Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YHR219C-A (YHR219C-A)

What is the putative UPF0479 protein YHR219C-A in Saccharomyces cerevisiae?

The YHR219C-A is a putative protein encoded by the YHR219C-A gene in Saccharomyces cerevisiae, classified in the UPF0479 protein family. S. cerevisiae, commonly known as baker's yeast or brewer's yeast, has been extensively studied as a model eukaryotic organism for molecular and cellular biology . The UPF0479 designation indicates an uncharacterized protein family with as-yet-unknown function. This protein is part of the extensive catalog of S. cerevisiae proteins that have been identified through genomic sequencing but whose functions remain to be fully elucidated.

Methodologically, researchers typically approach such uncharacterized proteins through comparative genomics, analyzing sequence homology with proteins of known function across species. For YHR219C-A, preliminary analyses suggest potential roles in cellular processes, though definitive functional characterization requires experimental validation through techniques such as gene knockout studies, localization experiments, and interaction analyses.

What expression systems are most suitable for recombinant production of YHR219C-A?

For recombinant production of YHR219C-A, researchers must carefully select expression systems based on experimental requirements. While E. coli systems offer rapid growth and high yields, they may present challenges for eukaryotic proteins like YHR219C-A . The selection between high-copy (pMB1') and low-copy (p15A) vectors significantly impacts protein production.

Methodologically, a balanced approach considering promoter strength and vector copy number is essential. Strong promoters (like P T7) combined with high-copy vectors may lead to metabolic burden, potentially decreasing soluble protein yields . For YHR219C-A, a moderate expression approach might involve:

When considering heterologous expression in E. coli, researchers should evaluate strains like BL21 or specialized derivatives such as BL21 ΔackA, which show reduced acetate production and potentially higher recombinant protein yields .

How can the function of YHR219C-A be preliminarily assessed based on S. cerevisiae biology?

Preliminary functional assessment of YHR219C-A can leverage S. cerevisiae's well-characterized biology. Since S. cerevisiae has been intensively studied as a eukaryotic model organism , researchers can employ several methodological approaches:

• Gene ontology analysis and computational prediction tools to suggest potential functions based on sequence motifs and structural elements.

• Microscopy-based localization studies using GFP-tagged YHR219C-A to determine subcellular localization, providing clues to function.

• Gene knockout or CRISPR-Cas9 editing techniques to create YHR219C-A deletion mutants, followed by phenotypic characterization during various growth conditions and cellular processes including budding, cytokinesis, and meiosis .

• Analysis of expression patterns during different cell cycle phases, particularly during cytokinesis and budding, where many S. cerevisiae proteins play critical roles. The putative protein might be involved in specific cellular processes like the formation of the actomyosin ring (AMR) or septum formation, which are critical for cell division in S. cerevisiae .

• Examination of potential roles in DNA repair mechanisms, given the importance of recombination repair during both meiosis and mitosis in S. cerevisiae .

How does promoter selection affect the expression efficiency and solubility of recombinant YHR219C-A?

Promoter selection significantly impacts expression efficiency and solubility of recombinant YHR219C-A. Research indicates that balancing transcriptional strength with the cell's metabolic capacity is crucial for obtaining soluble, properly folded protein .

Methodologically, comparative analysis of different promoters reveals distinct expression profiles:

Additional considerations include codon optimization for the expression host and fusion tags that may enhance solubility. Temperature reduction during induction (e.g., shifting from 37°C to 18-25°C) can further improve solubility by slowing translation and allowing more time for proper folding.

What are the implications of YHR219C-A's potential involvement in S. cerevisiae cytokinesis for research design?

Understanding YHR219C-A's potential role in S. cerevisiae cytokinesis has significant implications for research design. S. cerevisiae's asymmetric division process involves precisely coordinated events including actomyosin ring (AMR) constriction and primary septum formation .

If YHR219C-A is involved in cytokinesis, researchers should consider the following methodological approaches:

• Time-lapse fluorescence microscopy with labeled YHR219C-A to track its localization during cell division, particularly relative to the septin ring and AMR structures.

• Immunoprecipitation studies to identify interaction partners among known cytokinesis proteins, including septins, actins, myosins, and chitin synthases involved in septum formation.

• Synchronized culture experiments to precisely assess YHR219C-A expression and localization throughout the cell cycle, with particular focus on late G1 through cytokinesis.

• Conditional mutant strains with temperature-sensitive or degron-tagged YHR219C-A to observe phenotypic effects when the protein is rapidly inactivated at specific cell cycle stages.

• Electron microscopy studies of septum formation and AMR constriction in wild-type versus YHR219C-A mutant strains to detect ultrastructural abnormalities.

Research designs should account for the interdependence of AMR constriction and primary septum formation in S. cerevisiae , examining potential roles of YHR219C-A in either or both processes.

