Recombinant Schizosaccharomyces pombe Putative oxidoreductase C6G10.06 (SPAC6G10.06)

What are the optimal storage conditions for Recombinant S. pombe Putative oxidoreductase C6G10.06?

The shelf life and activity of Recombinant S. pombe Putative oxidoreductase C6G10.06 are influenced by multiple factors including buffer composition, storage temperature, and the intrinsic stability of the protein itself. For liquid formulations, the recommended storage period is up to 6 months at -20°C/-80°C, while lyophilized forms maintain stability for approximately 12 months at the same temperature range .

To maximize stability, aliquot the reconstituted protein to minimize freeze-thaw cycles, as repeated freezing and thawing significantly diminishes enzymatic activity. Working aliquots can be stored at 4°C for up to one week for ongoing experiments . For long-term storage, addition of glycerol to a final concentration between 5-50% is recommended before aliquoting and freezing at -20°C/-80°C, with 50% being the default concentration used by manufacturers .

What is the recommended reconstitution protocol for the lyophilized form of this oxidoreductase?

For optimal reconstitution of lyophilized Recombinant S. pombe Putative oxidoreductase C6G10.06, begin by centrifuging the vial briefly to ensure all material settles at the bottom. Reconstitute the protein using deionized sterile water to achieve a final concentration between 0.1-1.0 mg/mL .

The reconstitution process should be performed gently, avoiding vigorous shaking or vortexing which could lead to protein denaturation. After adding the appropriate volume of water, allow the protein to dissolve by gentle inversion or rotation for several minutes at room temperature. For enhanced long-term stability post-reconstitution, add glycerol to a final concentration of 5-50% before aliquoting and storing at -20°C/-80°C .

How is the purity of this recombinant protein determined and what level of purity can researchers expect?

The purity of Recombinant S. pombe Putative oxidoreductase C6G10.06 is primarily determined using SDS-PAGE analysis. The commercially available preparations typically demonstrate a purity level exceeding 85% as assessed by SDS-PAGE .

Researchers should note that this assessment reflects protein purity in terms of contaminant proteins, but does not necessarily indicate the percentage of properly folded, enzymatically active protein molecules. For applications requiring confirmation of enzymatic activity, it is advisable to perform specific oxidoreductase activity assays prior to experimental use. Additional purity assessments might include size exclusion chromatography or mass spectrometry to verify protein integrity and homogeneity.

What expression system is used for producing this recombinant oxidoreductase?

Recombinant S. pombe Putative oxidoreductase C6G10.06 is primarily produced using Escherichia coli expression systems . This prokaryotic expression platform offers several advantages for recombinant protein production, including rapid growth rates, high protein yields, and well-established genetic manipulation techniques.

What experimental approaches can be used to assess the enzymatic activity of S. pombe Putative oxidoreductase C6G10.06?

Assessing the enzymatic activity of S. pombe Putative oxidoreductase C6G10.06 requires specialized assays tailored to oxidoreductase function. Since this enzyme is classified as EC 1.-.-.- (indicating an oxidoreductase with incompletely characterized specificity) , researchers should consider a multi-faceted approach:

• Spectrophotometric Assays: Monitor oxidation of NADH/NADPH or reduction of artificial electron acceptors like 2,6-dichloroindophenol (DCIP) or cytochrome c at relevant wavelengths (340nm for NADH/NADPH).

• ROS Generation Analysis: Utilize H2DCFDA fluorescence assays similar to those employed in S. pombe studies to assess potential roles in reactive oxygen species (ROS) metabolism, as observed in other S. pombe oxidative stress research .

• Substrate Screening: Systematically test potential physiological substrates based on structural homology to characterized oxidoreductases.

• Oxygen Consumption Measurements: Using oxygen electrodes to measure enzyme-dependent oxygen consumption rates in the presence of different substrates.

The experimental conditions should mimic physiological S. pombe environments, maintaining pH between 5.0-7.0 and temperatures around 30°C, which represent optimal growth conditions for this organism.

How might S. pombe oxidoreductase C6G10.06 function in oxidative stress responses?

S. pombe oxidoreductase C6G10.06 may play a significant role in cellular defenses against oxidative stress. Research on S. pombe has shown that oxidative stress induces numerous defensive functions both directly (H2O2-responsive) and indirectly (cadmium-responsive) .

The oxidoreductase likely participates in redox homeostasis pathways that protect against reactive oxygen species (ROS) accumulation. In S. pombe, ROS accumulation has been observed in the nucleus and mitochondria under stress conditions, as demonstrated by H2DCFDA fluorescence studies . The oxidoreductase may catalyze reactions that either prevent ROS formation or facilitate their neutralization.

