Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YIL177W-A (YIL177W-A)

What is the structural characterization of Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YIL177W-A?

The Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YIL177W-A belongs to the UPF0479 protein family. Based on analysis of the related YLR467C-A protein, it is likely a small protein comprising approximately 150-160 amino acids. The protein family is characterized by conserved hydrophobic regions that may facilitate interaction with the yeast cell wall structure.

For research purposes, the recombinant version is typically expressed with affinity tags (commonly His-tag) to facilitate purification. The protein sequence likely contains structural elements similar to YLR467C-A, which has the sequence: "MMPAKLQLDVLRTLQSSARHGTQTLKNSNFLERFHKDRIVFCLPFFPALFLVPVQKVLQHLCLRFTQVAPYFIIQLFDLPSRHAENLAPLLASCRIQYTNCFSSSSNGQVPSIISLYLRVDLSPFYAKIFQISYRVPMIWLDVFQVFFVFLVISQHSLHS" .

What expression systems are most effective for producing Recombinant YIL177W-A?

Based on experimental protocols for similar S. cerevisiae recombinant proteins, E. coli expression systems have proven effective for producing recombinant YIL177W-A. The optimal expression parameters include:

For experimental applications, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided to maintain protein stability and activity.

What methodologies are most effective for studying YIL177W-A's role in protein secretion pathways?

Advanced study of YIL177W-A's role in protein secretion requires a multifaceted experimental approach:

• CRISPR-Cas9 Gene Disruption: Implementation of CRISPR-Cas9 technology to disrupt YIL177W-A, similar to the approach used for CWP2 and YGP1 disruption as described in the literature. This involves introducing a stop codon (TAA) in the open reading frame (ORF) of the gene .

• Comparative Transcriptomics: RNA-seq analysis comparing wild-type and YIL177W-A-disrupted strains can reveal downstream effects on gene expression, particularly those involved in protein secretion and cell wall organization.

• Secretome Analysis: Quantitative proteomics of the extracellular medium from wild-type and YIL177W-A-disrupted strains to identify differences in the profile of secreted proteins.

The following experimental design is recommended:

How does YIL177W-A expression correlate with stress response in Saccharomyces cerevisiae?

Based on studies of related cell wall proteins, YIL177W-A expression may be regulated in response to various stress conditions. For example, YGP1, another cell wall protein, has been shown to respond to acid stress and participate in flor formation in wine yeast .

To investigate YIL177W-A's role in stress response, researchers should:

• Expose S. cerevisiae to various stress conditions (oxidative, osmotic, temperature, pH) and measure YIL177W-A expression using RT-qPCR or Western blotting.

• Perform comparative growth assays of wild-type and YIL177W-A-disrupted strains under stress conditions.

• Conduct genetic interaction studies to identify synthetic lethal or synthetic sick interactions with known stress response genes.

Preliminary data from studies on similar proteins suggests that UPF0479 family proteins may be upregulated under conditions that challenge cell wall integrity, potentially as part of a compensatory mechanism to maintain cellular integrity during stress conditions.

What is the impact of simultaneous manipulation of YIL177W-A with other cell wall-related genes on recombinant protein production?

Studies on related cell wall proteins have demonstrated that simultaneous manipulation of multiple genes can have synergistic effects on recombinant protein secretion. For example, simultaneous disruption of YGP1 and overexpression of SED5 resulted in a remarkable 2.2-fold increase in extracellular cellobiohydrolase activity compared to the control strain .

Based on these findings, a systematic approach to studying YIL177W-A in combination with other genes should include:

• Creation of double mutants where YIL177W-A is disrupted along with genes like CWP2, YGP1, or UTH1.

• Combination of YIL177W-A disruption with overexpression of genes involved in secretory pathways, such as SED5 or PDI1.

• Assessment of recombinant protein production using a model protein such as cellobiohydrolase.

The following experimental design is recommended:

What purification strategies yield optimal results for recombinant YIL177W-A protein?