How can structural biology approaches be optimized for characterizing YHR219C-A?

Structural characterization of YHR219C-A requires methodological optimization across multiple techniques:

For X-ray crystallography:

• Expression screening in multiple systems (E. coli, S. cerevisiae, insect cells) to identify conditions yielding properly folded, homogeneous protein.

• Purification strategy development using affinity tags positioned to minimize structural interference.

• Limited proteolysis experiments to identify stable domains that may crystallize more readily than the full-length protein.

• Systematic crystallization trials with varying buffers, precipitants, and additives, potentially including surface entropy reduction mutations to promote crystal packing.

For NMR studies:

• Expression in minimal media supplemented with 15N-ammonium chloride and 13C-glucose for isotopic labeling.

• Optimization of sample conditions (buffer, pH, temperature) to maximize spectral quality and stability.

• Sequential backbone assignment followed by side-chain assignments for structural determination.

For cryo-electron microscopy:

• Expression and purification of sufficient quantities of homogeneous protein.

• Grid preparation optimization to achieve even particle distribution.

• Multi-particle averaging to enhance resolution, particularly if YHR219C-A forms oligomeric structures.

Each approach requires careful consideration of YHR219C-A's physicochemical properties, with pilot experiments to determine protein stability under various conditions. Integrating computational structure prediction with experimental data can provide valuable initial models to guide experimental design.

How should researchers optimize codon usage for YHR219C-A expression in heterologous systems?

Codon optimization is critical when expressing YHR219C-A in heterologous systems due to differences in codon bias between organisms. Methodologically, researchers should:

• Analyze the native codon usage of YHR219C-A in S. cerevisiae and compare it with the intended expression host.

• Generate a codon-optimized synthetic gene using algorithms that account for:

  • Host codon preference (frequency tables)

  • mRNA secondary structure prediction (avoiding stable structures near the 5' end)

  • GC content balancing

  • Removal of cryptic splice sites (for expression in eukaryotic hosts)

  • Elimination of sequence repeats that may cause recombination

• Consider "harmonized" rather than maximized codon usage, which preserves translational pauses that may be important for proper domain folding.

For E. coli expression, attention to rare codons (particularly for arginine, leucine, isoleucine, and proline) is essential. When using S. cerevisiae as the expression host, codon optimization is less critical but can still improve yields, especially for high-level expression .

What purification strategies are most effective for recombinant YHR219C-A?

Effective purification of recombinant YHR219C-A requires a multi-step strategy tailored to the protein's properties. Methodologically, researchers should:

• Begin with affinity chromatography using a fusion tag selected based on experimental requirements:

• Follow with ion exchange chromatography, selecting the appropriate resin based on theoretical isoelectric point (pI) calculation for YHR219C-A.

• Complete purification with size exclusion chromatography to:

  • Remove aggregates and oligomers

  • Confirm protein homogeneity

  • Exchange into final buffer conditions

• Consider specific contaminant removal strategies:

  • Nuclease treatment if DNA/RNA contamination is observed

  • Endotoxin removal columns for preparations intended for cellular studies

  • Additional chromatography steps if host cell protein contamination persists

Throughout purification, monitor protein quality using SDS-PAGE, Western blotting, and activity assays if available. Dynamic light scattering can assess sample homogeneity and detect aggregation tendencies, while circular dichroism provides information on secondary structure integrity.

How can researchers design experiments to elucidate YHR219C-A interactions with other cellular components?

To elucidate YHR219C-A interactions with other cellular components, researchers should employ a multi-faceted experimental approach:

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

  • Express epitope-tagged YHR219C-A in S. cerevisiae under native promoter control

  • Perform immunoprecipitation followed by LC-MS/MS to identify interaction partners

  • Include appropriate controls (untagged strains, tag-only constructs) to identify false positives

  • Consider SILAC labeling for quantitative comparison between specific and non-specific interactions

• Yeast two-hybrid (Y2H) screening:

  • Use YHR219C-A as both bait and prey to identify binary protein interactions

  • Screen against ordered libraries of S. cerevisiae proteins

  • Validate positive interactions with reverse Y2H and co-immunoprecipitation

• Proximity-dependent biotin identification (BioID):

  • Fuse BirA* to YHR219C-A and express in S. cerevisiae

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Compare results with classical AP-MS to distinguish stable versus transient interactions

• Fluorescence microscopy colocalization:

  • Generate fluorescently tagged YHR219C-A and candidate interactors

  • Perform live-cell imaging to assess colocalization patterns

  • Implement FRET or BiFC techniques to confirm direct interactions in vivo

• Genome-wide genetic interaction mapping:

  • Perform synthetic genetic array (SGA) analysis with YHR219C-A deletion strain

  • Identify genes with synthetic lethality or growth defects when combined with YHR219C-A deletion

  • Construct double mutants to validate key interactions

Integration of data from multiple methods provides the most robust interaction network, with each technique compensating for limitations of others. Bioinformatic analysis of the resulting interaction data can place YHR219C-A within functional pathways and biological processes.

