Recombinant Saccharomyces cerevisiae Uncharacterized protein YPL119C-A (YPL119C-A)

What is YPL119C-A and why is it classified as "uncharacterized"?

YPL119C-A is a gene locus in the Saccharomyces cerevisiae genome that encodes a protein of currently unknown function. It is designated as "uncharacterized" because despite its identification in the S. cerevisiae reference genome (derived from laboratory strain S288C), there is insufficient experimental evidence characterizing its biological role, molecular function, or biochemical properties . The protein is notably absent from detection in multiple proteomic studies, as evidenced by its inclusion in compiled lists of undetected proteins .

The "YPL" portion of the name indicates its chromosomal location (chromosome XVI), while "119C" denotes its specific position. The "-A" suffix typically indicates it was identified after the initial systematic gene naming. The "C" in "119C" signifies that the gene is located on the complementary strand of DNA.

What genomic and sequence information is available for YPL119C-A?

The genomic sequence of YPL119C-A is available through the Saccharomyces Genome Database (SGD), which maintains the reference sequence derived from strain S288C . Researchers can access this information to:

• Download the DNA or protein sequence

• View genomic context and coordinates

• Perform sequence analysis including BLASTN and BLASTP comparisons

• Design primers for amplification of the gene

• Generate restriction fragment maps for molecular cloning strategies

• View six-frame translations to confirm coding potential

For researchers initiating studies on YPL119C-A, sequence retrieval should be your first step, followed by in silico analysis to predict potential structural features, domains, and evolutionary relationships .

How can I design initial experiments to begin characterizing YPL119C-A?

When approaching an uncharacterized protein like YPL119C-A, a systematic experimental design is essential. Begin by defining your research variables and formulating specific, testable hypotheses . For initial characterization:

• Express and purify the recombinant protein using established S. cerevisiae expression systems

• Perform structural analyses (secondary structure predictions, hydrophobicity plots)

• Conduct localization studies using fluorescent protein fusions

• Generate knockout/knockdown strains to observe phenotypic effects

• Perform protein interaction studies to identify binding partners

Your experimental design should include appropriate controls, consider potential confounding variables, and use both between-subjects and within-subjects approaches where applicable . For example, when comparing wild-type and YPL119C-A mutant strains, ensure all other genetic and environmental variables are controlled.

What expression systems are most effective for producing recombinant YPL119C-A protein?

For recombinant expression of YPL119C-A, S. cerevisiae itself serves as an excellent host system, particularly for a yeast protein that may require specific post-translational modifications. Based on successful approaches with other yeast proteins, consider the following methodology:

• Vector selection: Use a high-copy 2μM expression plasmid like pGI-100 with a constitutive promoter such as the translation elongation factor 1-alpha (TEF2) promoter, which has been successfully used for other yeast proteins .

• Construct design: Engineer your expression construct to include:

  • Full-length YPL119C-A coding sequence

  • A purification tag (histidine tag is commonly used)

  • Appropriate restriction sites for cloning

• Expression conditions: Optimize temperature, induction time, and media composition to maximize protein yield while maintaining proper folding.

What methods can detect YPL119C-A when standard approaches have failed?

The presence of YPL119C-A in databases of undetected proteins suggests that conventional proteomic approaches may be insufficient for its detection . Consider these alternative approaches:

• Targeted mass spectrometry with enrichment techniques:

  • Develop specific reaction monitoring (SRM) assays

  • Use immunoprecipitation or affinity purification before MS analysis

  • Apply chemical crosslinking to capture transient interactions

• Enhanced expression systems:

  • Use stronger promoters to increase expression levels

  • Create fusion constructs with well-expressed partner proteins

  • Employ codon optimization for improved expression

• Alternative detection methods:

  • Develop specific antibodies against predicted epitopes

  • Utilize epitope tagging strategies (HA, FLAG, etc.)

  • Apply proximity labeling techniques (BioID, APEX)

• Specialized extraction protocols:

  • Test different cell lysis methods optimized for different cellular compartments

  • Use detergent combinations designed for hydrophobic or membrane-associated proteins

  • Apply specialized extraction buffers for different subcellular fractions

These approaches may overcome detection limitations reported in previous studies where YPL119C-A remained undetected .

How can I design experiments to determine if YPL119C-A has immunogenic properties?

