSTLU_28528 recombinant protein, store at -20°C for regular storage. For extended storage periods, it is recommended to conserve the protein at -20°C or -80°C. Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing cycles should be avoided as they can compromise protein stability and activity .

How should OSTLU_28528 recombinant protein be reconstituted for experimental use?

For proper reconstitution of OSTLU_28528, follow these methodological steps:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the default recommendation)

• Aliquot the reconstituted protein for long-term storage at -20°C/-80°C

This procedure helps maintain protein stability and prevents degradation during experimental procedures .

What is the expected shelf life of OSTLU_28528 recombinant protein?

The shelf life of OSTLU_28528 varies depending on storage form and conditions:

• Liquid form: Approximately 6 months when stored at -20°C/-80°C

• Lyophilized form: Approximately 12 months when stored at -20°C/-80°C

It's important to note that shelf life is influenced by multiple factors including:

• Storage state (liquid vs. lyophilized)

• Buffer ingredients

• Storage temperature

• Intrinsic stability of the protein itself

How should I design experiments to study OSTLU_28528 protein function?

When designing experiments to study OSTLU_28528 function, follow these methodological steps:

• Define your research question and hypotheses clearly (e.g., "How does OSTLU_28528 interact with other ribosomal proteins?")

• Identify your variables:

  • Independent variables: Factors you will manipulate (e.g., protein concentration, buffer conditions)

  • Dependent variables: Outcomes you will measure (e.g., binding affinity, structural changes)

  • Control variables: Factors you will keep constant

• Control for extraneous variables that might confound your results

• Select appropriate experimental methods based on your specific questions (e.g., pull-down assays, circular dichroism, thermal shift assays)

• Plan for statistical analysis of your results to ensure reliability and validity

This systematic approach allows for rigorous testing of hypotheses regarding OSTLU_28528 function while minimizing experimental bias and variability .

What experimental approaches are best for studying protein-protein interactions involving OSTLU_28528?

To investigate protein-protein interactions of OSTLU_28528, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Utilize the tag on the recombinant protein (tag type determined during manufacturing)

  • Pull down OSTLU_28528 and identify interacting partners via mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize OSTLU_28528 on a sensor chip

  • Measure real-time binding kinetics with potential interaction partners

• Proximity Ligation Assay (PLA):

  • Detect protein interactions in situ with high specificity

  • Particularly useful for confirming interactions identified through other methods

• Yeast Two-Hybrid (Y2H) screening:

  • Systematic screening for potential interaction partners

  • Requires subcloning OSTLU_28528 into appropriate vectors

Each method has specific advantages and limitations, and combining multiple approaches provides more robust evidence of genuine interactions .

How can I assess the impact of post-translational modifications on OSTLU_28528 function?

To evaluate the role of post-translational modifications (PTMs) on OSTLU_28528, implement these methodological strategies:

• Identification of PTM sites:

  • Perform mass spectrometry analysis to identify potential modification sites

  • Compare with predicted PTM sites using bioinformatics tools

• Site-directed mutagenesis:

  • Generate mutants where potential PTM sites are altered

  • Express and purify these mutants following the same protocols as wild-type

• Functional comparison:

  • Design assays comparing wild-type and mutant proteins

  • Measure parameters such as:

    • Binding affinities

    • Structural stability

    • Ribosomal incorporation efficiency

• In vivo validation:

  • Develop cellular assays to assess the biological relevance of identified PTMs

  • Consider using CRISPR/Cas9 to introduce mutations at PTM sites in the native gene

This systematic approach allows researchers to establish causal relationships between specific modifications and functional outcomes .

What challenges might arise when expressing OSTLU_28528 in different expression systems, and how can they be addressed?

Expression of OSTLU_28528 in different systems presents specific challenges that can be addressed through these methodological approaches:

For OSTLU_28528 specifically, E. coli expression has been successfully demonstrated, but other systems may be explored based on specific research needs and goals .

How can I design experiments to investigate the role of OSTLU_28528 in ribosome assembly and function?

To investigate OSTLU_28528's role in ribosome assembly and function, implement these methodological approaches:

• Reconstitution experiments:

  • Perform in vitro ribosome assembly with and without OSTLU_28528

  • Analyze assembly intermediates using sucrose gradient centrifugation

  • Quantify assembly efficiency under various conditions

• Structure-function studies:

  • Generate domain deletion or point mutation variants

  • Assess their impact on:

    • Ribosome assembly

    • Translation efficiency

    • Interaction with specific rRNAs or proteins

• Cryo-electron microscopy:

  • Determine structural changes in ribosomes with wild-type vs. mutant OSTLU_28528

  • Identify precise positioning and contacts within the ribosomal complex

• Translation assays:

  • Measure translation rates and fidelity using reporter systems

  • Compare systems with wild-type vs. depleted or mutant OSTLU_28528

These approaches provide complementary insights into both structural and functional aspects of OSTLU_28528's role in ribosome biology .

