Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YJL156W-A (YJL156W-A)

What is YJL156W-A and what are its basic properties?

YJL156W-A is a putative uncharacterized protein from the yeast Saccharomyces cerevisiae. This protein consists of 73 amino acids with the sequence: MLKIASLKKKDMQTKESCILKRPGLSCPPNKTKEVNESKQIFFLTWKNKATMKVSFIVAPTVMQVQCLFFFIL. It is classified under UniProt ID P0C5N9 and is often studied in recombinant form with tags such as His-tags to facilitate purification and analysis .

The protein's function remains largely uncharacterized, making it an interesting target for fundamental research into yeast genomics and proteomics. When working with this protein, researchers should note that commercially available recombinant versions are typically supplied as lyophilized powders with greater than 90% purity as determined by SDS-PAGE .

How should YJL156W-A be stored and handled in laboratory settings?

For optimal stability and activity, recombinant YJL156W-A should be stored at -20°C/-80°C upon receipt. Before opening, vials should be briefly centrifuged to bring the contents to the bottom. The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

To prevent protein degradation, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being a standard concentration) before making aliquots for long-term storage. Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity. For short-term use, working aliquots can be stored at 4°C for up to one week .

How should I design experiments to study the function of YJL156W-A in Saccharomyces cerevisiae?

When designing experiments to study YJL156W-A, follow these systematic steps for a robust experimental approach:

• Define your variables clearly: Identify your independent variables (what you will manipulate) and dependent variables (what you will measure) related to YJL156W-A function .

• Formulate a specific, testable hypothesis: Based on genomic location and any preliminary data about YJL156W-A, develop precise hypotheses about its potential functions.

• Design experimental treatments: Create experimental conditions that systematically manipulate relevant variables, including knockout/overexpression of YJL156W-A and various environmental conditions .

• Assign proper controls: Design both positive and negative controls, including wild-type strains and strains with mutations in related genes .

• Plan measurement methods: Select appropriate assays to measure phenotypic changes, protein interactions, or other relevant outcomes .

A well-designed experiment requires careful consideration of potential confounding variables. For example, if comparing growth rates between wild-type and YJL156W-A knockout strains, ensure that all other conditions (media composition, temperature, growth phase) are identical between groups .

What are the key considerations when designing knockout or gene modification experiments for YJL156W-A?

When designing genetic modifications for YJL156W-A, several critical factors must be considered:

• Genomic context awareness: The S. cerevisiae genome is very compact with relatively small intergenic regions. Since YJL156W-A is a small ORF, complete removal might affect regulatory sequences of adjacent genes. It is therefore recommended to confirm any observed effects through multiple independent approaches .

• Selection marker choice: Consider the available selectable markers in your experimental strain. Common prototrophic markers include URA3, LEU2, TRP1, and HIS3. If these are unavailable or already in use, consider dominant markers such as KanR (resistance to G418), NatR (resistance to nourseothricin), or HphR (resistance to hygromycin B) .

• Transformation method selection: The lithium acetate/single-stranded carrier DNA/PEG method is recommended for routine procedures due to its simplicity and efficiency. This method does not require specialized equipment and is suitable for high-efficiency transformation scenarios .

• Confirmation strategies: Given the potential for unintended effects on adjacent genes, use multiple verification approaches, such as:

  • RT-qPCR to confirm expression changes

  • Complementation tests with wild-type YJL156W-A

  • Phenotypic assays relevant to hypothesized functions

What is the most effective system for expressing recombinant YJL156W-A protein?

Based on available data, E. coli has been successfully used as an expression system for recombinant YJL156W-A with N-terminal His-tags . When selecting an expression system, consider these methodological approaches:

• E. coli expression optimization:

  • Codon optimization may be necessary since yeast and E. coli have different codon usage preferences

  • Evaluate multiple strains (BL21, Rosetta, etc.) for optimal expression

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

  • Consider fusion tags beyond His-tag (GST, MBP) if solubility issues arise

• Alternative expression systems:

  • Yeast-based expression might provide more native folding and post-translational modifications

  • Insect cell systems may be valuable for larger-scale production with eukaryotic processing

• Expression verification methods:

  • SDS-PAGE for protein size confirmation

  • Western blotting for tag detection

  • Mass spectrometry for sequence verification

The choice of expression system should align with your specific research goals, such as structural studies, functional assays, or antibody production.

