Recombinant Schizosaccharomyces pombe Uncharacterized protein C336.16 (SPBC336.16)

What is the structural composition of Recombinant Schizosaccharomyces pombe Uncharacterized protein C336.16?

The uncharacterized protein C336.16 (SPBC336.16) from S. pombe (strain 972/ATCC 24843) consists of 71 amino acids in its expression region (1-71). The full amino acid sequence is: MYHSYSHDLTNYLYNYFSSTTSWLVFIILSLDTINATFSNITFVDILMETGFTKNRSLDQTTCGIKFGFVN . This relatively short protein is identified in the UniProt database with the accession number G2TRR0.

For structural studies, researchers should consider using a combination of X-ray crystallography, NMR spectroscopy, and in silico modeling approaches. When working with recombinant versions, note that the tag type may vary during the production process, which could affect structural analyses.

What are the optimal storage conditions for maintaining protein C336.16 stability?

The recombinant protein is typically stored in a Tris-based buffer with 50% glycerol, which has been optimized specifically for this protein . For short-term storage (up to one week), working aliquots can be maintained at 4°C. For extended storage, the protein should be kept at -20°C or -80°C to preserve stability and function .

Researchers should avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity. When designing experiments, prepare single-use aliquots sized appropriately for your experimental needs to minimize freeze-thaw events.

How does protein C336.16 relate to the rec16 gene function in S. pombe?

While protein C336.16 (SPBC336.16) is currently uncharacterized, research on the rec16 gene in S. pombe provides context for understanding potential functional relationships. The rec16 gene product regulates multiple meiotic processes, including DNA synthesis, recombination, and transcript induction . Unlike other rec genes that primarily affect meiotic recombination, rec16 has broader impacts on meiotic processes.

When investigating potential functions of protein C336.16, researchers should consider whether it might participate in meiotic pathways. Comparative analyses with known rec proteins could identify structural or functional similarities. Co-immunoprecipitation experiments with rec16 protein or other meiotic factors would be valuable for determining potential interactions.

What expression systems are most effective for producing recombinant C336.16 protein?

For expression of S. pombe proteins like C336.16, several systems can be considered:

For an uncharacterized protein like C336.16, initial expression trials in E. coli with subsequent comparison to native protein from S. pombe extracts would establish whether simpler prokaryotic systems produce correctly folded protein.

What experimental approaches are most suitable for functional characterization of uncharacterized protein C336.16?

Given the limited knowledge about protein C336.16, a multifaceted approach combining genetic, biochemical, and computational methods is recommended:

• Genetic Approaches: Create knockout/knockdown strains in S. pombe to observe phenotypic changes. Based on findings related to rec16, which affects meiotic processes , examine meiotic phenotypes with particular attention to:

  • Meiotic DNA synthesis timing and efficiency

  • Recombination frequency

  • Transcript levels of meiotic genes

• Biochemical Approaches: Perform protein-protein interaction studies using:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation with known meiotic proteins

  • Chromatin immunoprecipitation if nuclear localization is suspected

• Computational Approaches: Utilize:

  • Structural prediction algorithms to identify functional domains

  • Phylogenetic analysis across yeast species

  • Comparative analysis with rec protein family members

What techniques can resolve contradictory data regarding C336.16 function in different experimental conditions?

When faced with contradictory data regarding protein C336.16 function, a systematic approach to reconciliation should include:

• Standardization of Experimental Conditions:

  • Control for strain background variations

  • Standardize growth media and culture conditions

  • Use consistent protein purification methods

• Multi-method Validation:

  • Verify findings using orthogonal techniques

  • Combine in vivo and in vitro approaches

  • Incorporate both genetic and biochemical methods

• Statistical Approaches:

  • Perform meta-analysis of multiple experimental datasets

  • Use Bayesian analysis to incorporate prior knowledge

  • Calculate effect sizes to quantify the magnitude of observed effects

• Context-dependent Analysis:

  • Consider whether contradictions reflect genuine biological context-dependency

  • Examine whether protein function varies with cell cycle stage or meiotic phase

  • Investigate potential post-translational modifications affecting function

A decision matrix for resolving contradictory findings might include:

How does C336.16 potentially interact with the meiotic recombination machinery in S. pombe?

Based on research on the rec16 gene in S. pombe, which is essential for normal meiotic replication, recombination, and transcript induction , protein C336.16 could potentially interact with the meiotic recombination machinery through several mechanisms:

• Direct DNA Interaction:
  The amino acid sequence of C336.16 (MYHSYSHDLTNYLYNYFSSTTSWLVFIILSLDTINATFSNITFVDILMETGFTKNRSLDQTTCGIKFGFVN) contains motifs that might enable DNA binding . Researchers should perform DNA binding assays with various DNA structures (single-stranded, double-stranded, and recombination intermediates).

