Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YDR545C-A (YDR545C-A)

What is YDR545C-A and where is it found?

YDR545C-A is a putative UPF0479 protein found in Saccharomyces cerevisiae (Baker's yeast), specifically strain ATCC 204508/S288c. The protein consists of 160 amino acids in its full-length form and is classified as part of the UPF0479 protein family . While the specific cellular location and detailed function of this protein remain under investigation, it exists natively in S. cerevisiae and can be expressed recombinantly in expression systems such as E. coli for research purposes. Understanding this protein's natural context is essential for properly designing experiments to elucidate its function and interactions within yeast cellular processes.

What are the structural features of YDR545C-A?

The YDR545C-A protein consists of 160 amino acids in its full-length form . While detailed structural information about this specific protein is limited in the available sources, as a member of the UPF0479 protein family, it likely shares structural motifs with other members of this group. Researchers working with this protein would typically need to employ techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy to determine its three-dimensional structure. This structural information would provide valuable insights into potential binding sites, functional domains, and possible interaction partners within the yeast cellular environment.

How can I obtain antibodies against YDR545C-A for my research?

Polyclonal antibodies against YDR545C-A are commercially available for research purposes. These antibodies are typically produced by immunizing rabbits with recombinant Saccharomyces cerevisiae YDR545C-A protein . When selecting an antibody for your research, consider factors such as:

• The application (ELISA, Western Blot, etc.)

• Specificity to the target protein

• Host species (commonly rabbit for polyclonal antibodies)

• Purification method (e.g., Antigen Affinity Purified)

• Storage requirements (-20°C or -80°C, avoiding repeated freeze-thaw cycles)

For instance, available antibodies against YDR545C-A are stored in preservative solutions (0.03% Proclin 300) with constituents like 50% Glycerol and 0.01M PBS at pH 7.4 . These antibodies are typically supplied in liquid form and should be handled according to the manufacturer's specifications to maintain their activity and specificity.

What expression systems are recommended for recombinant YDR545C-A production?

Based on available research data, E. coli has been successfully employed as an expression system for producing recombinant YDR545C-A protein with a His-tag . When designing an expression strategy, consider the following methodological aspects:

• Vector selection: Choose a vector with an appropriate promoter (constitutive or inducible)

• Affinity tag placement: N-terminal or C-terminal His-tag affects purification efficiency

• Expression conditions: Optimize temperature, induction time, and media composition

• Cell lysis methods: Sonication, homogenization, or enzymatic lysis

The choice of expression system should be guided by your specific research needs. While E. coli is commonly used due to its rapid growth and high protein yields, yeast expression systems may provide more native-like post-translational modifications for some applications. For structural studies or functional assays, the expression system should be chosen to maximize protein stability and proper folding.

What are the optimal purification strategies for His-tagged YDR545C-A?

Purification of His-tagged YDR545C-A typically employs immobilized metal affinity chromatography (IMAC) as the primary capture step. A methodological approach to purification would include:

• Cell lysis: Disruption of cells in an appropriate buffer containing protease inhibitors

• Clarification: Centrifugation or filtration to remove cell debris

• IMAC: Capture using Ni-NTA or similar resin

• Washing: Sequential washing with increasing imidazole concentrations

• Elution: Collection of purified protein with high imidazole buffer

• Buffer exchange: Dialysis or gel filtration to remove imidazole and adjust final buffer composition

For obtaining highly pure protein preparations suitable for structural studies or interaction analysis, additional purification steps such as ion exchange chromatography or size exclusion chromatography may be necessary. The purity of the final preparation should be confirmed using SDS-PAGE, and protein concentration can be determined using Bradford or BCA assays, with typical yields dependent on the specific expression conditions employed.

What are the known or predicted functions of YDR545C-A?

The specific functions of YDR545C-A remain largely uncharacterized in the current scientific literature. As a putative UPF0479 family protein, its biological role is still being investigated. When approaching functional studies, researchers should consider:

• Sequence homology analysis: Comparing with functionally characterized homologs

• Structural predictions: Using computational tools to infer potential activity

• Gene knockout studies: Examining phenotypic effects of YDR545C-A deletion

• Localization experiments: Determining subcellular distribution using tagged constructs

The limited information about biological pathways involving YDR545C-A suggests this as an area requiring further investigation . Researchers working on this protein have an opportunity to make significant contributions to our understanding of its role in yeast biology.

How can I identify potential interaction partners of YDR545C-A?

Identifying protein-protein interactions for YDR545C-A requires systematic experimental approaches. A comprehensive methodology would include:

• Yeast two-hybrid screening: Using YDR545C-A as bait to identify potential binding partners

• Co-immunoprecipitation: Pulling down protein complexes using anti-YDR545C-A antibodies

• Pull-down assays: Using recombinant His-tagged YDR545C-A as bait

• Mass spectrometry: Analyzing co-purified proteins to identify interaction partners

• Surface plasmon resonance: Quantifying binding kinetics with candidate interactors

Each method has specific advantages and limitations, and a combination of approaches is often necessary to establish reliable interaction networks. While current research data does not specify confirmed interaction partners for YDR545C-A , these methodological strategies would be appropriate for researchers seeking to expand knowledge in this area.

How can YDR545C-A be utilized in recombinant yeast vaccine development?