For efficient purification of His-tagged recombinant YIL177W-A, the following protocol is recommended:

• Cell Lysis: Use sonication or high-pressure homogenization in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors.

• Initial Purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient elution (10-250 mM imidazole).

• Secondary Purification: Size exclusion chromatography to remove aggregates and contaminants.

• Quality Control: SDS-PAGE and Western blotting to confirm purity (>90% as typically required for structural and functional studies) .

• Storage: Store at -20°C/-80°C in Tris/PBS-based buffer with 6% Trehalose at pH 8.0. For long-term storage, add glycerol to a final concentration of 50% .

Special considerations:

• Avoid repeated freeze-thaw cycles

• For functional studies, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Aliquot before freezing to minimize degradation from multiple thawing events

What analytical methods are recommended for assessing YIL177W-A's interaction with the cell wall?

To characterize YIL177W-A's interaction with the yeast cell wall, a combination of complementary techniques should be employed:

• Fluorescence Microscopy:

  • Express YIL177W-A fused to a fluorescent protein (e.g., GFP)

  • Use confocal microscopy to visualize localization to the cell wall

  • Co-localization studies with known cell wall markers

• Biochemical Fractionation:

  • Separate cell wall, membrane, and cytosolic fractions

  • Detect YIL177W-A in each fraction using Western blotting

  • Quantify relative distribution across cellular compartments

• Surface Plasmon Resonance (SPR):

  • Immobilize purified YIL177W-A on a sensor chip

  • Flow over isolated cell wall components

  • Measure binding kinetics and affinity constants

How can transcriptome analysis be optimized to understand YIL177W-A function in Saccharomyces cerevisiae?

Transcriptome analysis is a powerful approach to understand the functional implications of YIL177W-A. Based on the methodology described for related proteins, the following optimized protocol is recommended:

• Experimental Design:

  • Compare wild-type, YIL177W-A-disrupted, and YIL177W-A-overexpressing strains

  • Include relevant time points (e.g., exponential growth, early stationary phase)

  • Consider including stress conditions to identify condition-specific effects

• RNA Extraction and Quality Control:

  • Use methods optimized for yeast, such as hot phenol extraction

  • Verify RNA integrity using Bioanalyzer (RIN > 8.0)

  • Remove genomic DNA contamination with DNase treatment

• Library Preparation and Sequencing:

  • Use stranded library preparation to distinguish sense and antisense transcription

  • Aim for 20-30 million reads per sample for adequate coverage

  • Include technical and biological replicates (minimum n=3)

• Data Analysis Pipeline:

  • Quality control with FastQC

  • Alignment to S. cerevisiae reference genome using HISAT2 or STAR

  • Differential expression analysis with DESeq2 or edgeR

  • Functional enrichment analysis using GO, KEGG, or custom yeast databases

Based on studies of related proteins, the following gene expression patterns might be expected:

How can comparative genomics approaches illuminate the functional role of YIL177W-A?

Comparative genomics approaches can provide significant insights into YIL177W-A function by examining evolutionary conservation and variation:

• Ortholog Identification:

  • Identify YIL177W-A orthologs across different yeast species

  • Analyze sequence conservation and divergence

  • Map conserved domains that may indicate functional importance

• Synteny Analysis:

  • Examine gene neighborhood conservation across species

  • Identify co-evolving genes that may function in the same pathway

  • Assess whether YIL177W-A maintains consistent genomic context

• Evolutionary Rate Analysis:

  • Calculate the rate of sequence evolution (dN/dS ratio)

  • Identify positions under purifying or positive selection

  • Infer functional constraints from evolutionary conservation patterns

• Function Prediction:

  • Use phylogenetic profiling to predict functional associations

  • Employ computational methods to predict protein structure

  • Integrate with experimental data for function validation

Such analyses can reveal whether YIL177W-A's role in cell wall organization is conserved across species and identify specific structural features that have been maintained throughout evolution, providing insights into critical functional domains.