Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YDR467C (YDR467C)

What is YDR467C and what is known about its structure?

YDR467C is a putative uncharacterized protein from Saccharomyces cerevisiae, consisting of 108 amino acids. Based on available information, it has been identified as a protein of interest in yeast genomics, but its specific function remains largely unknown. The protein has been successfully expressed recombinantly with an N-terminal His-tag in E. coli expression systems, suggesting it is amenable to heterologous expression .

Structurally, YDR467C appears to contain hydrophobic regions based on its amino acid sequence, which includes multiple leucine and phenylalanine residues. This suggests potential membrane association or protein-protein interaction capabilities. Primary sequence analysis indicates the protein may have structural features common to regulatory proteins in yeast, though detailed three-dimensional structural studies would be required for confirmation.

What expression systems are recommended for recombinant YDR467C production?

E. coli expression systems have been successfully employed for the recombinant production of full-length YDR467C protein with an N-terminal His-tag . When using bacterial expression systems, researchers should consider the following methodological approach:

• Use a bacterial expression vector containing a strong inducible promoter (T7, tac)

• Incorporate an N-terminal His-tag for purification purposes

• Consider expression in specialized E. coli strains optimized for recombinant protein production (BL21(DE3), Rosetta)

• Optimize induction conditions: temperature (typically 16-30°C), IPTG concentration (0.1-1.0 mM), and duration (4-18 hours)

While E. coli has proven effective, for studies requiring post-translational modifications or native folding environment, researchers might consider:

• Yeast expression systems (particularly S. cerevisiae itself)

• Insect cell expression (for larger scale production)

• Cell-free expression systems (for rapid screening)

What are the optimal storage conditions for recombinant YDR467C protein?

For optimal stability and retention of biological activity, recombinant YDR467C protein should be stored according to these guidelines:

• Store lyophilized powder at -20°C/-80°C upon receipt

• Once reconstituted, store working aliquots at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is recommended) and store at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as they may compromise protein integrity

How should I reconstitute lyophilized YDR467C protein for experimental use?

The recommended reconstitution procedure for lyophilized YDR467C protein is as follows:

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

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

• For long-term storage, add glycerol to a final concentration of 50%

• Aliquot the reconstituted protein into small volumes to minimize freeze-thaw cycles

• The reconstituted protein is stored in Tris/PBS-based buffer at pH 8.0 with 6% Trehalose

This methodological approach ensures maximum retention of protein activity while minimizing degradation from repeated handling.

What experimental approaches can be used to characterize the function of YDR467C?

To characterize the function of an uncharacterized protein like YDR467C, researchers should consider a systematic approach:

• Genetic analysis:

  • Create deletion strains (YDR467C knockout) to observe phenotypic effects

  • Perform complementation studies to confirm phenotype rescue

  • Conduct synthetic lethality screens to identify genetic interactions

• Localization studies:

  • Generate GFP/fluorescent protein fusions to determine subcellular localization

  • Perform immunolocalization with antibodies against the recombinant protein

  • Use fractionation techniques to identify compartment-specific distribution

• Interaction studies:

  • Employ yeast two-hybrid (Y2H) assays to identify protein partners

  • Perform co-immunoprecipitation experiments to confirm interactions

  • Use microscale thermophoresis (MST) to quantify binding affinities

• Expression analysis:

  • Monitor expression under various stress conditions

  • Identify conditions that alter expression levels

  • Determine if the protein is regulated during specific cell cycle phases

Such multifaceted approaches, similar to those used in characterizing Rev7 protein in S. cerevisiae, would provide complementary data points for functional inference .

How can I design experiments to identify potential interaction partners of YDR467C?

To identify interaction partners of YDR467C, researchers can implement a multi-tiered experimental strategy:

• Yeast two-hybrid (Y2H) screening:

  • Use YDR467C as bait against a yeast genomic library

  • Perform targeted Y2H with suspected partners

  • Include appropriate controls to validate interactions specificity, as demonstrated in Rev7 interaction studies

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

  • Express tagged YDR467C in yeast

  • Purify protein complexes under native conditions

  • Identify co-purifying proteins by mass spectrometry

• Proximity-based labeling:

  • Fuse YDR467C to enzymes like BioID or APEX2

  • Allow in vivo labeling of proximal proteins

  • Purify and identify labeled proteins

• Microscale thermophoresis for quantitative analysis:

  • Purify YDR467C and potential binding partners

  • Label one protein (typically YDR467C) with a fluorescent dye

  • Measure binding affinity through thermophoretic mobility shifts

What phenotypes are associated with YDR467C deletion in S. cerevisiae?

