Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C1682.06 (SPCC1682.06)

How is recombinant SPCC1682.06 protein typically produced for research applications?

Recombinant SPCC1682.06 protein is commonly produced using E. coli expression systems. The methodology typically follows these steps:

• Gene cloning: The full-length coding sequence (1-238 amino acids) is PCR-amplified from S. pombe genomic DNA or cDNA.

• Vector construction: The amplified sequence is inserted into an expression vector containing an N-terminal His-tag for purification purposes.

• Transformation: The recombinant plasmid is transformed into an E. coli expression strain.

• Protein expression: Bacterial cultures are induced to express the recombinant protein.

• Purification: The His-tagged protein is purified using affinity chromatography.

• Quality control: The purified protein is assessed for purity (>90% by SDS-PAGE) .

For optimal results, expression conditions should be optimized for membrane proteins, potentially including lower induction temperatures (16-25°C) and specific E. coli strains designed for membrane protein expression.

What are the optimal storage and handling conditions for working with recombinant SPCC1682.06 protein?

Proper handling and storage of recombinant SPCC1682.06 protein is critical for maintaining its stability and functionality:

To minimize protein degradation, it is critical to avoid repeated freeze-thaw cycles. Working aliquots should be prepared during initial reconstitution . The protein is typically supplied as a lyophilized powder that requires proper reconstitution following manufacturer protocols to ensure optimal activity and stability.

How can researchers design experiments to determine the subcellular localization of SPCC1682.06?

Determining the subcellular localization of SPCC1682.06 requires a systematic experimental approach:

• Fluorescent protein tagging:

  • C-terminal or N-terminal GFP/mCherry fusion constructs

  • Expression under native promoter to avoid overexpression artifacts

  • Controls with known membrane protein localizations

• Immunofluorescence microscopy:

  • Production of specific antibodies against SPCC1682.06

  • Co-staining with markers for various cellular compartments (ER, Golgi, plasma membrane)

  • Super-resolution microscopy for detailed localization

• Subcellular fractionation:

  • Differential centrifugation to separate membrane fractions

  • Western blot analysis of fractions using anti-His antibodies (for recombinant protein) or specific antibodies

  • Mass spectrometry analysis of membrane proteome

• Electron microscopy:

  • Immunogold labeling for high-resolution localization

  • Freeze-fracture electron microscopy for membrane protein distribution

The experimental design should incorporate appropriate controls and multiple technical approaches to validate the localization findings. Since SPCC1682.06 is predicted to be a membrane protein, specific attention should be paid to distinguishing between different membrane compartments within the cell.

What strategies can be employed to generate and characterize SPCC1682.06 deletion mutants?

Generation and characterization of SPCC1682.06 deletion mutants requires careful experimental design, especially since S. pombe genome deletion projects have successfully deleted 99% of fission yeast open reading frames :

• Generation of deletion mutants:

  • PCR-based gene targeting (standard approach in S. pombe genome deletion project)

  • If PCR-based approach fails, use plasmid-based method with large homologous regions flanking the target gene

  • For heterozygous diploids, use a mating type-stable h+/h+ or h-/h- diploid strain background

• Confirmation of deletion:

  • PCR verification using primers outside the deletion cassette

  • Southern blot analysis for complex genomic regions

  • RT-PCR to confirm absence of transcript

• Phenotypic characterization:

  • Growth assays under various conditions (temperature, osmotic stress, nutrient limitation)

  • Microscopic analysis of cell morphology and division patterns

  • Sporulation and tetrad analysis to determine essentiality

• Complementation tests:

  • Reintroduction of SPCC1682.06 gene to confirm phenotype rescue

  • Expression of SPCC1682.06 under regulated promoters for functional studies

The S. pombe genome deletion project uses heterozygous diploid strains for initial deletions, followed by sporulation and tetrad analysis to determine if genes are essential for vegetative growth . This approach is particularly valuable for SPCC1682.06 whose function remains uncharacterized.

How can researchers design experiments to investigate potential protein-protein interactions involving SPCC1682.06?

Investigating protein-protein interactions for SPCC1682.06 requires specialized approaches for membrane proteins:

• Yeast two-hybrid (Y2H) adaptations:

  • Split-ubiquitin Y2H system designed specifically for membrane proteins

  • Modified membrane Y2H systems that allow for testing interactions in membrane environments

  • Controls with known membrane protein interactors

• Co-immunoprecipitation approaches:

  • Epitope tagging of SPCC1682.06 (His, FLAG, or HA tags)

  • Crosslinking before solubilization to capture transient interactions

  • Gentle detergent solubilization to maintain membrane protein complexes

  • Mass spectrometry analysis of precipitated complexes

• Proximity labeling methods:

  • BioID or TurboID fusion to SPCC1682.06

  • APEX2 proximity labeling

  • Identification of proximal proteins in native cellular context

• Förster Resonance Energy Transfer (FRET):

  • Dual fluorescent protein tagging (SPCC1682.06 and potential interactors)

  • Live-cell FRET measurements

  • Controls with non-interacting membrane proteins

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein fragments fused to SPCC1682.06 and candidate interactors

  • Visualization of reconstituted fluorescence upon interaction

  • Quantification of interaction strength

Each approach has advantages and limitations, particularly for membrane proteins. A multi-method strategy is recommended to validate any identified interactions. Proper experimental design must include appropriate controls to account for the hydrophobic nature of membrane proteins, which can lead to false positives in interaction studies.

What comparative genomics approaches can help elucidate the function of SPCC1682.06?

Comparative genomics provides valuable insights for uncharacterized proteins like SPCC1682.06:

• Ortholog identification:

  • BLAST searches against diverse fungal genomes

  • Reciprocal best hit analysis

  • Synteny conservation analysis across yeasts

• Phylogenetic profiling:

  • Construction of presence/absence patterns across species

  • Correlation with known functional categories

  • Identification of co-evolved gene clusters

• Structural prediction comparisons:

  • Secondary structure predictions across orthologs

  • Transmembrane domain conservation analysis

  • Identification of conserved motifs or domains

• Gene neighborhood analysis:

  • Examination of genomic context in S. pombe

  • Comparison of gene arrangements across species

  • Identification of conserved gene clusters suggesting functional relationships

• Expression correlation networks:

  • Analysis of co-expression patterns across conditions

  • Identification of genes consistently co-regulated with SPCC1682.06

  • Integration with protein-protein interaction data

By combining these approaches, researchers can generate testable hypotheses about SPCC1682.06 function based on evolutionary conservation patterns and genomic context. The identification of orthologs with known functions in other species would be particularly valuable for directing experimental investigations.

What experimental design approaches are most appropriate for functional characterization of SPCC1682.06?

Functional characterization of SPCC1682.06 requires a systematic experimental design following these steps :

• Define research variables:

  • Independent variable: Expression/activity level of SPCC1682.06

  • Dependent variables: Phenotypic outcomes (growth rate, stress response, membrane integrity)

  • Control variables: Growth conditions, genetic background, expression levels

• Generate specific testable hypotheses:

  • Based on bioinformatic predictions (e.g., membrane transport function)

  • Derived from phenotypic observations of deletion mutants

  • Informed by protein localization data

• Design experimental treatments:

  • Conditional expression systems (e.g., nmt1 promoter with thiamine regulation)

  • Site-directed mutagenesis of key predicted functional residues

  • Chimeric protein constructs to test domain functions

• Establish appropriate control groups:

  • Wild-type S. pombe strains

  • Strains expressing known membrane proteins of similar size/topology

  • Empty vector controls for expression studies

• Measurement methods for dependent variables:

  • Growth assays under various conditions

  • Membrane integrity tests (e.g., sensitivity to detergents or membrane-disrupting agents)

  • Metabolite transport assays if transporter function is suspected

  • Lipidomic analysis for membrane composition effects

What are the challenges and solutions in expressing and purifying SPCC1682.06 for structural studies?

Membrane proteins like SPCC1682.06 present specific challenges for structural studies:

For SPCC1682.06 specifically, the current E. coli expression system with His-tag purification may need further optimization for structural studies. Nanodiscs or amphipols could be employed to maintain protein stability in a membrane-like environment after purification. The systematic screening of conditions is essential for successful structural characterization of this membrane protein.

How can researchers investigate the essentiality and conditional phenotypes of SPCC1682.06?

Investigating essentiality and conditional phenotypes requires sophisticated genetic approaches:

• Tetrad analysis from heterozygous diploids:

  • Sporulation of heterozygous SPCC1682.06Δ/SPCC1682.06+ diploids

  • Dissection and analysis of tetrads on rich medium

  • Assessment of spore viability patterns (2:2 segregation suggests essentiality)

• Conditional mutant generation:

  • Temperature-sensitive alleles created by random mutagenesis

  • Auxin-inducible degron (AID) system for controlled protein depletion

  • Promoter replacement with regulatable promoters (nmt1, urg1)

• Synthetic genetic interactions:

  • Systematic genetic crosses with deletion collection strains

  • Synthetic genetic array (SGA) analysis

  • Identification of genetic interactions suggesting functional relationships

• High-throughput phenotyping:

  • Growth in presence of various stressors (osmotic, oxidative, pH)

  • Cell wall/membrane integrity stressors (calcofluor white, SDS)

  • Nutrient limitation responses

• Transcriptomic profiling:

  • RNA-seq analysis of deletion or depletion strains

  • Identification of compensatory gene expression changes

  • Integration with existing S. pombe transcriptome datasets

The S. pombe genome deletion project has established methods for determining gene essentiality through heterozygous diploid sporulation . For SPCC1682.06, a systematic approach combining these methods would provide comprehensive insights into its biological role and importance under various conditions.

What bioinformatic tools and resources are most valuable for analyzing SPCC1682.06?

Several specialized bioinformatic tools and resources are particularly useful for analyzing uncharacterized membrane proteins like SPCC1682.06:

• Protein structure and topology prediction:

  • TMHMM/Phobius for transmembrane domain prediction

  • PredictProtein for secondary structure analysis

  • AlphaFold2 for 3D structure prediction

• S. pombe-specific resources:

  • PomBase for genomic and functional information

  • S. pombe genome deletion project data

  • CYCLoPs for subcellular localization patterns

• Protein family and domain analysis:

  • InterPro for functional domains

  • PFAM for protein family classification

  • PROSITE for motif identification

• Membrane protein-specific tools:

  • MemGen for membrane protein family classification

  • OPM database for orientation of proteins in membranes

  • MESSA for integrated membrane protein annotation

• Functional prediction tools:

  • ProtFun for general function prediction

  • TransportDB for transporter classification

  • CELLO for subcellular localization prediction

Integration of predictions from multiple tools provides the most reliable functional hypotheses. Researchers should prioritize tools specifically designed for membrane proteins, as general protein analysis tools often have reduced accuracy for transmembrane proteins.

How can researchers design experiments to study potential post-translational modifications of SPCC1682.06?

Investigating post-translational modifications (PTMs) of SPCC1682.06 requires specialized experimental approaches:

• Mass spectrometry-based PTM identification:

  • Immunoprecipitation of tagged SPCC1682.06

  • Sample preparation optimized for membrane proteins

  • Enrichment strategies for specific modifications (phosphopeptides, glycopeptides)

  • Data analysis with PTM-specific search parameters

• Site-directed mutagenesis of predicted PTM sites:

  • Bioinformatic prediction of potential modification sites

  • Mutation of candidate residues (e.g., S/T/Y for phosphorylation)

  • Functional analysis of mutant proteins

• Specific modification detection methods:

  • Phospho-specific antibodies if available

  • ProQ Diamond staining for phosphoproteins

  • Glycoprotein-specific staining methods

  • Ubiquitination detection via western blotting

• PTM inhibitor studies:

  • Treatment with kinase/phosphatase inhibitors

  • Deglycosylation enzymes

  • Proteasome inhibitors (for ubiquitination)

  • Analysis of effects on protein function and stability

• Temporal dynamics of modifications:

  • Synchronized cultures to study cell cycle-dependent modifications

  • Stress responses to identify condition-specific modifications

  • Time-course analysis after stimulation

PTMs can significantly impact membrane protein localization, stability, and function. For SPCC1682.06, identifying relevant modifications could provide critical insights into its regulation and cellular role.