Recombinant Saccharomyces cerevisiae Putative uncharacterized membrane protein YBR064W (YBR064W)

What expression systems are most effective for recombinant YBR064W production?

While YBR064W is naturally found in S. cerevisiae, E. coli has been successfully used as an expression system for recombinant production. The recombinant form with an N-terminal His-tag expressed in E. coli provides good yield and maintains the protein's structural integrity .

For optimal expression in E. coli, induction conditions should be carefully controlled, with typical protocols using IPTG at lower temperatures (16-20°C) to allow proper folding of membrane proteins.

How stable is recombinant YBR064W under standard laboratory conditions?

Recombinant YBR064W demonstrates moderate stability under laboratory conditions. The lyophilized powder form provides excellent long-term stability, while reconstituted protein requires careful handling. Storage recommendations include keeping the protein at -20°C/-80°C, with working aliquots maintained at 4°C for up to one week . Repeated freeze-thaw cycles significantly reduce protein integrity and should be avoided.

The protein is typically stored in Tris/PBS-based buffer containing 6% Trehalose at pH 8, which helps maintain stability . Trehalose acts as a cryoprotectant and helps preserve protein structure during freezing and lyophilization processes. Researchers should monitor protein degradation using SDS-PAGE before critical experiments.

What purification strategies yield the highest purity for recombinant YBR064W?

The His-tagged recombinant YBR064W can be effectively purified using immobilized metal affinity chromatography (IMAC). A typical purification protocol involves:

• Cell lysis using mechanical disruption with appropriate detergents (e.g., n-dodecyl β-D-maltoside)

• Clarification by centrifugation (typically 20,000×g for 30 minutes)

• IMAC purification using Ni-NTA or similar resin

• Size exclusion chromatography for final polishing

This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE . For membrane proteins like YBR064W, maintaining the correct detergent concentration throughout purification is critical to prevent aggregation while preserving native-like structure.

What analytical methods are recommended for validating recombinant YBR064W identity and purity?

For researchers studying YBR064W, combining SDS-PAGE for routine purity assessment with at least one orthogonal method (typically mass spectrometry) is recommended for definitive protein validation before proceeding to functional studies.

How can researchers effectively reconstitute YBR064W for functional studies?

Since YBR064W is supplied as a lyophilized powder , proper reconstitution is critical for maintaining function. A methodological approach includes:

• Equilibrate the lyophilized protein to room temperature before opening

• Reconstitute using a buffer matching the final application (typically Tris/PBS at pH 8)

• For membrane protein studies, incorporate appropriate detergents or lipids:

  • For detergent micelles: 0.1-0.5% DDM or LMNG

  • For proteoliposomes: E. coli polar lipids or synthetic mixtures (POPC:POPE:POPG)

• Gentle mixing without vortexing to prevent denaturation

• Centrifugation (14,000×g, 10 min) to remove any insoluble material

• Validation of proper folding using circular dichroism or fluorescence spectroscopy

When designing reconstitution protocols, researchers should consider the membrane environment needed for the specific functional assay being performed.

What experimental designs are recommended for investigating YBR064W's potential functional role?

Given that YBR064W is a putative uncharacterized membrane protein, researchers should employ multiple complementary approaches to elucidate its function:

• Genetic approaches:

  • CRISPR-Cas9 knockouts in S. cerevisiae to observe phenotypic effects

  • Synthetic genetic arrays to identify genetic interactions

  • Complementation studies with mutant variants

• Biochemical approaches:

  • Protein-protein interaction studies using pull-downs with the His-tagged protein

  • Lipid binding assays to identify potential lipid interactions

  • Activity assays based on predicted functions from bioinformatic analysis

• Structural approaches:

  • Cryo-EM studies of the purified protein in detergent or lipid nanodiscs

  • X-ray crystallography (challenging for membrane proteins)

  • NMR for dynamics studies of specific domains

Quasi-experimental designs can help establish causality when randomized experiments are infeasible . For instance, regression discontinuity designs or instrumental variable approaches may help identify causal relationships between YBR064W function and cellular phenotypes in complex biological systems.

How can researchers employ comparative genomics to predict YBR064W function?

Comparative genomics provides valuable insights for uncharacterized proteins like YBR064W. A methodological approach includes:

• Perform sequence homology searches across species using BLAST and HMM-based methods

• Identify conserved domains and motifs through tools like InterPro and PFAM

• Examine genomic context in S. cerevisiae and related yeast species

• Analyze co-expression patterns with genes of known function

• Investigate protein structure prediction using AlphaFold2 or similar tools

This approach allows researchers to move beyond the "putative uncharacterized" designation by generating testable hypotheses about YBR064W function based on evolutionary conservation patterns.