While the provided search results do not specifically mention phenotypes associated with YDR467C deletion, researchers investigating this question should:

• Generate a YDR467C deletion strain:

  • Use homologous recombination to replace YDR467C with a selectable marker

  • Confirm deletion by PCR and/or Southern blotting

  • Ensure strain background compatibility with experimental goals

• Assess growth under various conditions:

  • Test growth on different carbon sources

  • Evaluate response to various stressors (temperature, oxidative, osmotic)

  • Examine sensitivity to DNA damaging agents (similar to analyses performed with Rev7)

• Examine cellular processes:

  • Analyze cell cycle progression

  • Evaluate DNA repair efficiency

  • Assess metabolic parameters

• Compare with related protein deletions:

  • Drawing parallels from Rev7 studies, which showed involvement in DNA repair processes, researchers might investigate whether YDR467C has any role in DNA damage response pathways

How do I design proper controls when studying an uncharacterized protein like YDR467C?

Designing appropriate controls is crucial for studies of uncharacterized proteins. For YDR467C research, implement these methodological controls:

• For genetic studies:

  • Include isogenic wild-type strains

  • Use empty vector controls in complementation experiments

  • Create a strain expressing an unrelated protein of similar size/characteristics

• For interaction studies:

  • Include known non-interacting proteins (like Sae2 used as negative control in Rev7 studies)

  • Test interactions in multiple strain backgrounds

  • Use truncated versions to identify interaction domains

• For biochemical assays:

  • Include buffer-only controls

  • Use denatured protein controls

  • Test with unrelated proteins of similar size/structure

• For functional complementation:

  • Test multiple expression levels

  • Create point mutations in conserved residues

  • Use heterologous proteins with similar predicted functions

The Rev7 interaction studies provide an excellent template, where interactions with Sae2 were used as negative controls to demonstrate specificity of interactions with MRX complex components .

What bioinformatics tools are most useful for predicting YDR467C function?

For computational prediction of YDR467C function, researchers should utilize multiple bioinformatics approaches:

• Sequence-based analysis:

  • BLAST, FASTA for sequence homology

  • Multiple sequence alignment tools (MUSCLE, Clustal Omega)

  • Hidden Markov Models for remote homology detection

• Structural prediction:

  • AlphaFold2 for 3D structure prediction

  • I-TASSER for template-based modeling

  • AF2-multimer for protein complex prediction (as used for Rev7-MRX complex modeling)

• Functional prediction:

  • InterPro for domain prediction

  • Gene Ontology term association

  • Conserved co-expression networks

• Evolutionary analysis:

  • Phylogenetic profiling

  • Analysis of conservation patterns

  • Synteny analysis across fungal species

By integrating predictions from these diverse computational approaches, researchers can generate testable hypotheses about YDR467C function to guide experimental design.

How can I use techniques like microscale thermophoresis (MST) to study YDR467C protein interactions?

Microscale thermophoresis (MST) provides a powerful approach for quantifying protein-protein interactions, as demonstrated in the Rev7 interaction studies . To implement MST for YDR467C interaction analysis:

• Protein preparation:

  • Express and purify YDR467C with an appropriate tag (His-tag as used in commercial preparations)

  • Ensure high purity (>90% by SDS-PAGE)

  • Label YDR467C with a fluorescent dye (typically on lysine residues or via tag)

• Experimental design:

  • Prepare serial dilutions of unlabeled potential binding partners

  • Keep labeled YDR467C concentration constant (typically 5-50 nM)

  • Include appropriate buffer controls and non-binding protein controls

• Data acquisition and analysis:

  • Measure thermophoretic mobility at constant temperature gradient

  • Plot normalized MST signals against ligand concentration

  • Fit data to appropriate binding models to determine dissociation constants (Kd)

  • Analyze Hill coefficients to assess binding cooperativity

The MST approach has successfully demonstrated binding between Rev7 and MRX complex components with sub-micromolar affinities (Kd values of 0.16-0.23 μM) , providing a methodological template for YDR467C interaction studies.

What is the role of YDR467C in DNA repair mechanisms based on current research?

