Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C27F1.10 (SPAC27F1.10)

What is SPAC27F1.10 and why is it of interest to researchers?

SPAC27F1.10 is an uncharacterized membrane protein encoded by the fission yeast Schizosaccharomyces pombe. It represents one of many putative membrane proteins whose biological functions remain experimentally unconfirmed. Such proteins are of considerable interest to researchers because they may reveal novel cellular pathways, structural motifs, or functional mechanisms relevant to membrane biology. The recombinant form is produced specifically to facilitate biochemical and structural studies that aim to elucidate its role in cellular processes.

How does SPAC27F1.10 compare with other uncharacterized membrane proteins in S. pombe?

SPAC27F1.10 is part of a larger category of uncharacterized S. pombe membrane proteins. Comparative analysis shows the following positioning within this category:

What expression systems are most effective for producing recombinant SPAC27F1.10?

The recombinant SPAC27F1.10 protein is typically synthesized in a Saccharomyces cerevisiae (budding yeast) expression system. This host is preferred because, as a eukaryotic expression system, it provides appropriate post-translational modifications and membrane insertion machinery that may be crucial for proper folding of this membrane protein. When designing expression protocols, researchers should consider codon optimization for S. cerevisiae and include appropriate regulatory elements for inducible expression. Alternative expression systems such as insect cells or mammalian cells might be considered for specific structural or functional studies, though these have not been widely reported for this particular protein.

What purification challenges are associated with SPAC27F1.10, and how can they be addressed?

As a small hydrophobic membrane protein, SPAC27F1.10 presents significant purification challenges related to insolubility and aggregation. A methodological approach to addressing these challenges includes:

• Detergent screening: Testing multiple detergents (DDM, LMNG, CHAPS) at varying concentrations to identify optimal solubilization conditions

• Buffer optimization: Including glycerol (5-50%) to enhance stability

• Tag selection: Utilizing His-tags or FLAG-tags for affinity purification

• Chromatography sequence: Implementing a multi-step purification protocol including affinity chromatography followed by size exclusion chromatography

• Sample validation: Confirming purity via SDS-PAGE (target >85%)

For particularly recalcitrant preparations, newer technologies such as lipid sponge droplets, which have shown success with other small membrane proteins like E. coli AcrZ, may facilitate improved yields of functional protein.

What structural determination methods are most suitable for SPAC27F1.10?

Given the challenges associated with membrane protein structural determination, a multi-technique approach is recommended for SPAC27F1.10:

• Cryo-electron microscopy (cryo-EM): Particularly useful if the protein can be stabilized in a detergent micelle or nanodisc

• X-ray crystallography: Requires extensive crystallization condition screening

• NMR spectroscopy: Suitable for mapping specific domains if isotope labeling can be achieved

• Computational prediction: AlphaFold2 or RoseTTAFold can provide preliminary structural models to guide experimental design

The partial sequence information available for SPAC27F1.10 allows for limited domain mapping and epitope analysis, which can inform targeted structural studies of specific regions. A combination of these approaches will likely provide the most comprehensive structural information.

How can researchers optimize membrane mimetics for structural studies of SPAC27F1.10?

Membrane mimetic selection is critical for structural and functional studies of SPAC27F1.10. Methodological optimization should include:

• Detergent screening (from mild to harsh): DDM → LMNG → OG → SDS

• Lipid nanodisc assembly with MSP1D1 or MSP1E3D1 scaffold proteins

• Bicelle formulation with DMPC/CHAPSO mixtures at varying q-ratios

• Amphipol (A8-35 or PMAL-C8) reconstitution for increased stability

• Testing of novel sponge-phase lipid systems that have shown promise for small membrane proteins

Researchers should systematically evaluate protein stability and homogeneity in each system using analytical size exclusion chromatography and negative-stain EM before proceeding to high-resolution structural studies.

What approaches can be used to identify potential binding partners of SPAC27F1.10?

Identifying interaction partners is a critical step toward understanding the function of SPAC27F1.10. Recommended methodological approaches include:

• Affinity purification followed by mass spectrometry (AP-MS)

• Yeast two-hybrid screening using the soluble domains of the protein

• Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling

• Co-immunoprecipitation with epitope-tagged versions expressed in S. pombe

• Pull-down assays using the recombinant protein as bait

How might SPAC27F1.10 relate to known membrane protein signaling pathways in S. pombe?

While the specific function of SPAC27F1.10 remains uncharacterized, examination of known S. pombe membrane signaling pathways provides relevant research directions. In S. pombe, the Rho1p signaling pathway plays essential roles in cell integrity and actin cytoskeleton polarization . This pathway involves:

• Rho1p binding to effector proteins when in GTP-bound state

• Interaction with protein kinase C homologues pck1p and pck2p

• Regulation of cell wall biosynthesis through (1,3)β-D-glucan synthase

• Maintenance of cell polarity and integrity

Given that SPAC27F1.10 is a membrane protein, investigating potential interactions with components of this pathway represents a logical research direction. Experimental approaches might include co-immunoprecipitation with Rho1p, pck1p, and pck2p, or genetic interaction studies examining phenotypes of SPAC27F1.10 deletion in combination with mutations in Rho pathway components.

What gene expression patterns might provide clues about SPAC27F1.10 function?

Analyzing gene expression patterns can provide valuable insights into the function of uncharacterized proteins. For S. pombe proteins, examination of expression changes under various stressors is particularly informative. For example, research has shown that nitrogen starvation induces significant changes in gene expression profiles, including membrane transporters and signaling proteins . Methodology for investigating SPAC27F1.10 expression patterns should include:

• Northern blot analysis under varied nutritional conditions

• RNA-seq profiling across growth phases and stress responses

• Comparison with expression patterns of genes at adjacent loci

• Analysis in genetic backgrounds with mutations in major signaling pathways (e.g., Δtsc1, Δtsc2)

• Reporter gene fusions to monitor expression in real-time

Notably, research has identified that many membrane transporters and signaling proteins show altered expression in Δtsc1 and Δtsc2 mutants during nitrogen starvation , providing a potential experimental framework for studying SPAC27F1.10 regulation.

How can SPAC27F1.10 be used to generate specific antibodies, and what controls are necessary?

The recombinant SPAC27F1.10 protein can serve as an immunogen for generating custom antibodies. A methodological approach would include:

• Protein preparation:

  • Expression with affinity tag for purification

  • Confirmation of >90% purity by SDS-PAGE

  • Verification of correct folding if possible

• Immunization protocol:

  • Selection of host species (rabbit, mouse, chicken)

  • Adjuvant selection (Freund's, Alum, TiterMax)

  • Immunization schedule (primary + 3 boosts)

• Essential controls:

  • Pre-immune serum testing

  • Antibody validation in SPAC27F1.10 knockout S. pombe

  • Peptide competition assays

  • Western blot against recombinant protein and S. pombe lysates

  • