Recombinant Schizosaccharomyces pombe Uncharacterized protein C1E8.03c (SPBC1E8.03c)

What is SPBC1E8.03c and what are its basic characteristics?

SPBC1E8.03c is a conserved fungal protein found in Schizosaccharomyces pombe (fission yeast). It is a protein-coding gene with Entrez Gene ID 2540688. The full-length protein consists of 477 amino acids and is currently classified as a hypothetical protein (NP_595801.1) in genomic databases . Despite being conserved, this protein remains largely uncharacterized, with limited information available about its specific functions in cellular processes. The gene was initially identified during the complete genome sequencing of S. pombe, published in Nature by Wood et al. .

How is recombinant SPBC1E8.03c typically produced for research purposes?

Recombinant SPBC1E8.03c is typically produced using Escherichia coli expression systems. The full-length coding sequence is cloned into appropriate expression vectors, often with an N- or C-terminal His-tag to facilitate purification . After transformation into E. coli and induction of protein expression, the recombinant protein is purified using affinity chromatography (typically Ni-NTA for His-tagged proteins), followed by additional purification steps such as ion exchange or size exclusion chromatography to achieve high purity.

For researchers seeking to express this protein, the following standard workflow is recommended:

• PCR amplification of the SPBC1E8.03c gene from S. pombe genomic DNA

• Restriction enzyme cloning into an expression vector with appropriate purification tags

• Transformation into an E. coli expression strain (such as BL21(DE3))

• Optimization of expression conditions (temperature, induction time, IPTG concentration)

• Cell lysis and protein purification via affinity chromatography

What expression systems have been validated for SPBC1E8.03c production?

Based on available research data, SPBC1E8.03c has been successfully expressed in E. coli systems, particularly for recombinant protein production with His-tags . For expression within S. pombe itself, several expression systems have been developed for studying fungal proteins. The following table summarizes validated expression systems:

For experiments requiring expression in S. pombe, the nmt1 promoter-based vectors can be particularly useful as demonstrated with other similar proteins . The pCAD1 vector allows for chromosomal integration, which can lead to more stable expression compared to episomal vectors.

What are the most effective methods for studying SPBC1E8.03c localization in S. pombe cells?

For studying the subcellular localization of SPBC1E8.03c in S. pombe cells, fluorescent tagging approaches have proven effective based on similar studies with other S. pombe proteins . The recommended methodology includes:

• Generation of fluorescently tagged SPBC1E8.03c:

  • Create C-terminal or N-terminal fusions with GFP or mCherry fluorescent proteins

  • Integration at the native genomic locus using homologous recombination

  • Verification of functional protein expression via Western blotting

• Microscopy analysis:

  • Live-cell imaging using confocal microscopy

  • Co-localization studies with known organelle markers

  • Time-lapse imaging to track dynamic changes during cell cycle progression

For optimal results, it's important to confirm that the fluorescent tag does not interfere with protein function, especially since SPBC1E8.03c's function remains largely unknown. Creating both N- and C-terminal tagged versions and comparing their localization patterns can help address potential artifacts caused by the tag position.

How can researchers effectively perform proteome analysis involving SPBC1E8.03c?

For comprehensive proteome analysis involving SPBC1E8.03c, researchers can adopt methodologies similar to those described for other S. pombe proteins . A recommended approach includes:

• Sample preparation:

  • Culture S. pombe strains under relevant conditions

  • Extract total protein using optimized lysis buffers

  • Perform fractionation to enrich for specific cellular compartments if needed

• Quantitative proteomics analysis:

  • Use two-dimensional liquid chromatography coupled to mass spectrometry

  • Apply isobaric labeling (iTRAQ or TMT) for quantitative comparison between conditions

  • Implement a global internal standard approach for normalization

• Data analysis:

  • Identify proteins using database searching

  • Perform statistical analysis to identify significantly changed proteins

  • Conduct pathway enrichment analysis to contextualize findings

This approach has been successfully applied in comparative proteome analysis in S. pombe and can reveal changes in protein levels across numerous biological pathways, potentially uncovering functional relationships involving SPBC1E8.03c .

What approaches can be used to identify potential interaction partners of SPBC1E8.03c?

