Recombinant Kluyveromyces marxianus 40S ribosomal protein S28 (RPS28)

What is the structural organization of K. marxianus RPS28 and how does it compare to other species?

K. marxianus RPS28 belongs to the family of small ribosomal proteins that are components of the 40S ribosomal subunit. Based on comparative analysis with other eukaryotic S28 proteins, it likely contains a globular region consisting of antiparallel β-strands arranged in a Greek-key topology, similar to the S28E structure determined for Methanobacterium thermoautotrophicum . The protein typically features:

• A core β-barrel structure that forms the globular region

• A C-terminal tail that protrudes from the core, which is often flexible and charged

• Surface features that facilitate RNA binding, including positively charged residues

The K. marxianus RPS28 protein comprises 67 amino acids, which is comparable to other yeast S28 proteins . Sequence analysis shows high conservation among archaeal and eukaryotic organisms, suggesting evolutionary importance in protein synthesis mechanisms .

What expression systems are optimal for producing recombinant K. marxianus RPS28?

For optimal expression of recombinant K. marxianus RPS28, researchers should consider:

Yeast Expression Systems:

• Homologous expression in K. marxianus itself may provide proper folding and post-translational modifications

• The promoter selection is critical; constitutive promoters like PDC1pr and TEF1pr provide strong expression at 30°C

• For temperature-regulated expression, heat-inducible promoters (HSP104pr, SSA2pr, TSA1pr) offer varying levels of induction at elevated temperatures

Expression Parameters Table:

For recombinant production, the combination of a strong promoter with the INU1 terminator has shown efficient expression in K. marxianus systems . Temperature induction offers a unique advantage for K. marxianus systems due to the organism's thermotolerance .

What purification strategies yield the highest purity and activity for recombinant K. marxianus RPS28?

Purification of recombinant K. marxianus RPS28 can be optimized using the following methodological approach:

• Affinity Chromatography:

  • His-tagged RPS28 can be efficiently purified using Ni-NTA or IMAC chromatography

  • Buffer optimization is critical; typically, phosphate or Tris buffers (pH 7.5-8.0) with 150-300 mM NaCl

  • Include protease inhibitors to prevent degradation during extraction

• Secondary Purification:

  • Ion exchange chromatography can further separate the protein from contaminants

  • Size exclusion chromatography helps remove aggregates and yields >90% purity

• Quality Assessment:

  • SDS-PAGE and Western blotting for purity verification

  • Mass spectrometry for confirmation of protein identity

  • Circular dichroism to assess proper folding

  • RNA binding assays to confirm functional activity

The purification protocol should be tailored to maintain the native structure, particularly the β-barrel and flexible C-terminal region that are critical for RNA binding functionality .

How can researchers use CRISPR/Cas9 technology to modify the RPS28 gene in K. marxianus?

Implementation of CRISPR/Cas9 for RPS28 gene modification in K. marxianus should follow this methodological framework:

• gRNA Design:

  • Design gRNAs targeting the RPS28 locus with high specificity

  • Use available K. marxianus genome data to ensure minimal off-target effects

  • Consider the target region's accessibility within the chromatin structure

• Delivery System:

  • Utilize a single-plasmid CRISPR/Cas9 platform optimized for K. marxianus

  • Transform approximately 300 ng of gRNA expression plasmid using the LiOAc/PEG method

• Screening Protocol:

  • After 48-72 hours of growth following transformation, screen hygromycin-resistant colonies

  • Use colony PCR and sequencing to identify mutations at the targeted locus

  • If creating an observable phenotype, replica-plate to appropriate medium for pre-screening

• Genetic Background Considerations:

  • NHEJ-deficient backgrounds improve efficiency; inactivating YKU80 or DNL4 eliminates multiple or random integration

  • Use auxotrophic strains to expand the range of selection markers available

When editing multiple genes, sequential mutation is recommended, with verification of each modification before proceeding to the next target . This approach maximizes efficiency while minimizing unintended genomic alterations.