To evaluate potential immunogenic properties of YPL119C-A, consider adapting methods used for studying other S. cerevisiae proteins in immunological contexts:

• In vivo vaccination models:

  • Express YPL119C-A in a recombinant S. cerevisiae construct

  • Vaccinate appropriate animal models

  • Assess both CD4+ and CD8+ T-cell responses using methods like ELISpot or flow cytometry

  • Consider multiple administration sites to enhance immune response

• Experimental design considerations:

  • Include proper controls (vector-only yeast constructs)

  • Measure responses after single and multiple administrations

  • Evaluate dose-dependent effects

  • Use appropriate statistical analyses to assess significance

• Epitope prediction and testing:

  • Use computational tools to predict potential T-cell and B-cell epitopes

  • Synthesize predicted epitope peptides

  • Test binding to MHC molecules and recognition by T-cells

This approach mirrors successful methodologies used for other recombinant S. cerevisiae-based immunological studies, where significant immune responses were observed even to self-antigens .

How can I resolve contradictory data regarding YPL119C-A function or expression?

Resolving contradictory data about uncharacterized proteins requires systematic investigation and methodological rigor:

• Establish a data tracking system:

  Study Type	Detection Method	Result	Experimental Conditions	Reference
  Proteomic	Mass Spec	Not detected	Standard extraction
  Transcriptomic	RNA-Seq	[Result]	[Conditions]	[Ref]
  Genetic	Deletion phenotype	[Result]	[Conditions]	[Ref]

• Identify potential sources of variability:

  • Strain differences (laboratory strains vs. wild isolates)

  • Growth conditions (media, temperature, growth phase)

  • Technical variations in detection methods

  • Post-translational modifications affecting detection

• Design controlled comparative experiments:

  • Use multiple detection methods in parallel

  • Test under identical conditions

  • Include appropriate positive and negative controls

  • Employ biological and technical replicates

• Apply statistical approaches to evaluate significance:

  • Meta-analysis of existing data

  • Bayesian approaches to reconcile conflicting evidence

  • Power analysis to determine appropriate sample sizes

Resolution of contradictory data often requires collaboration between laboratories using standardized protocols to minimize technical variables.

What computational approaches can predict YPL119C-A function when experimental data is limited?

When experimental characterization is challenging, computational approaches provide valuable insights:

• Comparative genomics analyses:

  • Ortholog identification across fungal species

  • Synteny analysis to identify conserved genomic contexts

  • Phylogenetic profiling to identify co-evolving genes

  • Identification of distant homologs using Position-Specific Scoring Matrices

• Structural prediction methods:

  • Ab initio structure prediction using algorithms like Rosetta or AlphaFold

  • Threading approaches to identify structural homologs

  • Molecular dynamics simulations to predict conformational properties

  • Binding site prediction to infer potential interactors or substrates

• Systems biology approaches:

  • Network analysis to predict functional associations

  • Gene co-expression analysis under various conditions

  • Integration with existing -omics datasets

  • Metabolic modeling to predict potential metabolic roles

• Text mining and literature-based discovery:

  • Natural language processing of scientific literature

  • Automated hypothesis generation from disconnected findings

  • Semantic relationship extraction from research articles

These computational strategies can generate testable hypotheses about YPL119C-A function even when direct experimental evidence is limited.

How can I design experiments to determine if YPL119C-A interacts with specific cellular pathways?

Investigating pathway interactions for uncharacterized proteins requires multifaceted approaches:

• Genetic interaction screening:

  • Synthetic genetic array (SGA) analysis

  • Suppressor/enhancer screening

  • CRISPR-based genetic interaction mapping

  • Chemical-genetic profiling

• Physical interaction studies:

  • Affinity purification coupled with mass spectrometry

  • Yeast two-hybrid screening

  • Protein complementation assays

  • FRET/BRET approaches for in vivo interaction detection

• Functional genomics approaches:

  • Transcriptomic profiling of YPL119C-A deletion/overexpression

  • ChIP-seq to identify potential DNA binding sites (if nuclear)

  • Ribosome profiling to assess translational impacts

  • Metabolomic analysis to identify affected metabolic pathways

• High-content screening:

  • Fluorescent reporter assays for pathway activation

  • Cell morphology phenotyping

  • Subcellular localization changes under different conditions

  • Quantitative phenotypic analysis under various stressors

These approaches should be designed with appropriate controls and statistical analyses to ensure reproducibility and meaningful interpretation of results.

What are best practices for generating and validating antibodies against uncharacterized proteins like YPL119C-A?

Developing antibodies against uncharacterized proteins presents unique challenges:

• Antigen design strategy:

  • Use computational epitope prediction to identify potentially immunogenic regions

  • Consider multiple peptide antigens targeting different regions

  • Express recombinant fragments with higher predicted antigenicity

  • Use both N-terminal and C-terminal regions when possible

• Validation methodology:

  • Western blot comparison using wild-type and knockout strains

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Immunofluorescence correlating with GFP-tagged versions

  • Preabsorption controls to demonstrate specificity

• Cross-reactivity assessment:

  • Testing against closely related yeast proteins

  • Validation in different strain backgrounds

  • Epitope mapping to confirm binding specificity

  • Quantitative affinity measurements

• Documentation requirements:

  • Detailed methods including host species, immunization protocol

  • Complete characterization of specificity and sensitivity

  • Batch-to-batch variation analysis

  • Optimal working conditions for different applications

Proper antibody development and validation is particularly critical for uncharacterized proteins where existing reagents and validation methods may be limited.

