Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YDR193W (YDR193W)

What is YDR193W and why is it significant for research?

YDR193W is a putative uncharacterized protein in the model organism Saccharomyces cerevisiae (budding yeast). Its significance stems from being part of the approximately 6-13% of proteins in the human proteome that have been detected but remain functionally uncharacterized . S. cerevisiae serves as an excellent model organism for studying such proteins due to its well-characterized genome, ease of genetic manipulation, and the high conservation of many fundamental biological processes between yeast and humans . The characterization of YDR193W could potentially reveal new insights into conserved cellular functions and contribute to our understanding of similar uncharacterized proteins in higher organisms, including humans.

What expression systems are recommended for producing recombinant YDR193W protein?

For recombinant YDR193W production, Escherichia coli is the most commonly used expression system due to its rapid growth, high protein yields, and established protocols . Specifically, available data shows that recombinant full-length YDR193W has been successfully expressed in E. coli with an N-terminal His-tag . When designing an expression system for YDR193W, researchers should consider:

• Strain selection: BL21(DE3) serves as a workhorse strain for non-toxic proteins, while BL21(DE3) pLysS or pLysE can repress basal expression levels for toxic proteins

• Promoter choice: Options include T7 promoter (high expression but strict regulation needed), trc and tac promoters (moderate expression), or BAD promoter (tightly regulated)

• Vector considerations: Plasmid copy number affects expression levels; high-copy pMB1' origin or low-copy p15A origin should be selected based on protein toxicity and desired yield

• Codon optimization: May increase expression up to 13-fold compared to non-optimized sequences

What detection methods can be used to verify YDR193W expression?

Multiple complementary methods can be employed to verify the expression of YDR193W:

• SDS-PAGE: The most basic method for confirming protein expression and approximate molecular weight. For YDR193W, which has a molecular weight of approximately 14-15 kDa plus any tag size, standard 12-15% gels are recommended .

• Western blotting: More specific detection using antibodies against the His-tag or against YDR193W directly if antibodies are available.

• Mass spectrometry: For precise identification and confirmation of the protein sequence:

  • Set mass tolerances to 10 ppm for precursors and 0.5 Da for fragments

  • Consider carbamidomethylation of cysteine residues as a fixed modification

  • Consider oxidation of methionine as a variable modification

  • Apply an FDR cut-off of 1% for both peptides and proteins

• Fluorescence detection: If expressed as a fusion with a fluorescent reporter such as YFP, expression can be monitored in real-time .

Is there existing expression data for YDR193W in S. cerevisiae?

According to the Saccharomyces Genome Database (SGD), no expression data is currently available for YDR193W . This lack of expression data suggests that YDR193W may be:

• Expressed at very low levels under standard laboratory conditions

• Expressed only under specific environmental conditions not commonly tested

• Expressed only during specific developmental or stress responses

• Potentially a misannotated open reading frame

Researchers interested in studying YDR193W expression in its native context should consider genome-wide techniques such as RNA-seq under various growth conditions or stress responses, or targeted approaches such as adding epitope tags to the endogenous locus to facilitate detection of the native protein. The SPELL (Serial Pattern of Expression Levels Locator) tool referenced in the SGD might help identify conditions under which similar genes are expressed, potentially guiding experimental design .

What experimental design strategies should be employed for functional characterization of uncharacterized proteins like YDR193W?

Functional characterization of uncharacterized proteins like YDR193W requires a multi-faceted experimental approach following these methodological steps:

Step 1: Define variables and controls

• Independent variables: Potential conditions affecting YDR193W function (e.g., nutrient availability, stress conditions, genetic background)

• Dependent variables: Measurable outputs (e.g., growth rate, biochemical activity, interaction profiles)

• Control for extraneous variables: Use isogenic strains differing only in YDR193W expression

Step 2: Design systematic experimental treatments
For YDR193W characterization, implement a matrix of approaches:

• Comparative genomics analysis:

  • Identify orthologs across species to infer evolutionary conservation

  • Compare to characterized proteins with similar domains or motifs

• Gene deletion and overexpression studies:

  • Create YDR193W deletion strains using homologous recombination

  • Employ controllable promoters (GAL1, TET) for conditional expression

  • Assess phenotypic changes across multiple conditions

• Protein localization studies:

  • Generate GFP/RFP fusions to determine subcellular localization

  • Perform fractionation studies coupled with Western blot analysis

• Interactome mapping:

  • Conduct yeast two-hybrid screens or affinity purification-mass spectrometry

  • Perform synthetic genetic array (SGA) analysis to identify genetic interactions

• High-throughput phenotypic screening:

  • Test growth under hundreds of environmental conditions and chemical stressors

  • Analyze metabolomic changes in deletion/overexpression strains

When analyzing results, ensure proper statistical analysis and validation through complementary methods to avoid artifacts from any single approach.