To identify potential interaction partners of SPBC1E8.03c, researchers can employ multiple complementary approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged SPBC1E8.03c (His, FLAG, or TAP tag) in S. pombe

  • Perform affinity purification under native conditions

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions by reciprocal pulldowns

• Yeast two-hybrid screening:

  • Use SPBC1E8.03c as bait against S. pombe genomic libraries

  • Screen for positive interactions and sequence positive clones

  • Validate interactions using co-immunoprecipitation in S. pombe

• Proximity-based labeling:

  • Create SPBC1E8.03c fusions with BioID or APEX2

  • Express in S. pombe and induce biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

These techniques should be applied with appropriate controls to distinguish true interactions from false positives. While currently no direct interaction partners of SPBC1E8.03c have been conclusively identified in the available literature , these methodologies provide a robust framework for discovering its protein interaction network.

What genetic approaches can be used to elucidate the function of SPBC1E8.03c?

Several genetic approaches can be employed to determine the function of SPBC1E8.03c:

• Gene knockout/deletion:

  • Create SPBC1E8.03c deletion strains using homologous recombination

  • Perform phenotypic characterization under various growth conditions

  • Test sensitivity to different stressors (temperature, osmotic stress, DNA damage)

  • Analyze cell morphology, cell cycle progression, and growth rates

• Conditional expression systems:

  • Place SPBC1E8.03c under the control of the nmt1 promoter for regulated expression

  • Create repressible or inducible systems to observe phenotypes upon depletion or overexpression

  • Monitor cellular responses using microscopy and biochemical assays

• Synthetic genetic array (SGA) analysis:

  • Cross SPBC1E8.03c deletion strain with a library of S. pombe deletion mutants

  • Identify synthetic lethal or synthetic sick interactions

  • Map genetic interaction networks to infer functional relationships

• Experimental evolution approaches:

  • Utilize S. pombe experimental evolution methods to understand gene function

  • Create fluorescently tagged strains to track population dynamics over time

  • Analyze adaptation patterns in wild-type versus SPBC1E8.03c mutant backgrounds

These approaches can provide complementary information about the biological role of SPBC1E8.03c, particularly when results from multiple methods are integrated.

How can researchers investigate the evolutionary conservation of SPBC1E8.03c across fungal species?

To investigate the evolutionary conservation of SPBC1E8.03c across fungal species, researchers should implement a comprehensive comparative genomics approach:

• Sequence homology analysis:

  • Perform BLAST, HMMer, or other sequence similarity searches against fungal genome databases

  • Identify orthologs and paralogs in related species

  • Generate multiple sequence alignments to identify conserved domains or motifs

• Phylogenetic analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Map the presence/absence of SPBC1E8.03c homologs across the fungal kingdom

  • Analyze rates of sequence evolution to identify conserved regions under purifying selection

• Functional complementation studies:

  • Express SPBC1E8.03c homologs from different species in S. pombe mutants

  • Test the ability of homologs to rescue phenotypes in SPBC1E8.03c deletion strains

  • Compare biochemical properties of recombinant proteins from different species

Given that SPBC1E8.03c is described as a "conserved fungal protein" , this suggests evolutionary conservation across fungal species, but the extent and pattern of this conservation would provide valuable insights into its functional importance.

What proteomics approaches can identify post-translational modifications of SPBC1E8.03c?

To identify post-translational modifications (PTMs) of SPBC1E8.03c, several specialized proteomics approaches should be considered:

• Enrichment-based strategies:

  • Phosphorylation: Immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) enrichment

  • Ubiquitination: Antibody-based enrichment of diglycine remnants

  • Glycosylation: Lectin affinity chromatography or hydrazide chemistry

  • Acetylation: Anti-acetyllysine antibody immunoprecipitation

• Mass spectrometry analysis:

  • High-resolution MS/MS using electron transfer dissociation (ETD) or higher-energy collisional dissociation (HCD)

  • Data-dependent acquisition (DDA) focusing on modified peptides

  • Parallel reaction monitoring (PRM) for targeted analysis of specific modifications

• Data analysis workflow:

  • Search MS data against the S. pombe proteome with variable modification options

  • Apply appropriate false discovery rate controls

  • Validate PTM sites using synthetic peptide standards

  • Map identified PTMs onto protein structure (if available) or homology models

• Functional characterization of PTMs:

  • Generate site-specific mutants (e.g., phosphomimetic or phosphodeficient)

  • Assess the impact of mutations on protein localization, interactions, and function

  • Compare PTM profiles under different physiological conditions

These approaches, particularly when integrated with the two-dimensional LC coupled to MALDI MS methodology described for S. pombe proteome analysis , can provide comprehensive insights into the post-translational regulation of SPBC1E8.03c.

