Recombinant Schizosaccharomyces pombe Uncharacterized protein C1450.16c (SPCC1450.16c)

How is SPCC1450.16c classified in genomic databases?

In genomic databases, SPCC1450.16c is classified as follows:

What experimental evidence exists for SPCC1450.16c expression?

Several studies have detected SPCC1450.16c expression in various experimental conditions:

• Gene expression profiling studies have identified SPCC1450.16c among the differentially expressed genes in S. pombe under various stress conditions .

• In Upf1 target identification studies, SPCC1450.16c (listed as SPCC1450.01c in Table 3) was identified as a pseudogene that is potentially regulated by the nonsense-mediated mRNA decay (NMD) pathway .

• Microarray analyses of S. pombe under various conditions have detected expression of this gene, suggesting it is transcriptionally active in certain cellular states .

How does SPCC1450.16c expression change under different experimental conditions?

Analysis of global gene expression profiles has revealed that SPCC1450.16c expression changes under various experimental conditions:

• Stress Response: While not specifically highlighted in the provided search results, studies of stress responses in S. pombe have identified numerous genes with altered expression, including potentially SPCC1450.16c .

• Cell Cycle Regulation: The expression may be cell cycle-regulated, as extensive studies have been performed to identify periodically expressed genes in S. pombe .

• Nutrient Conditions: Some genes related to SPCC1450.16c function have shown differential expression under nitrogen starvation conditions, suggesting potential involvement in metabolic adaptation .

• Genetic Perturbations: Experiments using mutant strains such as the Spc1 kinase overexpression have identified numerous differentially expressed genes in the S. pombe genome, which could include SPCC1450.16c depending on its function in relevant pathways .

What is known about the potential enzymatic activity of SPCC1450.16c?

Key points about its predicted enzymatic activity:

• The presence of conserved catalytic domains typical of lipases suggests it may catalyze the hydrolysis of ester bonds, particularly in lipids.

• The protein likely participates in lipid metabolism pathways, potentially involving the breakdown of storage lipids or modification of membrane lipids.

• Experimental validation of this enzymatic activity would require:

  • Expression and purification of the recombinant protein

  • Enzyme activity assays using appropriate lipid substrates

  • Determination of substrate specificity and kinetic parameters

  • Analysis of the effects of pH, temperature, and potential inhibitors

What are the predicted interaction partners of SPCC1450.16c?

• Lipid metabolism enzymes and regulators

• Membrane-associated protein complexes

• Lipid transport proteins

• Signaling pathways related to metabolic regulation

To identify interaction partners experimentally, researchers could employ:

• Yeast two-hybrid screening

• Co-immunoprecipitation followed by mass spectrometry

• Proximity-dependent biotin labeling methods

• Genetic interaction screens using S. pombe deletion libraries

What are the optimal conditions for expression and purification of recombinant SPCC1450.16c?

Based on the available information for commercially produced recombinant SPCC1450.16c:

Expression Systems:

• E. coli has been successfully used for heterologous expression

• Yeast expression systems may also be suitable, particularly for obtaining post-translational modifications

Expression Constructs:

• His-tagged versions (typically N-terminal) have been reported

• Full-length constructs (1-513 amino acids) have been successfully expressed

Purification Approach:

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

• Buffer optimization, typically using Tris-based buffers (pH 8.0)

• Inclusion of glycerol (6-50%) for stability during storage

Recommended Storage:

• Store at -20°C/-80°C upon receipt

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

What methods are available for studying SPCC1450.16c function in vivo?

Several methodological approaches can be employed to study SPCC1450.16c function in vivo:

• Gene Deletion/Knockout:

  • CRISPR-Cas9 editing in S. pombe

  • Homologous recombination-based gene replacement

  • Analysis of resulting phenotypes under various conditions

• Expression Modulation:

  • Regulated promoter systems like nmt1 (thiamine-repressible) or urg1 (rapidly inducible)

  • Analysis of the effects of overexpression or repression

• Localization Studies:

  • Fluorescent protein tagging (GFP, mCherry)

  • Immunofluorescence microscopy with specific antibodies

• Genetic Interaction Screens:

  • Synthetic genetic array (SGA) analysis

  • Suppressor screens to identify functional relationships

• Transcriptome Analysis:

  • RNA-seq or microarray analysis of mutant strains

  • Identification of genes affected by SPCC1450.16c manipulation

How can researchers detect and quantify SPCC1450.16c in experimental samples?