What are the optimal purification strategies for recombinant YDR193W protein?

For efficient purification of recombinant YDR193W, a systematic approach should be followed based on its physicochemical properties:

• Expression optimization:

  • Test multiple E. coli strains including BL21(DE3), BL21(DE3)pLysS, and BL21(DE3) Codon Plus RIPL

  • Evaluate different induction conditions (IPTG concentration, temperature, duration)

  • Consider testing both rich (LB) and defined media (M9)

• Solubility assessment and enhancement:

  • Perform small-scale expression tests with varied conditions (16-37°C induction)

  • If inclusion bodies form, test solubilization strategies:

    • Co-expression with chaperones (GroEL/ES, DnaK)

    • Fusion tags (MBP, SUMO, Trx) beyond the His-tag

    • Lower expression temperature (16°C) and IPTG concentration

• Purification protocol:

  • Primary capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA

  • Secondary purification: Size exclusion chromatography to remove aggregates

  • Buffer optimization: Screen multiple buffers varying pH (6.5-8.5), salt (50-500mM NaCl), and additives (glycerol, reducing agents)

• Quality control:

  • Assess purity by SDS-PAGE (>90% purity recommended)

  • Verify identity by mass spectrometry

  • Evaluate protein stability through thermal shift assays

  • Check for proper folding via circular dichroism

• Storage optimization:

  • Test stability in different buffer conditions

  • Aliquot and store at -80°C to avoid freeze-thaw cycles

  • Consider lyophilization with 6% trehalose as a stabilizer

How can computational approaches predict potential functions of YDR193W?

Computational prediction of YDR193W function should follow a multi-layered approach:

• Sequence-based analysis:

  • Homology detection: Search for remote homologs using PSI-BLAST, HHpred, or HMMER

  • Domain and motif identification: Scan InterPro, PFAM, PROSITE databases

  • Disorder prediction: Use PONDR, IUPred to identify structurally disordered regions

  • Secondary structure prediction: Apply PSIPRED, JPred for structural elements

• Structural prediction:

  • Generate 3D structural models using AlphaFold2 or RoseTTAFold

  • Perform structural similarity searches against PDB using DALI or TM-align

  • Identify potential binding pockets using CASTp or SiteMap

• Systems biology integration:

  • Construct protein-protein interaction networks using available yeast interactome data

  • Analyze co-expression patterns with genes of known function

  • Identify synthetic genetic interactions to place in biological pathways

• Functional prediction:

  • Employ Gene Ontology term prediction tools (PANNZER, DeepGOPlus)

  • Use pathway mapping tools to predict metabolic or signaling roles

  • Apply clustering algorithms to group with functionally characterized proteins

• Validation planning:

  • Design experiments to test top computational predictions

  • Prioritize predictions with highest confidence scores

  • Focus on predictions supported by multiple methods

This systematic approach has successfully reduced the number of uncharacterized proteins (uPE1) in the human proteome from 13% to 6% in recent years , demonstrating its effectiveness when applied rigorously.

What techniques are most effective for studying the potential interaction partners of YDR193W?

To comprehensively identify and validate interaction partners of YDR193W, employ a combination of complementary techniques:

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

  • Express tagged YDR193W (His-tag or TAP-tag) in yeast

  • Perform gentle lysis preserving protein complexes

  • Capture complexes via affinity purification

  • Identify components by mass spectrometry

  • Use SILAC or TMT labeling for quantitative assessment

  • Compare results with control purifications to filter non-specific interactions

• Yeast two-hybrid screening:

  • Create both DNA-binding domain and activation domain fusions

  • Screen against yeast genomic or cDNA libraries

  • Verify positive interactions through reverse Y2H and co-immunoprecipitation

  • Use N- and C-terminal fusions to minimize false negatives

• Proximity-based labeling:

  • Fuse YDR193W to BioID or APEX2 enzymes

  • Express fusion protein in yeast cells

  • Allow proximity-dependent labeling to occur

  • Purify biotinylated proteins and identify by MS

  • This approach captures transient and weak interactions

• Genetic interaction mapping:

  • Create YDR193W deletion strain

  • Perform synthetic genetic array (SGA) analysis

  • Identify genes with synthetic lethal/sick phenotypes

  • These often represent parallel pathways or complex members

• Advanced techniques:

  • Crosslinking mass spectrometry (XL-MS): To map interaction interfaces

  • Fluorescence resonance energy transfer (FRET): For direct protein-protein interactions in vivo

  • Microfluidics platforms: For high-throughput interaction studies

Data integration should follow using Cytoscape or similar platforms to construct interaction networks and identify key functional clusters.

What experimental approaches can address the challenges of expressing YDR193W in heterologous systems?

Expression of uncharacterized proteins like YDR193W in heterologous systems presents several challenges that can be systematically addressed:

• Challenge: Low expression levels
  Solution approaches:

  • Codon optimization: Design sequence adapted to host codon usage, which can increase yield up to 13-fold

  • Promoter selection: Test multiple promoters in parallel (P T7, Plac trc, P tac, P BAD) to identify optimal expression control

  • Vector backbone modification: Compare high-copy (pMB1') vs. low-copy (p15A) origins of replication

  • Host strain engineering: Test expression in an E. coli BL21 ΔackA strain, which shows higher recombinant protein production due to reduced acetate production

• Challenge: Inclusion body formation
  Solution approaches:

  • Temperature optimization: Lower induction temperature to 16-20°C

  • Fusion tags: Test solubility enhancement tags (MBP, SUMO, GST)

  • Co-expression strategies: Include molecular chaperones (GroEL/ES)

  • Strain selection: Use Origami™ 2(DE3) for enhancing disulfide bond formation in cytoplasm

• Challenge: Protein toxicity to host
  Solution approaches:

  • Tight expression control: Use pLysS/pLysE systems to reduce basal expression

  • Induction optimization: Titrate inducer concentration for minimal toxicity

  • Specialized growth media: Test autoinduction media for gradual protein expression

  • Metabolic burden reduction: Balance plasmid copy number and promoter strength

• Challenge: Improper folding
  Solution approaches:

  • Expression in yeast: Return to native host or use Pichia pastoris

  • Periplasmic expression: Direct protein to E. coli periplasm using signal sequences

  • Cell-free protein synthesis: Bypass cellular toxicity entirely

• Challenge: Post-translational modifications
  Solution approaches:

  • Eukaryotic expression systems: Switch to insect or mammalian cells if modifications are crucial

  • Co-expression of modifying enzymes: Introduce necessary kinases, glycosyltransferases

A systematic experimental design testing multiple conditions in parallel is recommended to efficiently identify optimal expression parameters.

How should researchers approach the structural characterization of YDR193W?

A comprehensive structural characterization of YDR193W requires a multi-technique approach:

• Computational structure prediction and analysis:

  • Generate models using AlphaFold2 and RoseTTAFold

  • Analyze predicted structure for functional clues:

    • Identify potential binding pockets

    • Search for structural homologs

    • Predict potential membrane-spanning regions

  • Use these predictions to guide experimental approaches

• X-ray crystallography workflow:

  • Protein engineering: Design constructs removing flexible regions identified computationally

  • Crystallization screening: Test thousands of conditions using nanoliter-scale robotics

  • Optimization: Refine promising conditions by varying pH, precipitant, additives

  • Data collection: Collect diffraction data at synchrotron facilities

  • Structure determination: Use molecular replacement or experimental phasing methods

• NMR spectroscopy approach:

  • Isotopic labeling: Express protein with 15N, 13C enrichment

  • Sample optimization: Screen buffers for optimal peak dispersion

  • Spectral acquisition: Collect 2D and 3D NMR spectra

  • Structure calculation: Assign resonances and derive distance constraints

  • Particularly suitable if YDR193W is under 20 kDa in size

• Cryo-electron microscopy:

  • More suitable if YDR193W forms larger complexes

  • Sample preparation: Optimize grid freezing conditions

  • Data collection: Acquire thousands of particle images

  • Processing: Perform 2D classification and 3D reconstruction

• Hybrid methods:

  • Small-angle X-ray scattering (SAXS): For solution structure

  • HDX-MS: To probe dynamics and ligand interactions

  • Integrative modeling: Combine data from multiple techniques

Before investing in high-resolution techniques, preliminary characterization using circular dichroism and thermal shift assays is recommended to confirm proper folding and stability of the purified protein.