What purification strategies are most effective for YJL156W-A?

For His-tagged YJL156W-A protein, the following purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or cobalt-based resins provides high selectivity for His-tagged proteins.

• Buffer optimization: Since little is known about YJL156W-A's properties, test multiple buffer conditions:

  • pH range: 6.0-8.0

  • Salt concentration: 150-500 mM NaCl

  • Stabilizing agents: glycerol (5-10%)

  • Reducing agents: DTT or β-mercaptoethanol if cysteine residues are present

• Secondary purification: Consider size exclusion chromatography (SEC) or ion exchange chromatography as polishing steps to achieve higher purity.

• Quality control assessment:

  • SDS-PAGE analysis (>90% purity should be achievable)

  • Western blot verification

  • Activity assays (if function becomes known)

For storage, maintaining the protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0 has been documented to preserve stability .

What techniques are most appropriate for studying YJL156W-A's potential function?

To elucidate the function of this uncharacterized protein, a multi-omics approach is recommended:

• Computational prediction methods:

  • Sequence homology analysis to identify conserved domains

  • Structural prediction and modeling

  • Protein-protein interaction prediction

• Experimental approaches:

  • Yeast two-hybrid screening to identify interacting proteins

  • Co-immunoprecipitation followed by mass spectrometry

  • Phenotypic analysis of knockout strains under various stress conditions

  • Subcellular localization studies using fluorescent tags

  • Transcriptomic analysis (RNA-seq) of knockout vs. wild-type strains

• Functional genomics techniques:

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

  • CRISPR-based screens for functional relationships

  • Comparative genomics across fungal species

When analyzing data from these approaches, it's crucial to integrate multiple lines of evidence rather than relying on a single technique, as functional predictions for uncharacterized proteins often benefit from converging methodologies.

How can I design appropriate qPCR experiments to study YJL156W-A expression?

When designing qPCR experiments to study YJL156W-A expression, follow these methodological guidelines:

• Primer design considerations:

  • Design primers specific to YJL156W-A, avoiding regions with homology to other yeast genes

  • Optimal amplicon size: 80-150 bp

  • Primer melting temperatures: 58-62°C

  • GC content: 40-60%

  • Check for secondary structures and primer-dimer formation

• Reference gene selection:

  • Use multiple reference genes for normalization (e.g., ACT1, TDH3, ALG9)

  • Validate reference gene stability under your specific experimental conditions

• RNA extraction and quality control:

  • Use methods optimized for yeast cells (which require breaking the cell wall)

  • Verify RNA integrity using microfluidic analyzers or gel electrophoresis

  • Perform DNase treatment to remove genomic DNA contamination

• Data analysis approach:

  • Use the comparative Ct (2^-ΔΔCt) method for relative quantification

  • Include technical and biological replicates (minimum triplicate)

  • Apply appropriate statistical tests based on experimental design

Proper validation of qPCR methods is essential for reliable results, including standard curves to determine efficiency, melt curve analysis, and no-template controls.

How does the genomic context of YJL156W-A influence its functional analysis?

The genomic context of YJL156W-A presents specific challenges for functional analysis due to the compact nature of the S. cerevisiae genome. When investigating this protein, consider:

• Adjacent gene effects: The S. cerevisiae genome has relatively small intergenic regions, creating potential for regulatory overlaps. Any manipulation of YJL156W-A might affect adjacent genes, leading to confounding phenotypes .

• Transcriptional interference: Insertion of marker genes or expression cassettes may disrupt local chromatin structure or introduce new regulatory elements that affect nearby genes.