• Protein Complex Formation:
  Investigate whether C336.16 forms complexes with known recombination proteins in S. pombe. Key experimental approaches include:

  • Affinity purification followed by mass spectrometry

  • Yeast two-hybrid screening against known recombination proteins

  • Co-localization studies during meiotic prophase

• Transcriptional Regulation:
  As rec16 mutations affect transcript levels of some meiotic genes , C336.16 might function in transcriptional regulation. Researchers should perform:

  • RNA-seq in wild-type versus C336.16 mutant strains during meiosis

  • ChIP-seq to identify potential binding sites

  • Reporter assays to test transcriptional activation/repression activity

What is the evolutionary conservation of C336.16 across yeast species and what does this suggest about its function?

Evolutionary conservation analysis can provide valuable insights into protein function. For C336.16, researchers should:

• Perform BLAST searches against protein databases of related yeast species and other fungi

• Conduct multiple sequence alignments to identify conserved domains

• Generate phylogenetic trees to visualize evolutionary relationships

A hypothetical conservation analysis might yield results like:

Higher conservation in the Schizosaccharomyces genus would suggest a specialized function in these species, while broader conservation would indicate a more fundamental role. Conserved domains might point to functional regions worth targeting in mutational studies.

What are the most effective protocols for purifying recombinant C336.16 protein for structural studies?

For structural studies of recombinant C336.16 protein, a comprehensive purification strategy should include:

• Expression Optimization:

  • Test multiple affinity tags (His, GST, MBP) to determine optimal solubility

  • Evaluate expression temperatures (16°C, 25°C, 30°C) and induction conditions

  • Consider co-expression with chaperones if folding issues arise

• Purification Protocol:

  • Initial capture: Affinity chromatography based on selected tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing step: Size exclusion chromatography

  • Buffer optimization: Screen buffers with varying pH, salt concentration, and additives

• Quality Control Checkpoints:

  • SDS-PAGE and western blotting to confirm protein identity and purity

  • Dynamic light scattering to assess homogeneity

  • Mass spectrometry to verify molecular weight and post-translational modifications

  • Circular dichroism to evaluate secondary structure formation

How can researchers design effective knockout and knockdown experiments for C336.16 in S. pombe?

Designing effective genetic manipulation experiments for C336.16 requires careful consideration of several factors:

• Complete Knockout Strategy:

  • CRISPR-Cas9 targeting with homology-directed repair

  • Homologous recombination with selection markers

  • Verification by PCR, sequencing, and western blotting

• Conditional Knockdown Approaches:

  • Tetracycline-regulatable promoters

  • Auxin-inducible degron system

  • Temperature-sensitive mutants

• Experimental Design Considerations:
  Based on quasi-experimental design principles , implement:

  • One-group pretest-posttest design using a double pretest (Design A3)

  • Untreated control group with dependent pretest and posttest samples (Design C1)

  • Multiple time-point observations (Design D1)

• Phenotypic Analysis Pipeline:

  • Growth rate measurements in various media conditions

  • Cell cycle analysis by flow cytometry

  • Meiotic efficiency and spore viability assessment

  • Transcriptomic analysis before and after meiotic induction

When phenotyping, particular attention should be paid to meiotic processes, given the potential relationship to rec16 function , with measurements taken at multiple timepoints to capture dynamic changes during meiosis.

What are the key future research directions for understanding protein C336.16 function?

Based on current knowledge and gaps identified in this FAQ collection, several key research directions emerge for understanding protein C336.16 function:

• Comprehensive Characterization:

  • Determine the three-dimensional structure through X-ray crystallography or NMR

  • Identify binding partners through proteome-wide interaction studies

  • Establish subcellular localization throughout the cell cycle and meiosis

• Functional Analysis:

  • Create and analyze knockout phenotypes under various conditions

  • Perform complementation studies with orthologs from related species

  • Investigate potential roles in meiotic processes based on similarities to rec16 function

• Systems Biology Approaches:

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Develop network models of protein interactions during meiosis

  • Apply machine learning to predict function from sequence and structural data

• Translational Applications:

  • Explore potential as a model for understanding eukaryotic protein evolution

  • Investigate relevance to fungal biology and potential antifungal targets

  • Develop tools for controlling meiotic processes in research applications

Researchers should prioritize establishing the basic functional characteristics of C336.16 while designing experiments with sufficient controls and replication to generate robust, reproducible results using appropriate quasi-experimental designs .