Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YJL225W-A (YJL225W-A)

What is YJL225W-A and what are its key characteristics?

YJL225W-A is a putative protein belonging to the UPF0479 family in Saccharomyces cerevisiae. According to available data, it is a relatively small protein consisting of 160 amino acids in full length . While comprehensive functional characterization remains limited, structural analysis indicates it belongs to a conserved protein family with potential functional significance in yeast cellular processes.

For initial characterization, researchers should employ:

• Sequence homology analysis against characterized proteins

• Bioinformatic prediction of functional domains

• Expression profiling under various growth conditions

• Preliminary structural prediction analysis

What expression systems are optimal for recombinant production of YJL225W-A?

Based on available data, YJL225W-A has been successfully expressed as a recombinant protein in E. coli with a His-tag fusion for purification . When selecting an expression system, consider these methodological approaches:

For experimental design considerations:

• Optimize codon usage for the selected expression system

• Test multiple fusion tags (His, GST, MBP) for improved solubility

• Evaluate expression under various induction conditions

• Implement quality control measures via SDS-PAGE and Western blot analysis

How should researchers design experimental controls when studying YJL225W-A function?

When designing experiments to investigate YJL225W-A function, proper controls are essential for result validation:

• Negative controls:

  • Empty vector transformants

  • Expression of an unrelated protein of similar size

  • Wild-type strains without genetic modification

• Positive controls:

  • Well-characterized proteins from the same family (if available)

  • Tagged version of a known protein using identical methodology

  • Validated assay systems with established outcomes

• Experimental validation approaches:

  • Multiple independent transformants/clones analysis

  • Replicate experiments with varied conditions

  • Complementary methodologies to confirm key findings

Following experimental design principles from established literature will ensure rigorous and reproducible results .

What purification strategy is most effective for obtaining high-purity YJL225W-A protein?

For optimal purification of recombinant YJL225W-A, implement a multi-step approach:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) utilizing the His-tag

  • Gentle cell lysis conditions to maintain protein structure

  • Inclusion of protease inhibitors during extraction

• Secondary purification:

  • Size exclusion chromatography for homogeneity

  • Ion exchange chromatography for charge variant separation

  • Affinity chromatography with specific ligands if applicable

• Buffer optimization matrix:

• Quality assessment:

  • SDS-PAGE for purity evaluation (>95% recommended)

  • Western blot for identity confirmation

  • Mass spectrometry for accurate mass determination

  • Dynamic light scattering for homogeneity analysis

How can researchers effectively design CRISPR-based experiments to study YJL225W-A function?

For CRISPR-based functional studies of YJL225W-A, implement the following methodological workflow:

• gRNA design considerations:

  • Target sequences with minimal off-target potential

  • Design multiple gRNAs targeting different regions

  • Consider target accessibility in chromatin context

  • Evaluate potential for homology-directed repair if inserting tags

• Experimental approaches:

  • Complete gene deletion for loss-of-function analysis

  • N- or C-terminal tagging for localization studies

  • Point mutations for structure-function analysis

  • Conditional expression systems for essential gene studies

• Validation strategy:

  • PCR confirmation of intended modifications

  • Sequencing to verify precise editing

  • Expression analysis via RT-qPCR and Western blotting

  • Phenotypic characterization under multiple conditions

• Analysis of complex phenotypes:

  • Growth rate measurements under various conditions

  • Metabolic profiling using mass spectrometry

  • Transcriptomic analysis to identify downstream effects

  • Microscopy to assess morphological changes

This approach follows established experimental design principles for genetic manipulation in yeast systems .

What methodological considerations are important when implementing SMART designs for studying adaptive interventions in YJL225W-A research?