What techniques are most effective for studying RPS28-RNA interactions in K. marxianus?

To effectively study RPS28-RNA interactions in K. marxianus, researchers should employ these methodological approaches:

• In Vitro RNA Binding Assays:

  • Electrophoretic Mobility Shift Assays (EMSA) to detect protein-RNA complexes

  • Filter binding assays to determine binding constants

  • Surface Plasmon Resonance (SPR) for real-time binding kinetics

• Structural Studies:

  • Nuclear Magnetic Resonance (NMR) spectroscopy, as successfully used for S28E from M. thermoautotrophicum

  • X-ray crystallography of RPS28-RNA complexes

  • Cryo-electron microscopy of intact ribosomes to visualize RPS28 in its native context

• Cross-linking Approaches:

  • UV cross-linking followed by immunoprecipitation (CLIP)

  • Chemical cross-linking coupled with mass spectrometry (CXMS)

  • Ribosome profiling to identify RPS28-associated mRNAs

When designing these experiments, it's important to consider that RPS28 contains a positively charged surface that extends over the β-barrel and into the flexible C-terminus, which likely represents the RNA binding site . The C-terminal tail contains a conserved signature sequence motif that may form an α-helix upon RNA interaction , making this region of particular interest.

How can researchers assess the impact of RPS28 mutations on ribosome assembly and function?

To systematically evaluate the effects of RPS28 mutations on ribosome assembly and function in K. marxianus, implement this methodological framework:

These approaches enable comprehensive characterization of how specific residues in RPS28 contribute to ribosome assembly and function, providing insights into the molecular mechanisms underlying its role in translation.

What are the best strategies for integrating K. marxianus RPS28 into synthetic biology applications?

For effective integration of K. marxianus RPS28 into synthetic biology applications, researchers should consider:

• Standardized Assembly Systems:

  • Use the MoClo/Yeast Tool Kit standard for cloning and rapid construction

  • Store parts according to established standards for quick sharing and reproducibility

  • Utilize characterized promoters and terminators for fine-tuned expression control

• Expression Optimization:

  • Select appropriate promoters based on desired expression patterns:

    • Constitutive expression: PDC1pr and TEF1pr

    • Temperature-inducible expression: HSP104pr, SSA2pr, and TSA1pr

  • Pair with efficient terminators such as INU1 terminator for optimal expression

• Integration Approaches:

  • Use characterized chromosomal integration sites selected for efficiency or visible phenotypes

  • Consider centromeric plasmids with kanMX cassettes to minimize variations in expression due to copy number

  • Implement CRISPR/Cas9 platform for precise genome engineering

• Functional Applications:

  • Design RPS28 variants with enhanced RNA binding properties

  • Explore potential roles in specialized ribosome development

  • Investigate applications related to thermotolerance and aging mechanisms

K. marxianus offers unique advantages as a host organism due to its thermotolerance and potential as a next-generation cell factory for bio-based chemicals . By combining rigorous characterization of RPS28 with standardized synthetic biology approaches, researchers can develop innovative applications in metabolic engineering and biotechnology.

How should researchers design experiments to compare the function of K. marxianus RPS28 with homologs from other species?

To effectively compare K. marxianus RPS28 with homologs from other species, implement this systematic experimental design:

• Sequence and Structural Analysis:

  • Perform multiple sequence alignment of RPS28 proteins across species

  • Generate phylogenetic trees to understand evolutionary relationships

  • Create comparative structural models based on known structures such as S28E from M. thermoautotrophicum

• Functional Complementation Assays:

  • Design a complementation system using RPS28-depleted cells

  • Express RPS28 from different species (e.g., K. marxianus, S. cerevisiae, mammals)

  • Assess growth rates, translation efficiency, and ribosome assembly

  • Measure rescue of phenotypes associated with RPS28 deficiency

• Chimeric Protein Analysis:

  • Create chimeric proteins swapping domains between K. marxianus RPS28 and homologs

  • Focus on the β-barrel region and the C-terminal tail separately

  • Assess which regions confer species-specific functions

• Environmental Response Assessment:

  • Test function under different conditions (temperature, pH, stress)

  • Determine if K. marxianus RPS28 confers thermotolerance advantages

  • Compare expression patterns during stress response or aging

This comprehensive approach allows identification of conserved and species-specific aspects of RPS28 function, providing insights into ribosomal protein evolution and specialized adaptations in K. marxianus.

