Recombinant Chlamydomonas reinhardtii 40S ribosomal protein S18 (RPS18)

What is Chlamydomonas reinhardtii RPS18 and what is its significance in molecular research?

Chlamydomonas reinhardtii RPS18 (Ribosomal Protein S18) is a core component of the 40S ribosomal subunit in this model green alga. The protein consists of 153 amino acids and plays an essential role in ribosome assembly and protein translation. The significance of RPS18 extends beyond its structural role in ribosomes, as evidenced by differential expression patterns during stress responses. In the hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5), RPS18 shows significant upregulation (3.07-fold) compared to wild type, suggesting its involvement in adaptive responses to stress conditions . For researchers, this makes RPS18 an interesting target for studying translation regulation during environmental adaptation in photosynthetic organisms.

How does Chlamydomonas reinhardtii RPS18 compare to RPS18 from other organisms?

The search results indicate that RPS18 proteins from various organisms including human, rat, pig, goat, E. coli, Cyanophora paradoxa, and Cuscuta reflexa are available as recombinant proteins . These RPS18 variants exhibit different amino acid lengths and host expression systems, allowing researchers to select the most appropriate version for comparative studies. When designing experiments involving multiple RPS18 orthologs, researchers should account for these variations in experimental design and data interpretation.

What expression systems are most effective for producing recombinant Chlamydomonas reinhardtii RPS18?

The yeast protein expression system is generally considered the most economical and efficient eukaryotic system for expression of Chlamydomonas proteins, including RPS18 . This system provides several advantages for researchers:

• It maintains eukaryotic post-translational modifications that may be important for protein function

• It offers good protein folding environment for complex proteins

• It allows for both secretion and intracellular expression

• It provides high yield-to-cost ratio compared to mammalian systems

For Chlamydomonas reinhardtii RPS18 specifically, yeast expression has been successfully employed to produce recombinant protein with >90% purity, suitable for ELISA and other applications . Alternative expression systems include E. coli, which may be preferred when rapid production is prioritized over eukaryotic modifications. When higher protein quality is required (though at greater expense), mammalian cell systems can be considered, particularly for functional studies where native-like properties are essential.

What purification strategies yield the highest purity for recombinant Chlamydomonas RPS18?

For high-purity recombinant Chlamydomonas reinhardtii RPS18, affinity chromatography using tagged protein versions offers the most efficient purification strategy. Based on the available information, His-tagged RPS18 can be purified to >90% purity using immobilized metal affinity chromatography (IMAC) . The purification workflow typically involves:

• Cell lysis under optimized buffer conditions to maintain protein stability

• Clarification of lysate by centrifugation and/or filtration

• IMAC purification using Ni-NTA or similar resin

• Washing steps with gradually increasing imidazole concentrations

• Elution with high imidazole concentration

• Buffer exchange to remove imidazole and stabilize the purified protein

For applications requiring even higher purity, researchers should consider implementing additional purification steps such as ion exchange chromatography or size exclusion chromatography. The choice between His-tag and GST-tag versions should be based on specific experimental requirements, with His-tagged versions typically preferred for structural studies due to the smaller tag size, while GST-tagged versions may offer better solubility for certain applications.

What analytical methods are recommended for verifying the identity and quality of recombinant RPS18?

A comprehensive quality assessment of recombinant Chlamydomonas reinhardtii RPS18 should employ multiple complementary analytical techniques:

• SDS-PAGE analysis: For purity assessment and approximate molecular weight confirmation (approximately 18 kDa plus tag size)

• Western blotting: Using anti-His or anti-RPS18 antibodies to confirm identity

• Mass spectrometry: For precise molecular weight determination and sequence verification through peptide mapping

• Circular dichroism: To evaluate secondary structure and proper folding

• Dynamic light scattering: To assess homogeneity and detect aggregation

• Functional assays: Such as RNA binding assays to confirm biological activity

Researchers should establish acceptance criteria for each analytical method based on their specific application requirements. For instance, structural studies may require >95% purity and monodispersity, while certain biochemical assays might be successful with >90% purity . Batch-to-batch consistency is crucial for reproducible experiments, so researchers should maintain detailed records of analytical results for each preparation.