What research design is appropriate for investigating how YDR193W may contribute to cellular pathways in S. cerevisiae?

To systematically investigate YDR193W's role in cellular pathways, implement a multi-tiered experimental design:

• Phenotypic profiling under varied conditions:

  • Create precision deletion strains (ΔYDR193W) and tagged/overexpression variants

  • Subject strains to comprehensive phenotypic array testing:

    • Growth in different carbon sources and nitrogen sources

    • Response to various stressors (oxidative, heat, osmotic)

    • Sensitivity to drugs with known mechanisms of action

  • Analyze growth kinetics using automated plate readers for quantitative assessment

  • Design as a full factorial experiment to identify condition-specific functions

• Transcriptomic and proteomic analysis:

  • Perform RNA-seq comparing ΔYDR193W to wild-type under standard and stress conditions

  • Apply SILAC or TMT proteomics to identify affected proteins

  • Analyze data using pathway enrichment tools to identify perturbed processes

  • Validate key findings with RT-qPCR and Western blotting

• Metabolomic investigation:

  • Measure metabolite changes in ΔYDR193W vs. wild-type using LC-MS/MS

  • Focus on central carbon metabolism, lipid metabolism, and amino acid pathways

  • Integrate with transcriptomic/proteomic data for pathway mapping

• Genetic interaction mapping:

  • Perform synthetic genetic array (SGA) analysis or CRISPR-based screens

  • Create double mutants with genes in major cellular pathways

  • Identify synthetic lethal, suppressive, or epistatic relationships

  • Map YDR193W to specific pathways based on interaction profiles

• Localization and dynamics studies:

  • Track YDR193W-GFP localization during cell cycle and stress responses

  • Perform time-lapse microscopy to detect dynamic relocalization

  • Co-localize with markers of cellular compartments

This systematic approach allows for unbiased hypothesis generation followed by focused validation experiments, leveraging the power of S. cerevisiae as a model organism where "processes that one can study with relative ease in yeast are identical in most respects to the ways in which human cells repair DNA damage and generate genetic diversity" .

How can researchers design experiments to determine if YDR193W has orthologs with conserved functions in other organisms?

To identify and confirm functional conservation of YDR193W orthologs across species, implement this methodological framework:

• Computational identification of putative orthologs:

  • Create clusters of orthologs (ScCOGs) and homologs (ScCHGs) across species

  • Employ reciprocal BLAST, OrthoMCL, and HMMer for sensitive detection

  • Generate phylogenetic trees to distinguish orthologs from paralogs

  • Calculate sequence conservation metrics and identify conserved motifs

  • Apply this process across 700+ organisms as done in previous studies

• Functional complementation testing:

  • Clone identified orthologs from other species (starting with closely related fungi)

  • Express these orthologs in ΔYDR193W S. cerevisiae strain

  • Test for phenotypic rescue under conditions where YDR193W deletion shows defects

  • Design appropriate controls:

    • Empty vector

    • Wild-type YDR193W (positive control)

    • Unrelated protein (negative control)

• Domain swapping experiments:

  • Create chimeric proteins with domains from different species' orthologs

  • Express in ΔYDR193W strain and assess functionality

  • Map functionally conserved regions through systematic domain analysis

• Comparative phenotypic analysis:

  • Create knockout/knockdown of orthologs in model organisms:

    • S. pombe (fission yeast)

    • C. elegans (nematode)

    • D. melanogaster (fruit fly)

    • Human cell lines (if ortholog identified)

  • Compare phenotypes across species for functional conservation patterns

• Biochemical characterization across species:

  • Express and purify recombinant proteins from multiple species

  • Compare biochemical properties (substrate specificity, binding partners)

  • Perform structural studies to identify conserved structural elements

This approach has been validated in previous studies where "S. cerevisiae is predicted to be a good model in which to study a significant fraction of common biological processes" across species . The methods enable researchers to determine both the extent of conservation and the evolutionary trajectory of the protein function.

Data Table: YDR193W Protein Characteristics