How can researchers develop an S. pombe strain with conditional expression of SPBC1E8.03c for functional studies?

Developing S. pombe strains with conditional expression of SPBC1E8.03c requires strategic genetic engineering approaches:

• Promoter replacement strategy:

  • Replace the native SPBC1E8.03c promoter with the nmt1 promoter or its derivatives (nmt41, nmt81) with varying expression levels

  • The nmt1 promoter is repressed by thiamine, allowing for controlled gene expression

  • Integration can be achieved using the following steps:
    a. PCR amplification of the nmt1 promoter with flanking homology to the SPBC1E8.03c locus
    b. Transformation of S. pombe using the lithium acetate method
    c. Selection of transformants and verification by PCR and sequencing

• Degron-based systems:

  • Fuse an auxin-inducible degron (AID) tag to SPBC1E8.03c

  • Express the TIR1 F-box protein in the same strain

  • Addition of auxin triggers rapid protein degradation

  • This allows for fast depletion of the protein without affecting transcription

• Expression vectors approach:

  • Create both integrative (pCAD1-based) and episomal (pREP1-based) expression constructs

  • Transform these into SPBC1E8.03c deletion strains

  • This dual approach allows for stable expression from the chromosome and potentially higher expression from the episomal vector

For optimal experimental control, it is recommended to develop multiple strains with different systems, as demonstrated in studies of other S. pombe proteins . This redundancy helps validate phenotypes and ensures robustness of functional analyses.

What are common challenges in purifying recombinant SPBC1E8.03c protein and how can they be addressed?

Purification of recombinant SPBC1E8.03c may present several challenges common to uncharacterized proteins. Here are key issues and solutions:

• Protein solubility issues:

  • Challenge: Formation of inclusion bodies in E. coli expression systems

  • Solutions:
    a. Reduce expression temperature to 16-20°C
    b. Use solubility-enhancing fusion tags (SUMO, MBP, GST)
    c. Optimize induction conditions (lower IPTG concentration)
    d. Consider co-expression with molecular chaperones (GroEL/GroES)

• Protein stability problems:

  • Challenge: Rapid degradation during purification

  • Solutions:
    a. Include protease inhibitor cocktails in all buffers
    b. Optimize buffer conditions (pH, salt concentration, glycerol)
    c. Perform purification at 4°C
    d. Add stabilizing agents such as arginine or trehalose

• Low expression yield:

  • Challenge: Poor expression levels in standard systems

  • Solutions:
    a. Optimize codon usage for E. coli
    b. Try different E. coli expression strains (BL21, Rosetta, Arctic Express)
    c. Consider expression in S. pombe using nmt1 promoter systems
    d. Scale up culture volume to compensate for low yield

• Purification specificity:

  • Challenge: Co-purification of contaminants with His-tagged protein

  • Solutions:
    a. Increase imidazole concentration in wash buffers
    b. Add secondary purification steps (ion exchange, size exclusion)
    c. Consider alternative affinity tags or dual tagging strategies
    d. Validate protein identity by mass spectrometry

Since SPBC1E8.03c is a full-length protein of 477 amino acids , it may benefit from expression as domains if the full-length protein proves challenging to express in soluble form.

How can researchers troubleshoot inconsistent results in SPBC1E8.03c localization studies?