Based on observations of S. pombe stress responses, this oxidoreductase might be transcriptionally induced along with other H2O2-responsive proteins during oxidative stress . Experimental approaches to verify this role could include:

• Comparing wild-type and oxidoreductase-deficient S. pombe strains for survival under H2O2 challenge

• Measuring cellular antioxidant metabolites like glutathione (GSH) and ergothioneine in response to oxidoreductase expression levels

• Assessing protein levels in response to oxidative stressors using quantitative proteomics approaches

What is the relationship between S. pombe oxidoreductase C6G10.06 and mitochondrial function?

The relationship between S. pombe oxidoreductase C6G10.06 and mitochondrial function likely involves redox regulation and protection against oxidative damage. Studies in S. pombe have demonstrated that mitochondria are significant sites of ROS accumulation, particularly under stress conditions .

The putative oxidoreductase may function in:

• Mitochondrial Redox Balance: Catalyzing reactions that maintain proper electron flow through respiratory complexes, preventing electron leakage that leads to ROS formation.

• Mitochondrial Quality Control: Contributing to mechanisms that identify and repair oxidatively damaged mitochondrial components.

• Metabolic Adaptation: Facilitating metabolic shifts in response to changing redox states within the cell.

Research in S. pombe has shown that mitochondrial proteins like Sdh2 (succinate dehydrogenase) are subject to regulation during stress responses . The oxidoreductase may interact with such respiratory chain components to modulate their activity or participate in their quality control.

Experimental approaches to investigate this relationship could include:

• Subcellular localization studies using fluorescently tagged oxidoreductase

• Mitochondrial functional assays in oxidoreductase-deficient strains

• Assessment of mitochondrial ROS levels using indicators like MitoSOX or H2DCFDA in relation to oxidoreductase activity

What methods can be used to study potential protein-protein interactions involving S. pombe oxidoreductase C6G10.06?

Investigating protein-protein interactions involving S. pombe oxidoreductase C6G10.06 requires multi-faceted approaches:

• Co-Immunoprecipitation (Co-IP): Using antibodies against tagged versions of the oxidoreductase to pull down protein complexes from S. pombe lysates, followed by mass spectrometry-based identification of interacting partners.

• Yeast Two-Hybrid Screening: Creating fusion constructs of the oxidoreductase with DNA-binding domains and screening against S. pombe cDNA libraries to identify potential interactors.

• Bimolecular Fluorescence Complementation (BiFC): Expressing the oxidoreductase fused to one half of a fluorescent protein and candidate partners fused to the complementary half, allowing visualization of interactions in living cells.

• Proximity-Dependent Biotin Identification (BioID): Fusing the oxidoreductase to a biotin ligase to biotinylate proximal proteins, which can then be isolated and identified.

• Quantitative Proteomics: Comparing protein abundance in wild-type versus oxidoreductase-deficient strains under various conditions, similar to the emPAI value analysis methods used in S. pombe research .

When planning these experiments, researchers should consider potential challenges in preserving weak or transient interactions that may be physiologically significant. Sample preparation should be optimized to maintain native protein conformations and interaction networks.

How can S. pombe oxidoreductase C6G10.06 be used to study chronological lifespan in yeast models?

S. pombe oxidoreductase C6G10.06 represents a valuable tool for investigating chronological lifespan in yeast models, particularly through its potential roles in redox regulation and stress response. Research has established that oxidative stress management is critical for G0 phase maintenance and chronological lifespan in S. pombe .

Methodological approaches include:

• Gene Deletion/Mutation Studies: Creating C6G10.06 deletion or point mutation strains to assess the impact on chronological lifespan under various nutritional and stress conditions.

• Complementation Experiments: Reintroducing wild-type or mutant versions of the oxidoreductase into deletion strains to identify functionally important domains or residues.

• Integration with Other Pathways: Combining oxidoreductase mutations with defects in other longevity pathways, such as proteasome function or autophagy, to understand potential collaborative roles. For example, research has shown that proteasome dysfunction in S. pombe G0 phase leads to ROS accumulation that can be partially mitigated by autophagy .

• Metabolomic Analysis: Measuring changes in metabolites related to oxidative stress protection, such as glutathione and ergothioneine, in response to oxidoreductase manipulation .

• Viability Assessments: Using standard techniques like colony-forming unit counts to track chronological survival curves of strains with varying oxidoreductase activity levels.

This research could provide insights into fundamental mechanisms of cellular aging and stress resistance applicable beyond yeast models.