• Methodological approaches to address these challenges:

  • Use precise genome editing techniques like CRISPR-Cas9 for minimal disruption

  • Employ multiple independent knockout/modification strategies

  • Perform complementation tests with ectopically expressed YJL156W-A

  • Conduct transcriptional analysis of neighboring genes after YJL156W-A manipulation

• Comparative genomics considerations:

  • Examine conservation and synteny of YJL156W-A across fungal species

  • Investigate co-evolution patterns with functionally related genes

Understanding the genomic neighborhood of YJL156W-A provides context for interpreting experimental results and may offer clues to its functional relationships.

What strategies can resolve contradictory experimental results when studying YJL156W-A?

When faced with contradictory results in YJL156W-A research, implement these systematic troubleshooting approaches:

• Validation through methodological triangulation:

  • Confirm findings using multiple independent techniques

  • Vary experimental conditions systematically to identify context-dependent effects

  • Use both gain-of-function and loss-of-function approaches

• Strain background considerations:

  • Verify that the same S. cerevisiae strain background was used across experiments

  • Repeat key experiments in multiple strain backgrounds

  • Check for suppressor mutations that might have arisen during strain construction

• Environmental and experimental variables:

  • Standardize growth conditions (media composition, temperature, growth phase)

  • Control for batch effects in reagents and materials

  • Document detailed experimental protocols to enable precise replication

• Data analysis refinement:

  • Reanalyze raw data using multiple statistical approaches

  • Increase sample size to improve statistical power

  • Consider consulting statistical experts for complex experimental designs

• Collaborative verification:

  • Engage with other laboratories to independently verify key findings

  • Compare protocols in detail to identify subtle methodological differences

Contradictory results often reveal important biological insights about context-dependency or complex regulatory mechanisms, rather than simply representing experimental errors .

How can I design experiments to investigate potential interaction partners of YJL156W-A?

To systematically identify and validate interaction partners of YJL156W-A, implement these methodological approaches:

• High-throughput screening methods:

  • Yeast two-hybrid (Y2H) screening against genomic or cDNA libraries

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

  • Protein microarray analysis with purified YJL156W-A

• Validation techniques for identified interactions:

  • Co-immunoprecipitation experiments

  • Bimolecular fluorescence complementation (BiFC)

  • Fluorescence resonance energy transfer (FRET)

  • Surface plasmon resonance (SPR) for quantitative binding analysis

• Functional validation strategies:

  • Double knockout/knockdown experiments

  • Synthetic genetic interactions analysis

  • Co-localization studies using fluorescently tagged proteins

  • Mutational analysis of interaction domains

• Data integration and network analysis:

  • Connect identified interactions with existing protein interaction networks

  • Perform Gene Ontology enrichment analysis of interacting proteins

  • Use algorithms to predict functional relationships based on interaction patterns

Design your validation experiments with appropriate controls, including:

• Proteins known not to interact with YJL156W-A (negative controls)

• Proteins with partial homology to test specificity

• Mutated versions of YJL156W-A to map interaction domains

What considerations are important when designing complementation studies with YJL156W-A?

Complementation studies are essential for validating phenotypes associated with YJL156W-A mutations. Follow these methodological guidelines:

• Vector selection and design:

  • Choose between integrative or episomal vectors based on expression needs

  • Consider promoter strength (native vs. constitutive vs. inducible)

  • Include appropriate selectable markers different from those used in the knockout strain

  • Add epitope tags if needed for detection, but verify they don't interfere with function

• Controls for rigorous interpretation:

  • Empty vector control

  • Wild-type YJL156W-A expression

  • Point mutants to identify critical residues

  • Expression of homologs from related species for evolutionary analysis

• Expression verification methods:

  • RT-qPCR to confirm transcript levels

  • Western blotting to verify protein expression

  • Fluorescent tagging to assess localization if relevant

• Phenotypic analysis approaches:

  • Design assays specifically targeting the phenotypes observed in knockout strains

  • Include quantitative measurements rather than just qualitative observations

  • Perform time-course experiments to capture dynamic phenotypes

  • Test multiple environmental conditions to identify context-dependent effects

Successful complementation provides strong evidence for direct causality between YJL156W-A and observed phenotypes, ruling out indirect effects from neighboring genes or spontaneous mutations.