When implementing Sequential Multiple Assignment Randomized Trial (SMART) designs for studying adaptive intervention strategies in YJL225W-A research, consider these methodological principles:

• Key design elements:

  • Define clear tailoring variables (e.g., expression level thresholds)

  • Determine decision points based on measurable outcomes

  • Establish intervention options at each stage

  • Identify primary and secondary endpoints

• Implementation framework:

  • Stage 1: Initial intervention (e.g., different expression conditions)

  • Assessment of intermediate outcomes

  • Stage 2: Adaptive intervention based on response to initial treatment

  • Final outcome measurement

• Analysis considerations:

  • Compare effectiveness of intervention options at different stages

  • Evaluate embedded adaptive interventions

  • Account for sequential randomization in statistical models

  • Consider potential time-varying confounders

This approach adapts the SMART methodology from clinical research to molecular biology applications, allowing for systematic optimization of experimental conditions for YJL225W-A characterization .

How can researchers identify and characterize the interaction network of YJL225W-A?

For comprehensive mapping of YJL225W-A protein interactions, implement these complementary approaches:

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

  • Express tagged YJL225W-A under native conditions

  • Perform gentle lysis and immunoprecipitation

  • Identify co-purifying proteins by mass spectrometry

  • Apply statistical filters to distinguish specific interactions from contaminants

• Yeast two-hybrid screening:

  • Construct bait plasmids with YJL225W-A

  • Screen against genomic or ORFeome prey libraries

  • Validate interactions through secondary assays

  • Map interaction domains through deletion constructs

• Proximity-based labeling approaches:

  • Generate BioID or TurboID fusion constructs

  • Identify proteins in proximal space through biotinylation

  • Compare with control samples for specificity

  • Map subcellular interaction domains

• Interaction network analysis:

What strategies can researchers employ to investigate potential post-translational modifications of YJL225W-A?

For comprehensive analysis of YJL225W-A post-translational modifications (PTMs), implement this systematic approach:

• Sample preparation considerations:

  • Isolate protein under native conditions to preserve in vivo modifications

  • Include phosphatase and deacetylase inhibitors during extraction

  • Prepare samples from various growth conditions and stress responses

  • Generate both bottom-up (peptide) and top-down (intact protein) preparations

• Mass spectrometry-based identification:

  • Employ enrichment strategies for specific PTM types

  • Use fragmentation methods optimized for PTM detection

  • Implement database searching with variable modification parameters

  • Quantify modification stoichiometry when possible

• PTM-specific analytical approaches:

• Functional validation:

  • Generate site-directed mutants of modified residues

  • Analyze phenotypic consequences of mutation

  • Investigate temporal dynamics of modifications

  • Identify enzymes responsible for modification/demodification

How should researchers design experiments to determine the structure-function relationship of YJL225W-A?

To systematically investigate structure-function relationships in YJL225W-A, implement this comprehensive strategy:

• Structural determination approaches:

  • X-ray crystallography of purified protein

  • NMR spectroscopy for solution structure

  • Cryo-EM for larger complexes

  • Computational prediction and modeling as preliminary guide

• Structure-guided mutagenesis:

  • Conserved residue identification through multiple sequence alignment

  • Domain boundary determination through limited proteolysis

  • Alanine-scanning mutagenesis of potential functional sites

  • Chimeric constructs with homologous proteins

• Functional assay development:

  • Growth phenotype analysis under various conditions

  • Protein-protein interaction assessment before and after mutation

  • Subcellular localization determination

  • Biochemical activity assays if enzymatic function is suspected

• Advanced structural analysis techniques:

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Cross-linking mass spectrometry for interaction interfaces

  • SAXS for solution conformation

  • Molecular dynamics simulations for conformational flexibility

Proteinase A from S. cerevisiae provides an excellent model for structural characterization methodology that can be applied to YJL225W-A research .

How can YJL225W-A be utilized in recombinant yeast-based immunotherapy research?