Several methods are available for detection and quantification of SPCC1450.16c:

• Antibody-Based Methods:

  • Western blotting using specific antibodies against SPCC1450.16c or epitope tags

  • Immunoprecipitation for enrichment and detection

  • ELISA for quantitative measurement

• Mass Spectrometry:

  • Targeted proteomics approaches such as selected reaction monitoring (SRM)

  • Label-free quantification or isotope labeling methods (SILAC, TMT)

• mRNA Quantification:

  • RT-qPCR for transcript level quantification

  • RNA-seq for genome-wide expression analysis and comparison

• Recombinant Protein Standards:

  • Using purified recombinant protein as standards for quantification curves

  • Ensuring proper normalization and controls

How can researchers determine if SPCC1450.16c is involved in specific cellular pathways?

To determine the involvement of SPCC1450.16c in specific cellular pathways, researchers can employ multiple complementary approaches:

• Functional Genomics:

  • Analysis of genetic interactions using deletion libraries or synthetic genetic arrays

  • Phenotypic profiling under various conditions (stress, nutrient limitation)

• Transcriptomics:

  • RNA-seq or microarray analysis of SPCC1450.16c deletion or overexpression strains

  • Identification of co-regulated genes and pathway enrichment analysis

• Metabolomics:

  • Analysis of lipid profiles if SPCC1450.16c functions as a lipase

  • Comparison of metabolite levels between wild-type and mutant strains

• Integration with Existing Data:

  • Comparison with known stress response pathways in S. pombe

  • Analysis of potential connections to cell cycle regulation

  • Examination of relationships to RNA processing pathways, given associations with gene regulation

• Bioinformatic Analysis:

  • Protein domain analysis and functional prediction

  • Comparison with characterized proteins in other organisms

What approaches can resolve contradictory findings about SPCC1450.16c function?

When facing contradictory findings about SPCC1450.16c function, researchers should consider:

• Experimental Conditions:

  • Different growth conditions might reveal different aspects of protein function

  • Stress conditions versus normal growth conditions may affect results

  • Temperature, nutrient availability, and growth phase should be controlled

• Strain Background Effects:

  • Genetic background differences between laboratory strains

  • Potential suppressor mutations or genetic modifiers

• Methodology Validation:

  • Independent verification using multiple techniques

  • Controls for antibody specificity or tag interference

  • Validation of knockout/knockdown efficiency

• Temporal and Spatial Considerations:

  • Cell cycle-dependent effects

  • Subcellular localization may affect function

  • Protein activity might be regulated post-translationally

• Integrated Analysis:

  • Combining genetic, biochemical, and cell biological approaches

  • Meta-analysis of multiple independent studies

  • Consultation with S. pombe research community and databases

How can genome-wide methods be applied to study SPCC1450.16c function in context?

Genome-wide approaches provide powerful tools for understanding SPCC1450.16c function in broader cellular contexts:

• Transcriptome Analysis:

  • RNA-seq or microarray studies comparing wild-type and SPCC1450.16c mutant strains

  • Analysis under various conditions to identify condition-specific effects

• Chromatin Immunoprecipitation (ChIP) Studies:

  • If SPCC1450.16c affects gene expression, ChIP-seq can identify genomic binding sites

  • Analysis of histone modifications in mutant backgrounds if the protein affects chromatin

• Genetic Interaction Mapping:

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

  • E-MAP (Epistatic Miniarray Profile) for quantitative interaction mapping

• Proteomics Approaches:

  • Mass spectrometry-based identification of interaction partners

  • Global proteome changes in response to SPCC1450.16c manipulation

• Metabolomics:

  • Comprehensive lipid profiling if SPCC1450.16c functions as predicted lipase

  • Metabolic flux analysis to determine effects on cellular metabolism

• Comparative Genomics:

  • Analysis of conservation and divergence across yeast species

  • Identification of functional homologs in other organisms