While the provided search results do not directly address YDR467C's role in DNA repair, researchers investigating this question can:

• Draw parallels from related yeast proteins:

  • Rev7 in S. cerevisiae plays a crucial role in DNA double-strand break (DSB) repair by promoting non-homologous end joining (NHEJ) while inhibiting homologous recombination (HR)

  • The methodology used to characterize Rev7's function in DNA repair provides a roadmap for similar studies with YDR467C

• Design experiments to test DNA repair involvement:

  • Assess sensitivity of YDR467C deletion strains to DNA damaging agents (HU, MMS, IR)

  • Analyze genetic interactions with known DNA repair genes

  • Measure DNA repair efficiency in YDR467C mutants

• Investigate protein-protein interactions:

  • Test for interactions with established DNA repair complexes (like MRX)

  • Examine potential recruitment to sites of DNA damage

  • Use chromatin immunoprecipitation to detect DNA binding

If YDR467C shows properties similar to Rev7, researchers might investigate whether it interacts with the MRX complex or influences the choice between NHEJ and HR repair pathways .

How can I design CRISPR/Cas9 experiments to study YDR467C function in vivo?

For CRISPR/Cas9-based functional studies of YDR467C:

• Guide RNA design and validation:

  • Design sgRNAs targeting YDR467C coding sequence or regulatory regions

  • Ensure specificity through in silico prediction tools

  • Validate cutting efficiency in vitro before cellular experiments

• Experimental approaches:

  • Gene knockout: Create complete YDR467C deletion through NHEJ repair

  • Point mutations: Introduce specific mutations using HDR with donor templates

  • Tagging: Create fluorescent protein fusions for localization studies

  • CRISPRi/CRISPRa: Modulate expression without altering sequence

• Delivery methods for S. cerevisiae:

  • Plasmid-based expression of Cas9 and sgRNA

  • Transformation with ribonucleoprotein (RNP) complexes

  • Integration of Cas9 into the genome for stable expression

• Phenotypic analysis:

  • Growth assays under various conditions

  • Specific functional assays based on hypothesized function

  • High-throughput screens when function is unknown

This methodological approach allows precise genetic manipulation to probe YDR467C function in its native cellular context.

What are the best approaches to resolve contradictory data about YDR467C function?

When faced with contradictory data regarding YDR467C function, researchers should implement these methodological approaches:

• Systematic validation:

  • Repeat experiments with standardized conditions

  • Use multiple experimental approaches to test the same hypothesis

  • Implement blind experimental design when possible

• Control for strain background effects:

  • Test phenotypes in multiple S. cerevisiae strain backgrounds

  • Create isogenic strains differing only in YDR467C status

  • Consider genetic interactions that might mask or enhance phenotypes

• Address technical variability:

  • Standardize protein preparation methods

  • Validate antibody specificity

  • Use quantitative approaches with appropriate statistical analysis

• Integrate diverse data types:

  • Combine genetic, biochemical, and structural approaches

  • Use computational modeling to reconcile seemingly contradictory results

  • Consider condition-specific functions that might explain discrepancies

This methodological framework, similar to the multi-faceted approach used in characterizing Rev7 function , helps resolve contradictions and build a more comprehensive understanding of protein function.

How can I integrate proteomics and transcriptomics data to elucidate YDR467C function?

Integrating multi-omics data provides powerful insights into protein function. For YDR467C:

• Data generation and processing:

  • Transcriptomics: Perform RNA-seq on YDR467C deletion vs. wild-type strains

  • Proteomics: Use quantitative proteomics (TMT, SILAC) to identify protein abundance changes

  • Interactomics: Generate protein-protein interaction data through AP-MS or BioID

• Integrative analysis approaches:

  • Correlate transcript and protein level changes

  • Identify enriched pathways and processes using GO term analysis

  • Construct regulatory networks incorporating YDR467C

• Functional validation pipeline:

  • Select top candidates from integrated analysis for validation

  • Design targeted genetic interaction experiments

  • Test specific biochemical activities suggested by omics data

• Visualization and interpretation:

  • Create integrated heatmaps showing coordination between datasets

  • Use network visualization tools to represent functional relationships

  • Develop predictive models of YDR467C function

This integrated approach provides a comprehensive view of YDR467C function within the cellular context, similar to the multi-faceted characterization performed for Rev7 .