What controls and validation steps are essential when characterizing recombinant K. marxianus RPS28 activity?

Rigorous characterization of recombinant K. marxianus RPS28 requires these essential controls and validation steps:

• Expression and Purification Controls:

  • Empty vector controls to account for host cell background

  • Untagged protein controls to assess tag interference

  • Alternative tag positions (N-terminal vs. C-terminal) to minimize functional disruption

  • Purification from different expression systems to compare post-translational modifications

• Structural Validation:

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Size exclusion chromatography to verify monodispersity

  • Limited proteolysis to assess domain organization

  • Mass spectrometry to confirm protein integrity and modifications

• Functional Validation:

  • RNA binding assays with specific and non-specific RNA substrates

  • Ribosome incorporation assays to confirm assembly competence

  • In vitro translation assays to assess functional impact

  • Comparative analysis with commercially available RPS28 proteins (>90% purity)

• Negative Controls for Specificity:

  • Mutated RPS28 lacking key functional residues

  • Heterologous ribosomal proteins of similar size

  • Heat-denatured protein samples

  • Competition assays with unlabeled proteins

These controls and validation steps ensure that observed activities are specifically attributable to properly folded and functional K. marxianus RPS28, minimizing artifacts and enabling confident interpretation of experimental results.

What are common challenges in expressing recombinant K. marxianus RPS28 and how can they be addressed?

Researchers frequently encounter these challenges when expressing recombinant K. marxianus RPS28, along with recommended solutions:

• Low Expression Levels:

  • Problem: Ribosomal proteins often have low expression due to tight regulation

  • Solution: Optimize codon usage for the expression host

  • Solution: Use strong promoters such as PDC1pr or TEF1pr for constitutive expression

  • Solution: Consider inducible systems like heat-inducible promoters (HSP104pr, SSA2pr, TSA1pr)

• Protein Solubility Issues:

  • Problem: Aggregation due to exposure of RNA-binding surfaces

  • Solution: Express with solubility-enhancing tags (SUMO, MBP)

  • Solution: Optimize buffer conditions (add low concentrations of RNA)

  • Solution: Use lower induction temperatures for heterologous expression

• Proteolytic Degradation:

  • Problem: Small ribosomal proteins are often targets for proteases

  • Solution: Include protease inhibitor cocktails during purification

  • Solution: Use protease-deficient expression strains

  • Solution: Optimize purification workflow to minimize handling time

• Improper Folding:

  • Problem: Incorrect disulfide bond formation or protein misfolding

  • Solution: Express in yeast rather than bacterial systems for proper eukaryotic folding

  • Solution: Consider addition of molecular chaperones

  • Solution: Use native K. marxianus expression system for authentic folding

• Purification Challenges:

  • Problem: Co-purification with endogenous RNA

  • Solution: Include high-salt washes (500-750 mM NaCl) during purification

  • Solution: Add RNase treatment steps where appropriate

  • Solution: Use size exclusion chromatography as a final polishing step

Implementing these troubleshooting strategies will significantly improve the yield and quality of recombinant K. marxianus RPS28 for research applications.

How can researchers resolve discrepancies in experimental results when studying K. marxianus RPS28?