How can researchers address challenges in maintaining S. cerevisiae expression strains for YHR219C-A production?

Maintaining stable S. cerevisiae expression strains for YHR219C-A production presents several challenges requiring methodological solutions:

• Plasmid stability issues:

  • Implement auxotrophic selection markers (URA3, LEU2, HIS3) appropriate for host strain

  • Consider genomic integration for long-term stable expression

  • Maintain selective pressure throughout cultivation

  • Monitor plasmid retention via replica plating on selective and non-selective media

• Genetic drift concerns:

  • Establish master and working cell banks with minimal passages

  • Implement strict quality control procedures including growth rate monitoring and expression level verification

  • Limit the number of generations between thawing and production

  • Periodically sequence verify key genetic elements

• Culture contamination management:

  • Use sterile technique and dedicated equipment

  • Add low concentrations of antibiotics to media where appropriate

  • Regularly check culture purity through microscopy and selective plating

  • Consider implementing PCR-based strain verification

• Expression level variability:

  • Control induction conditions precisely (temperature, inducer concentration, cell density at induction)

  • Standardize media preparation and culture conditions

  • Monitor expression levels by Western blot or fluorescence

  • Consider chromosomal integration under control of constitutive promoters for consistent expression

For long-term projects, genomic integration of YHR219C-A expression cassettes provides greater stability than episomal vectors. Integration can be achieved through homologous recombination targeting specific loci like HO or URA3, with confirmation by PCR and sequencing to verify proper integration.

What approaches should be used to troubleshoot inclusion body formation during YHR219C-A expression?

Inclusion body formation during recombinant YHR219C-A expression requires systematic troubleshooting approaches:

• Expression condition optimization:

  • Reduce induction temperature (typically to 15-25°C)

  • Decrease inducer concentration for titratable promoters

  • Lower expression rate by using weaker promoters or lower copy number vectors

  • Induce at higher cell densities to reduce expression per cell

• Vector design modifications:

  • Incorporate solubility-enhancing fusion partners (MBP, SUMO, Trx)

  • Try different affinity tag positions (N-terminal vs. C-terminal)

  • Include flexible linkers between domains

  • Consider domain-based expression if full-length protein is problematic

• Host strain selection:

  • Evaluate specialized strains (e.g., BL21 derivatives with enhanced disulfide bond formation)

  • Test strains co-expressing chaperones (GroEL/ES, DnaK/DnaJ/GrpE)

  • Consider S. cerevisiae expression to maintain native folding environment

• Media and additive optimization:

  • Add osmolytes (sorbitol, betaine) to stabilize folding intermediates

  • Supplement with cofactors or ligands that may stabilize the native structure

  • Explore chemical chaperones (DMSO, glycerol at low percentages)

If inclusion bodies persist despite optimization efforts, researchers may develop refolding protocols from purified inclusion bodies, using step-wise dialysis to gradually remove denaturants while providing an appropriate redox environment for disulfide bond formation.

How should researchers analyze contradictory data about YHR219C-A function across different experimental approaches?

When faced with contradictory data about YHR219C-A function from different experimental approaches, researchers should implement a systematic analysis methodology:

• Evaluate experimental variables across studies:

  • Strain background differences (laboratory vs. wild isolates of S. cerevisiae)

  • Growth conditions and media composition

  • Protein tagging strategies that may affect function

  • Expression levels (overexpression vs. native expression)

  • Assay sensitivity and specificity

• Assess technical validity of conflicting results:

  • Replicate number and statistical power

  • Appropriate controls (positive, negative, technical)

  • Methodology validation with known positive examples

  • Potential artifacts from experimental manipulation

• Consider biological explanations for discrepancies:

  • Multifunctional properties of YHR219C-A in different cellular contexts

  • Condition-specific functions (stress response, cell cycle phase)

  • Redundant pathways or compensatory mechanisms

  • Indirect versus direct effects

• Design decisive experiments to resolve contradictions:

  • Create conditional alleles to distinguish primary from secondary effects

  • Use orthogonal techniques to validate key findings

  • Perform epistasis analysis to place YHR219C-A in pathways

  • Implement time-resolved studies to capture dynamic functions

• Integrate data using systematic frameworks:

  • Weight evidence based on methodological strength

  • Develop testable models that accommodate seemingly contradictory observations

  • Consider protein moonlighting functions as explanation for diverse phenotypes

  • Use computational approaches to integrate diverse datasets

When reporting results, researchers should transparently address contradictions, avoiding selective citation of supportive evidence. Presentation of alternative interpretations with qualifying statements about confidence levels fosters scientific progress and identifies areas for further investigation.