How can I optimize primers and PCR conditions for amplifying YPL119C-A?

Successful amplification of YPL119C-A requires careful primer design and PCR optimization:

• PCR optimization strategy:

  • Test multiple annealing temperatures (gradient PCR)

  • Optimize magnesium concentration

  • Try different polymerases (high-fidelity for cloning applications)

  • Add PCR enhancers if dealing with high GC content

  • Consider touchdown PCR protocols to increase specificity

• Template preparation:

  • Use high-quality genomic DNA from S. cerevisiae S288C strain

  • Consider using colony PCR for rapid screening

  • For difficult templates, try alternative extraction methods

• Validation of PCR products:

  • Sequence verification of cloned products

  • Restriction digestion analysis

  • Size verification on agarose gels

These guidelines will help ensure successful amplification of YPL119C-A for subsequent cloning and expression studies.

What purification strategies are most effective for recombinant YPL119C-A?

Purifying an uncharacterized protein requires a systematic approach:

• Expression construct design:

  • Include a purification tag (6xHis, GST, MBP)

  • Consider tag position (N-terminal or C-terminal) based on predicted structure

  • Include a protease cleavage site for tag removal

  • Example construct: [Promoter]-[YPL119C-A]-[6xHis]-[Stop]

• Initial small-scale tests:

  • Test expression in different conditions (temperature, time, media)

  • Perform solubility assessment (compare soluble vs. insoluble fractions)

  • Evaluate different lysis methods (chemical, sonication, pressure)

  • Test binding efficiency to purification resin

• Chromatography strategy:

  • Primary purification: Affinity chromatography (Ni-NTA for His-tagged protein)

  • Secondary purification: Size exclusion chromatography

  • Optional tertiary step: Ion exchange chromatography

  • Consider orthogonal approaches if purity is insufficient

• Quality control assessment:

  • SDS-PAGE for purity evaluation

  • Mass spectrometry for identity confirmation

  • Dynamic light scattering for aggregation analysis

  • Functional assays (once developed) for activity confirmation

This systematic approach maximizes the chances of obtaining pure, active recombinant YPL119C-A protein for subsequent functional and structural studies.

How might characterization of YPL119C-A contribute to broader understanding of uncharacterized proteomes?

Studying YPL119C-A has implications beyond this specific protein:

• Methodological advancement:

  • Development of improved detection methods for low-abundance proteins

  • Refinement of computational prediction algorithms

  • Establishment of integrated workflows for uncharacterized protein characterization

  • Creation of standardized reporting formats for negative results

• Systems biology insights:

  • Better understanding of protein interaction networks

  • Identification of previously unknown cellular pathways

  • Improved genome annotation and curation

  • Enhanced evolutionary models of protein function development

• Translational potential:

  • Identification of novel targets for antifungal development

  • Discovery of unique protein families with biotechnological applications

  • Better understanding of fundamental eukaryotic cellular processes

  • Development of yeast as improved recombinant protein production systems

Characterization of proteins like YPL119C-A contributes to closing the gap between genome sequencing and functional understanding of encoded proteomes.

What approaches can assess if YPL119C-A has potential as a vaccine carrier or immunotherapeutic agent?

Building on successful use of S. cerevisiae in vaccination studies, assessment of YPL119C-A would include:

• Immunogenicity evaluation:

  • Express YPL119C-A in yeast expression systems

  • Assess immune responses in appropriate animal models

  • Measure both CD4+ and CD8+ T-cell responses using standardized assays

  • Compare immune responses when administered at single versus multiple sites

• Antigen fusion constructs:

  • Create fusion proteins with model antigens

  • Evaluate processing and presentation of fusion antigens

  • Compare immunogenicity with established carriers

  • Assess potential adjuvant properties

• Safety assessment:

  • Evaluate potential cross-reactivity with human proteins

  • Assess inflammatory responses at injection sites

  • Monitor for unintended immune effects

  • Establish dose-dependent safety profiles

• Mechanistic studies:

  • Determine uptake by antigen-presenting cells

  • Evaluate activation of innate immune pathways

  • Assess cross-presentation efficiency

  • Determine memory response generation

These approaches would determine if YPL119C-A shares properties with other S. cerevisiae proteins that have shown promise as vaccine vehicles .