What techniques are recommended for structural characterization of S. pombe oxidoreductase C6G10.06?

Comprehensive structural characterization of S. pombe oxidoreductase C6G10.06 requires multiple complementary techniques:

These approaches can be complemented by bioinformatic analyses such as:

• Sequence alignment with structurally characterized oxidoreductases

• Secondary structure prediction

• Domain organization analysis

• Evolutionary conservation mapping

The recombinant protein's purity (>85% by SDS-PAGE) would need to be improved for most structural biology applications, typically requiring >95% homogeneity.

What are common challenges in maintaining enzymatic activity of S. pombe oxidoreductase C6G10.06 during experimental procedures?

Maintaining enzymatic activity of S. pombe oxidoreductase C6G10.06 during experimental procedures presents several challenges researchers should address:

• Oxidation Sensitivity: As an oxidoreductase, the protein may be particularly susceptible to oxidative inactivation. Including reducing agents like DTT or β-mercaptoethanol (1-5 mM) in buffers can help preserve activity.

• Metal Ion Requirements: Many oxidoreductases require specific metal cofactors for activity. Buffers should be supplemented with physiologically relevant concentrations of potential cofactors (e.g., Fe²⁺, Zn²⁺, Cu²⁺) or EDTA should be avoided in buffers if metal dependency is suspected.

• Temperature Stability: While the protein may be stored at -20°C/-80°C long-term , activity assays should be performed at temperatures reflecting physiological conditions for S. pombe (25-30°C).

• Buffer Optimization: The pH, ionic strength, and specific buffer components can significantly impact enzymatic activity. A range of conditions should be tested systematically (pH 5.0-8.0, 50-200 mM salt).

• Protein Concentration Effects: Oxidoreductases may display concentration-dependent activity profiles due to potential oligomerization. Activity should be assessed across a range of protein concentrations (0.1-10 μg/mL).

• Substrate Availability: Ensuring appropriate substrate concentrations and preventing substrate degradation during extended assays is critical for accurate activity measurement.

Implementing a quality control workflow that includes activity checks before each experimental session will help ensure reproducibility and reliability of results.

How can researchers distinguish between different isoforms or closely related oxidoreductases when studying S. pombe C6G10.06?

Distinguishing between S. pombe oxidoreductase C6G10.06 and related enzymes requires a strategic approach combining multiple techniques:

• Specific Antibody Development: Generate antibodies against unique peptide regions of C6G10.06 identified through sequence alignment with related S. pombe oxidoreductases.

• Mass Spectrometry-Based Identification: Use targeted proteomics approaches like Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) to monitor peptides unique to C6G10.06.

• Substrate Specificity Profiling: Systematically characterize kinetic parameters (Km, Vmax, kcat) with various substrates to create a "fingerprint" of activity that distinguishes C6G10.06 from related enzymes.

• Gene Deletion Verification: In genetic studies, confirm successful deletion or mutation of specifically C6G10.06 through both genomic PCR and RT-PCR to verify absence of the targeted gene and its transcript.

• Isoform-Specific Inhibition: Develop or identify inhibitors with differential potency against C6G10.06 versus related oxidoreductases.

• Expression Pattern Analysis: Monitor tissue-specific or condition-dependent expression patterns that may differ between C6G10.06 and related enzymes.

When analyzing experimental data, researchers should always consider the possibility of functional redundancy among related oxidoreductases, which may mask phenotypes in single-gene deletion studies.

What are promising future research directions regarding S. pombe oxidoreductase C6G10.06?

Research on S. pombe oxidoreductase C6G10.06 presents several promising avenues for future investigation:

• Comprehensive Substrate Identification: Employing metabolomics approaches to identify physiological substrates, potentially revealing previously unknown metabolic pathways in S. pombe.

• Integration with Stress Response Networks: Exploring how this oxidoreductase coordinates with other cellular defenses against oxidative stress, particularly in relation to proteasome function and autophagy pathways already implicated in S. pombe stress responses .

• Translational Applications: Investigating whether insights from this S. pombe oxidoreductase can inform understanding of related human enzymes and their roles in disease states characterized by redox imbalance.

• Evolutionary Conservation Analysis: Comparing the function of this oxidoreductase across different yeast species and more complex eukaryotes to understand conservation of redox regulatory mechanisms.

• Synthetic Biology Applications: Exploring potential biotechnological applications, such as using modified versions of the oxidoreductase for biocatalysis or biosensor development.

• Structural Biology and Drug Design: Determining the three-dimensional structure to facilitate rational design of specific modulators that could serve as research tools or potential therapeutic leads.