What systems biology approaches can integrate YBR064W into cellular pathway models?

For understanding YBR064W in a broader cellular context:

• Transcriptomics:

  • RNA-seq analysis comparing wild-type and YBR064W deletion strains

  • Identification of differentially expressed genes to infer pathway involvement

• Proteomics:

  • Proximity labeling approaches (BioID, APEX) using YBR064W as bait

  • Quantitative proteomics to identify proteins affected by YBR064W deletion

• Metabolomics:

  • Untargeted metabolomics to identify metabolic alterations in YBR064W mutants

  • Flux analysis to determine impact on specific metabolic pathways

• Network analysis:

  • Integration of multi-omics data to position YBR064W in cellular networks

  • Pathway enrichment analysis of interacting partners

These systems approaches can reveal unexpected connections between YBR064W and cellular processes, generating novel hypotheses for targeted experimental validation.

What are common challenges in working with recombinant YBR064W and how can they be addressed?

For membrane proteins like YBR064W, protein engineering approaches such as fusion to solubility-enhancing tags (MBP, SUMO) or truncation of highly hydrophobic regions may improve handling while preserving essential structural features for study.

How should researchers address contradictory data regarding YBR064W localization or function?

When confronted with contradictory data:

• Methodological validation:

  • Verify antibody specificity using knockout controls

  • Employ multiple orthogonal techniques for localization (fluorescence microscopy, subcellular fractionation)

  • Use different epitope tags (N-terminal vs. C-terminal) to control for tagging artifacts

• Condition-dependent analysis:

  • Test different growth conditions and stress responses

  • Examine cell-cycle dependence of localization/function

  • Investigate strain-specific differences

• Technical reconciliation:

  • Develop a unified experimental framework to test competing hypotheses

  • Use collaborative blind testing to eliminate experimenter bias

  • Apply statistical approaches like meta-analysis to resolve contradictions

The quasi-experimental design principles discussed in search result can be valuable when designing experiments to resolve conflicting data, particularly by carefully controlling for confounding variables and establishing appropriate control groups.

What controls are essential when studying potential interactions of YBR064W with other cellular components?

Rigorous controls are essential for interaction studies involving YBR064W:

• For pull-down experiments:

  • Empty vector/tag-only negative control

  • Known non-interacting protein negative control

  • Competitive binding with untagged protein

  • RNase/DNase treatment to eliminate nucleic acid-mediated interactions

• For localization studies:

  • Marker proteins for different cellular compartments

  • Multiple fixation and permeabilization methods

  • Live cell imaging to eliminate fixation artifacts

• For functional assays:

  • Temperature-sensitive mutants as positive controls

  • Proper wild-type and deletion controls

  • Complementation controls to verify specificity

Implementing these controls helps distinguish true biological interactions from technical artifacts, a critical consideration when working with putative uncharacterized proteins like YBR064W where functional information is limited.

How might YBR064W research connect to broader studies of membrane proteins in S. cerevisiae?

YBR064W research can contribute to broader membrane protein biology through:

• Serving as a model for studying membrane protein folding and topology in eukaryotes

• Providing insights into membrane protein quality control mechanisms

• Expanding our understanding of the approximately 30% of proteins encoded by the S. cerevisiae genome that remain functionally uncharacterized

Future studies could leverage approaches like those used with other recombinant S. cerevisiae systems, where whole recombinant yeast has been engineered to express target proteins for immunological studies . Such approaches might reveal unexpected functions for YBR064W in cellular processes beyond current predictions.

What emerging technologies show promise for structural and functional characterization of YBR064W?

Several cutting-edge technologies hold particular promise:

• Cryo-electron tomography for visualizing YBR064W in its native membrane environment

• Single-particle cryo-EM for high-resolution structural determination

• AlphaFold2 and RoseTTAFold for computational structure prediction

• Native mass spectrometry for analyzing intact membrane protein complexes

• Hydrogen-deuterium exchange mass spectrometry for dynamics and interaction mapping

• Single-molecule FRET for conformational dynamics studies

These technologies, when applied to the recombinant His-tagged YBR064W described in the literature , could provide unprecedented insights into this uncharacterized protein's structure and function.