YJL225W-A can be incorporated into yeast-based immunotherapy platforms through these methodological approaches:

• Expression system design:

  • Whole recombinant Saccharomyces cerevisiae expressing YJL225W-A

  • Heat-killed yeast preparations maintaining antigenic epitopes

  • Surface display systems for enhanced immune recognition

  • Multi-epitope constructs combining YJL225W-A with other antigens

• Immunological assessment:

  • T-cell proliferation assays in response to yeast-expressed YJL225W-A

  • Cytokine profiling to characterize immune response quality

  • Antibody generation and characterization

  • Antigen presentation analysis with dendritic cells

• Experimental design considerations:

  • Dose escalation studies to determine optimal immunogenic dose

  • Repeated administration protocols to assess boosting effects

  • Evaluation in relevant animal models

  • Assessment of mutation-specific responses if applicable

This approach adapts established yeast-based immunotherapy methodologies that have shown promise in cancer immunotherapy applications .

What analytical methods are most appropriate for evaluating immune responses to YJL225W-A in experimental models?

For comprehensive immune response evaluation to YJL225W-A, implement these analytical approaches:

• T cell response assessment:

  • Proliferation assays using tritiated thymidine incorporation

  • ELISPOT for cytokine-producing cells quantification

  • Flow cytometry for phenotypic characterization

  • TCR repertoire analysis for clonal expansion

• Antibody response evaluation:

  • ELISA for antibody titer determination

  • Western blot for epitope recognition patterns

  • Avidity assessment through chaotropic ELISAs

  • Functional antibody assays if applicable

• Antigen presentation analysis:

  • MHC-peptide complex detection

  • Dendritic cell activation status measurement

  • Cross-presentation assessment

  • In vitro T cell stimulation with loaded APCs

• Data analysis considerations:

This comprehensive analytical framework adapts methodologies from immunological studies of yeast-based therapeutics .

What approaches should researchers use to analyze evolutionary conservation of YJL225W-A across fungal species?

To investigate evolutionary aspects of YJL225W-A, implement this systematic comparative genomics approach:

• Homology identification:

  • BLAST/PSI-BLAST searches against fungal genome databases

  • Hidden Markov Model construction for remote homolog detection

  • Synteny analysis to identify positional orthologs

  • Paralog identification within S. cerevisiae

• Evolutionary rate analysis:

  • dN/dS calculations to detect selection pressure

  • Sliding window analysis for domain-specific evolution

  • Branch-site models for lineage-specific selection

  • Coevolution analysis with interacting partners

• Structural conservation assessment:

  • Multiple sequence alignment of identified homologs

  • Secondary structure prediction comparison

  • Tertiary structure modeling and comparison

  • Functional site conservation mapping

• Functional divergence investigation:

  • Complementation studies across species

  • Heterologous expression phenotypic analysis

  • Domain swapping experiments

  • Transcriptional regulation comparison

This approach can reveal important insights into the evolutionary history and functional constraints of YJL225W-A, providing context for experimental findings.

How can adaptive intervention methodologies be applied to optimize YJL225W-A expression and purification?

Applying adaptive intervention principles to YJL225W-A expression and purification enables systematic optimization:

• Sequential decision points:

  • Initial expression system selection

  • Mid-course evaluation of expression levels

  • Secondary intervention based on protein solubility

  • Final optimization based on functional activity

• Tailoring variables:

  • Expression level thresholds

  • Solubility percentages

  • Purity metrics

  • Activity measurements

• Stage-specific interventions:

  Stage	Assessment Criteria	Potential Interventions
  Initial expression	Detectable protein	Change vector, host strain, fusion tag
  Solubility evaluation	>30% soluble fraction	Modify lysis conditions, add solubilizing agents
  Purification assessment	>90% purity	Additional chromatography steps, buffer optimization
  Activity determination	Functional assay results	Refolding protocols, stabilizing additives

• Analysis approach:

  • Compare effectiveness of intervention sequences

  • Identify optimal decision rules at each stage

  • Develop adaptive protocols for future production

This methodology adapts principles from sequential multiple assignment randomized trials (SMART) to protein production optimization .