How is RPS18 expression altered during stress responses in Chlamydomonas reinhardtii?

Proteomic analyses reveal that RPS18 expression undergoes significant modulation during stress responses in Chlamydomonas reinhardtii. In the PGR5-deficient hpm91 mutant under conditions promoting hydrogen photoproduction, RPS18 shows a 3.07-fold upregulation compared to wild type (p-value 4.63E-02) . This differential expression pattern is part of a broader ribosomal protein response, as shown in the following table:

The coordinated upregulation of these ribosomal proteins suggests a reprogramming of the translation machinery during stress adaptation. Researchers investigating stress responses should consider examining RPS18 as part of a broader ribosomal protein signature. Time-course experiments monitoring RPS18 expression at multiple points during stress application can provide insights into the dynamics of translational adaptation mechanisms.

What role might RPS18 play in hydrogen production pathways in Chlamydomonas reinhardtii?

The significant upregulation of RPS18 in the hydrogen-producing hpm91 mutant suggests potential involvement in pathways supporting sustained hydrogen photoproduction. Several hypotheses merit investigation:

• RPS18 may participate in selective translation of proteins required for hydrogen metabolism

• Elevated RPS18 levels might contribute to modifying ribosome composition for stress adaptation

• RPS18 upregulation could be part of a mechanism to maintain protein synthesis capacity during sulfur deprivation

Research data shows that hpm91 mutants maintain lower reactive oxygen species (ROS) levels compared to wild type during hydrogen production . The connection between elevated ribosomal proteins like RPS18 and ROS management represents an intriguing research question. Researchers could design experiments using RPS18 knockdown or overexpression to directly test its impact on hydrogen production efficiency and ROS accumulation.

How can researchers utilize recombinant RPS18 to study ribosome assembly and function in Chlamydomonas?

Recombinant Chlamydomonas reinhardtii RPS18 provides a valuable tool for in vitro studies of ribosome assembly and function. Researchers can employ the following methodological approaches:

• Reconstitution studies: Using purified recombinant RPS18 along with other ribosomal proteins and rRNA to study assembly intermediates and order of assembly

• Binding partner identification: Employing His-tagged RPS18 in pull-down assays followed by mass spectrometry to identify interaction partners

• Structural studies: Utilizing purified RPS18 for crystallography or cryo-EM studies of subcomplex assembly

• Translation assays: Developing in vitro translation systems with native or modified RPS18 to assess functional impacts

When designing these experiments, researchers should consider using both wild-type RPS18 and strategic mutants based on conserved residues identified through sequence alignments. Comparative studies between Chlamydomonas RPS18 and orthologs from other species can provide evolutionary insights into ribosome specialization in photosynthetic organisms.

What are common challenges when working with recombinant Chlamydomonas reinhardtii RPS18 and how can they be addressed?

Researchers working with recombinant Chlamydomonas reinhardtii RPS18 may encounter several technical challenges that can be addressed through strategic optimization:

• Solubility issues: RPS18 may show limited solubility when expressed without its ribosomal RNA partners. This can be improved by:

  • Optimizing expression temperature (typically lowering to 16-18°C)

  • Adding solubility enhancers like sorbitol or arginine to lysis buffers

  • Using fusion tags like GST that enhance solubility

  • Co-expressing with binding partners or chaperones

• Aggregation during storage: To minimize aggregation:

  • Include 5-10% glycerol in storage buffers

  • Maintain moderate ionic strength (150-300 mM NaCl)

  • Store at concentrations below 1 mg/ml

  • Add reducing agents like DTT or TCEP if cysteine residues are present

• Functional activity assessment: As a ribosomal protein, RPS18's natural function depends on interactions with rRNA and other proteins. Researchers can develop surrogate activity assays focusing on:

  • RNA binding capacity using fluorescence anisotropy

  • Correct folding assessment through limited proteolysis

  • Interaction studies with known binding partners

When troubleshooting expression or purification issues, systematic documentation of conditions and outcomes will facilitate optimization. The expression system choice significantly impacts results - while yeast systems provide good balance for Chlamydomonas proteins, E. coli systems may require more extensive optimization but can ultimately yield higher quantities .