When troubleshooting inconsistent results in localization studies of SPBC1E8.03c, researchers should consider the following factors and solutions:

• Tag interference issues:

  • Problem: Fluorescent tags affecting protein localization

  • Solutions:
    a. Compare N-terminal and C-terminal tagging approaches
    b. Use smaller tags (e.g., small epitope tags followed by immunofluorescence)
    c. Verify functionality of tagged protein via complementation tests
    d. Ensure the tag doesn't disrupt targeting sequences or domains

• Expression level artifacts:

  • Problem: Overexpression causing mislocalization

  • Solutions:
    a. Express from the native promoter at the endogenous locus
    b. Use weak or regulated promoters (like nmt81) if native expression is too low
    c. Compare localization at different expression levels
    d. Validate with antibody staining of endogenous protein if available

• Cell cycle-dependent localization:

  • Problem: Variable localization depending on cell cycle stage

  • Solutions:
    a. Synchronize cells using methods appropriate for S. pombe
    b. Track localization through the cell cycle using time-lapse imaging
    c. Use cell cycle markers to correlate localization with cell cycle stages
    d. Analyze localization in cell cycle mutants arrested at specific stages

• Technical microscopy issues:

  • Problem: Inconsistent imaging results

  • Solutions:
    a. Standardize sample preparation protocols
    b. Use consistent imaging parameters across experiments
    c. Include positive controls with known localization patterns
    d. Implement quantitative image analysis to measure localization objectively

The fluorescent tagging approaches described for studying S. pombe strains provide a good foundation for addressing these challenges, particularly when combined with careful controls and standardized protocols.

What bioinformatic approaches can predict potential functions of SPBC1E8.03c?

To predict potential functions of the uncharacterized SPBC1E8.03c protein, researchers should employ multiple complementary bioinformatic approaches:

• Sequence-based predictions:

  • Protein domain identification using InterPro, Pfam, and SMART databases

  • Motif recognition using PROSITE, ELM, and other motif databases

  • Secondary structure prediction using PSIPRED, JPred, or similar tools

  • Disorder prediction using IUPred2A or PONDR

• Structure-based predictions:

  • Template-based structure modeling using I-TASSER or SWISS-MODEL

  • Ab initio structure prediction using AlphaFold2 or RoseTTAFold

  • Structure-based function prediction using ProFunc or COFACTOR

  • Binding site prediction using SiteEngine or FTSite

• Network-based approaches:

  • Guilt-by-association analysis using available S. pombe expression data

  • Co-expression network analysis to identify functionally related genes

  • Phylogenetic profiling to identify genes with similar evolutionary patterns

  • Integration of diverse data types using machine learning approaches

• Gene ontology enrichment:

  • Identify ontology terms enriched among similar proteins

  • Map potential functions based on closest characterized homologs

  • Integrate results from multiple prediction methods to generate consensus hypotheses

Since SPBC1E8.03c is described as a conserved fungal protein , comparative analysis across fungal species may be particularly informative for functional prediction.

How should researchers interpret proteomics data to understand SPBC1E8.03c function in different cellular contexts?

When interpreting proteomics data related to SPBC1E8.03c, researchers should follow these analytical principles:

• Differential expression analysis:

  • Compare SPBC1E8.03c levels across different conditions using quantitative proteomics

  • Identify co-regulated proteins that follow similar expression patterns

  • Analyze changes in SPBC1E8.03c abundance in response to environmental stressors

  • Look for patterns in wild-type versus mutant backgrounds

• Protein interaction network analysis:

  • Map SPBC1E8.03c within the context of the S. pombe interactome

  • Identify interaction clusters or protein complexes containing SPBC1E8.03c

  • Analyze the dynamics of these interactions under different conditions

  • Integrate interaction data with gene expression and phenotypic data

• Post-translational modification (PTM) analysis:

  • Map identified PTMs to regulatory motifs within the protein sequence

  • Track changes in PTM patterns across different conditions

  • Correlate PTM changes with alterations in protein function or localization

  • Identify kinases, phosphatases, or other enzymes that may regulate SPBC1E8.03c

• Pathway enrichment analysis:

  • Use tools like KEGG, Reactome, or Gene Ontology to identify enriched pathways

  • Look for enrichment of specific cellular processes among co-regulated proteins

  • Consider both direct and indirect effects when interpreting pathway changes

  • Apply the global internal standard approach for proteome analysis as described for S. pombe

The analytical framework described for comparative proteome analysis in S. pombe provides a solid foundation for interpreting proteomics data related to SPBC1E8.03c, particularly when examining changes across numerous biological pathways within the cell.