When faced with discrepant results in K. marxianus RPS28 research, implement this systematic troubleshooting approach:

• Source of Variation Identification:

  • Protein Heterogeneity: Verify protein batch consistency using SDS-PAGE and mass spectrometry

  • Strain Variations: Confirm K. marxianus strain identity and genetic background

  • Environmental Conditions: Standardize growth temperature, media composition, and induction parameters

  • Assay Conditions: Document and control buffer compositions, incubation times, and temperatures

• Methodological Standardization:

  • Protein Quantification: Use multiple methods (Bradford, BCA, A280) to verify concentrations

  • Activity Assays: Include internal controls and standards in each experiment

  • Data Analysis: Apply consistent normalization and statistical methods

  • Technical Replicates: Ensure sufficient replication to assess experimental variability

• Systematic Validation Approaches:

  • Independent Methods: Confirm key findings using orthogonal techniques

  • Positive and Negative Controls: Include well-characterized controls in all experiments

  • Biological Replicates: Test from independent protein preparations and cell cultures

  • Blind Analysis: Conduct critical experiments with blinded samples to reduce bias

• Comparative Analysis:

  • Related Species: Compare with RPS28 from well-studied organisms like S. cerevisiae

  • Published Data: Benchmark against available literature data

  • Commercial Standards: Use commercially available RPS28 proteins (>90% purity) as reference points

  • Computational Validation: Compare experimental results with predictions from structural models

This systematic approach enables identification of sources of variability, validation of reproducible findings, and resolution of experimental discrepancies in K. marxianus RPS28 research.

What emerging technologies could advance our understanding of K. marxianus RPS28 function?

Several cutting-edge technologies hold promise for elucidating K. marxianus RPS28 function:

• Cryo-Electron Microscopy Advances:

  • High-resolution structural determination of entire K. marxianus ribosomes

  • Visualization of RPS28 interactions within the ribosomal complex

  • Time-resolved cryo-EM to capture dynamic states during translation

• Ribosome Profiling with Long-Read Sequencing:

  • Precise mapping of RPS28-associated mRNAs during translation

  • Identification of specialized translation programs associated with RPS28 variants

  • Integration with proteomics to correlate ribosome occupancy with protein output

• In Situ Structural Biology:

  • Cryo-electron tomography to visualize ribosomes in their cellular context

  • Correlative light and electron microscopy to track RPS28-containing ribosomes

  • Integrative structural biology combining multiple data sources

• Synthetic Biology Approaches:

  • Engineering specialized ribosomes with modified RPS28 variants

  • Development of orthogonal translation systems in K. marxianus

  • Creation of minimal ribosomes to determine essential RPS28 functions

• AI-Driven Structure Prediction and Design:

  • AlphaFold and RoseTTAFold for accurate prediction of RPS28 structures and interactions

  • Machine learning to predict functional effects of mutations

  • Computational design of RPS28 variants with enhanced properties

These technologies will enable researchers to move beyond static views of RPS28 function toward understanding its dynamic roles in translation regulation, potentially revealing specialized functions related to stress response, aging, and thermotolerance in K. marxianus.

How might research on K. marxianus RPS28 contribute to our understanding of specialized ribosomes?

Research on K. marxianus RPS28 could significantly advance our understanding of specialized ribosomes through these promising avenues:

• Heterogeneity in Ribosome Composition:

  • Investigation of potential RPS28 variants in K. marxianus similar to the RpS28a and RpS28-like variants found in Drosophila

  • Characterization of differentially modified RPS28 populations within cells

  • Correlation between RPS28 variants and translation of specific mRNA subsets

• Stress-Responsive Translation Regulation:

  • Analysis of RPS28's role in thermotolerant translation, leveraging K. marxianus's unique temperature resistance

  • Identification of mRNAs preferentially translated by RPS28-containing ribosomes under stress

  • Comparative studies with stress-sensitive species to identify adaptive mechanisms

• Aging and Longevity Connections:

  • Investigation of whether K. marxianus RPS28 expression changes during cellular aging

  • Determination if RPS28 overexpression affects lifespan or stress resistance, as observed in other species

  • Identification of proteins with anti-aging roles that might be regulated by RPS28-specialized ribosomes

• Evolutionary Adaptations in Translation:

  • Comparative analysis of RPS28 across species with different ecological niches

  • Identification of specific adaptations in K. marxianus RPS28 that may contribute to its industrial robustness

  • Investigation of how RPS28 variations contribute to translational specialization across evolution