How can researchers optimize experimental conditions for functional studies of RPS18 in photosynthetic research?

For functional studies integrating RPS18 into broader photosynthetic research contexts, several optimization strategies should be considered:

• Buffer composition: When studying RPS18 in relation to photosynthetic complexes, buffers should be designed to maintain stability of both ribosomal and photosynthetic components:

  • Consider using buffers containing glycerol (10-15%)

  • Include mild non-ionic detergents when membrane components are involved

  • Maintain physiologically relevant magnesium concentrations (5-10 mM)

  • Test both reducing and non-reducing conditions depending on the experimental context

• Experimental timing: For studies examining RPS18's role during stress responses:

  • Establish clear time-course sampling points based on known stress response phases

  • Consider multiple stress intensities to capture threshold effects

  • Include recovery periods to assess reversibility of observed changes

• Control selection: Proper controls are critical for interpreting RPS18 functional data:

  • Include both wild-type cells and PGR5-deficient mutants as in the study showing RPS18 upregulation

  • Consider multiple reference genes when performing expression analysis

  • Include appropriate negative controls for interaction studies (unrelated proteins with similar properties)

The integration of RPS18 studies with photosynthetic research benefits from combined approaches examining both protein level changes (as seen in the proteomic data showing 3.07-fold upregulation in hpm91 ) and functional consequences through physiological measurements.

What emerging techniques could advance understanding of RPS18 function in Chlamydomonas reinhardtii?

Several cutting-edge techniques show promise for deepening our understanding of RPS18 functions in Chlamydomonas reinhardtii:

• Ribosome profiling: This technique can reveal how changes in RPS18 expression affect translation of specific mRNAs during stress responses like those observed in hydrogen-producing conditions. This would provide insights beyond the observed 3.07-fold upregulation to identify downstream impacts on cellular physiology.

• Cryo-electron microscopy: High-resolution structural studies of Chlamydomonas ribosomes with and without stress conditions could reveal conformational changes or compositional differences involving RPS18, potentially explaining the functional significance of its upregulation during stress.

• CRISPR-Cas9 genome editing: Creation of precise RPS18 mutants in Chlamydomonas could allow structure-function studies in vivo, particularly to test hypotheses about its role in stress adaptation and hydrogen production pathways.

• Proximity labeling approaches: Techniques like BioID or APEX2 fused to RPS18 could identify stress-specific interaction partners that change during conditions like those in the hpm91 mutant with enhanced hydrogen production.

• Single-cell proteomics: This emerging technology could reveal cell-to-cell variability in RPS18 expression and its correlation with stress resistance heterogeneity within populations.

Researchers adopting these approaches should design experiments that connect molecular-level RPS18 properties with broader cellular phenotypes, particularly focusing on the relationship between ribosomal adaptation and stress response mechanisms.

How might RPS18 contribute to biotechnology applications involving Chlamydomonas reinhardtii?

The differential expression of RPS18 in stress-resistant phenotypes suggests several potential biotechnology applications:

• Biomarker development: The 3.07-fold upregulation of RPS18 in hydrogen-producing strains suggests its potential use as a molecular marker for screening high-efficiency hydrogen production strains. Researchers could develop rapid assays for RPS18 levels as a proxy for stress adaptation capacity.

• Synthetic biology platforms: Engineering strains with modified RPS18 expression could potentially enhance translation efficiency of desired proteins during industrial applications, particularly under bioreactor stress conditions.

• Stress-resistant strain development: Understanding the relationship between RPS18 upregulation and lower ROS levels observed in hpm91 strains could guide genetic engineering approaches to develop algal strains with enhanced tolerance to industrial conditions.

• Biofuel optimization: The connection between ribosomal protein expression patterns and hydrogen production efficiency suggests that manipulating translation through RPS18 and related proteins might offer novel approaches to improving biofuel yields.

These applications require integration of basic research findings about RPS18 regulation with applied biotechnology approaches. Researchers should consider both direct genetic manipulation of RPS18 and broader approaches targeting the regulatory networks controlling ribosomal protein expression during stress responses.