While direct evidence for using YDR545C-A in vaccine development is not present in the search results, the principles of using Saccharomyces cerevisiae as a vaccine vehicle provide a framework for potential applications. Recombinant S. cerevisiae has been successfully used to express heterologous antigens for immune response induction . For researchers interested in exploring YDR545C-A in this context, consider:

• Antigen presentation: Investigating whether YDR545C-A can act as a carrier protein

• Immunogenicity studies: Determining immune responses elicited by YDR545C-A

• Adjuvant properties: Assessing if the protein enhances immune responses to co-delivered antigens

• Safety profiling: Evaluating potential adverse effects in model systems

The success of S. cerevisiae as a vaccine vehicle stems from its ability to induce dendritic cell maturation and facilitate antigen presentation via both MHC class I and II pathways . Researchers could explore whether YDR545C-A possesses unique properties that could be leveraged in vaccine design, particularly for applications requiring immune responses against yeast-derived antigens.

What considerations are important when designing CRISPR/Cas9 experiments to study YDR545C-A function?

When employing CRISPR/Cas9 technology to investigate YDR545C-A function in S. cerevisiae, researchers should implement a methodical approach:

• Guide RNA design: Select target sequences with minimal off-target effects

• Delivery method: Optimize transformation protocols for S. cerevisiae

• Screening strategy: Develop efficient methods to identify successful gene edits

• Phenotypic analysis: Establish assays to detect functional consequences of YDR545C-A modification

• Complementation studies: Reintroduce wild-type or mutant variants to confirm specificity

The compact genome of S. cerevisiae makes it particularly amenable to CRISPR/Cas9 editing, but care must be taken to avoid disrupting adjacent genes or regulatory elements. When analyzing phenotypes, researchers should examine growth rates, stress responses, and metabolic profiles under various conditions to identify subtle effects of YDR545C-A modification that might reveal functional insights.

What are common difficulties in detecting YDR545C-A expression and how can they be overcome?

Researchers may encounter challenges when attempting to detect YDR545C-A expression, particularly due to potential low abundance or antibody specificity issues. A methodological approach to troubleshooting includes:

• Antibody validation: Confirm antibody specificity using positive and negative controls

• Protein extraction optimization: Modify lysis buffers to improve solubilization

• Detection enhancement: Employ more sensitive techniques like chemiluminescence

• Enrichment strategies: Consider immunoprecipitation before Western blotting

• Alternative detection methods: Use mass spectrometry for antibody-independent verification

For Western blot applications, researchers should optimize primary antibody concentration and incubation conditions. The polyclonal antibodies available for YDR545C-A have been validated for ELISA and Western blot applications , but specific experimental conditions may require further optimization. When troubleshooting, systematically modify one variable at a time and maintain appropriate controls to identify the source of technical difficulties.

How can I address solubility issues when working with recombinant YDR545C-A?

Recombinant proteins often present solubility challenges during expression and purification. For YDR545C-A, consider the following methodological approaches:

• Expression temperature modulation: Lower temperatures (16-20°C) often improve folding

• Buffer optimization: Screen different pH values, salt concentrations, and additives

• Solubility tags: Consider fusion partners like MBP, SUMO, or GST

• Refolding protocols: Develop strategies for solubilizing and refolding inclusion bodies

• Co-expression with chaperones: Introduce molecular chaperones to assist folding

The choice of purification buffer components can significantly impact protein solubility. Common additives that may improve YDR545C-A solubility include glycerol (5-10%), low concentrations of non-ionic detergents, and stabilizing agents like arginine or proline. Screening a matrix of buffer conditions using small-scale expression tests can identify optimal parameters before scaling up production.

How can structural studies of YDR545C-A contribute to understanding UPF0479 protein family function?

Structural characterization of YDR545C-A could provide significant insights into the UPF0479 protein family. A comprehensive research approach would include:

• X-ray crystallography: Determining high-resolution structure through protein crystallization

• NMR spectroscopy: Analyzing dynamic properties and potential conformational changes

• Cryo-electron microscopy: Visualizing larger complexes involving YDR545C-A

• Computational modeling: Predicting functional sites based on structural features

• Structure-function correlations: Testing predictions through mutagenesis studies

The resulting structural data would allow researchers to identify conserved domains, potential active sites, or binding interfaces that might suggest functional roles. Comparative analysis with structures of other UPF0479 family members could reveal evolutionary relationships and functional adaptations. Such knowledge would advance our understanding of this protein family and potentially uncover novel biological functions or therapeutic targets.

What role might YDR545C-A play in Saccharomyces cerevisiae stress responses?

Investigating the potential role of YDR545C-A in yeast stress responses represents an important research direction. A methodological approach would include:

• Gene expression analysis: Examining YDR545C-A regulation under various stress conditions

• Knockout phenotyping: Assessing stress sensitivity of YDR545C-A deletion strains

• Localization studies: Tracking protein redistribution during stress exposure

• Interaction mapping: Identifying stress-dependent binding partners

• Biochemical assays: Measuring activity changes in response to stress conditions

Researchers could expose wild-type and YDR545C-A mutant yeast to stressors such as oxidative agents, heat shock, osmotic pressure, or nutrient deprivation, and compare growth rates, viability, and molecular responses. Such studies would provide insights into whether YDR545C-A contributes to specific stress adaptation mechanisms and could reveal novel aspects of yeast cellular physiology.