What statistical approaches should I use when analyzing data from experiments with OSTLU_28528?

For robust statistical analysis of OSTLU_28528 experimental data, follow these methodological guidelines:

• Experimental design considerations:

  • Ensure adequate replication (minimum n=3 for most experiments)

  • Include appropriate controls (positive, negative, vehicle)

  • Consider power analysis to determine sample size

• Data preprocessing:

  • Assess normality of data distribution (Shapiro-Wilk or Kolmogorov-Smirnov tests)

  • Check for outliers and determine appropriate handling

  • Transform data if necessary to meet assumptions of parametric tests

• Statistical test selection:

  • For comparing two groups: t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple groups: ANOVA with appropriate post-hoc tests (Tukey's, Bonferroni, etc.)

  • For correlation analysis: Pearson's (parametric) or Spearman's (non-parametric)

• Advanced analyses for complex experiments:

  • Repeated measures designs: RM-ANOVA or mixed models

  • Dose-response experiments: Nonlinear regression analysis

  • High-dimensional data: Principal component analysis or other dimensionality reduction methods

How can I interpret contradictory results in OSTLU_28528 functional studies?

When faced with contradictory results in OSTLU_28528 studies, apply this systematic framework:

• Methodological differences assessment:

  • Compare experimental conditions (pH, temperature, buffer composition)

  • Evaluate protein preparation methods (tags, purification protocols)

  • Assess expression systems used (bacterial vs. eukaryotic)

• Technical validation:

  • Verify protein quality (purity, aggregation state, activity)

  • Confirm antibody specificity if immunological methods were used

  • Check for batch-to-batch variations in reagents

• Biological context considerations:

  • Examine cell/tissue type differences

  • Consider developmental or physiological states

  • Evaluate potential species-specific differences

• Integration of multiple techniques:

  • Compare in vitro vs. in vivo findings

  • Triangulate results using orthogonal methods

  • Weight evidence based on methodological rigor

• Computational analysis:

  • Perform meta-analysis if multiple datasets are available

  • Use machine learning approaches to identify patterns not obvious in individual experiments

This comprehensive approach helps resolve apparent contradictions and develops a more nuanced understanding of OSTLU_28528 biology .

How might OSTLU_28528 be used in structural biology studies of the eukaryotic ribosome?

OSTLU_28528 can be strategically employed in structural biology through these methodological approaches:

• Cryo-EM studies:

  • Use purified OSTLU_28528 in ribosome reconstitution experiments

  • Generate ribosomes with labeled or modified S3a for localization

  • Compare structures with wild-type vs. mutant proteins to identify conformational changes

• X-ray crystallography:

  • Attempt crystallization of OSTLU_28528 alone or in complex with interacting partners

  • Use selenomethionine-labeled protein for phase determination

  • Employ surface entropy reduction to improve crystal quality

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map dynamic regions and binding interfaces

  • Compare solvent accessibility patterns in free vs. ribosome-bound states

  • Identify allosteric effects of ligand binding

• Integrative structural biology:

  • Combine data from multiple techniques (cryo-EM, NMR, SAXS, etc.)

  • Develop computational models incorporating experimental constraints

  • Validate models through directed mutagenesis and functional assays

These approaches can reveal insights into both the structure of OSTLU_28528 itself and its contribution to ribosome architecture and function .

What are the considerations for designing target engagement studies for OSTLU_28528?

When designing target engagement studies for OSTLU_28528, implement these methodological principles:

• Assay development:

  • Establish quantitative methods to measure free vs. bound OSTLU_28528

  • Consider fluorescence polarization, thermal shift, or ELISA-based approaches

  • Validate assays using known interaction partners

• Experimental design:

  • Define clear hypotheses about target binding

  • Include appropriate controls (non-binding mutants, competitive inhibitors)

  • Plan for concentration-response relationships

• Sample collection and preparation:

  • For cellular studies, optimize lysis conditions to preserve interactions

  • For tissue studies, consider rapid preservation methods

  • For in vivo studies, develop appropriate sampling timelines

• Analysis considerations:

  • Calculate target engagement parameters (Kd, Bmax, etc.)

  • Assess engagement kinetics if time-course data is available

  • Compare engagement across different experimental conditions

These approaches allow researchers to quantitatively assess how OSTLU_28528 interacts with its biological targets under various conditions .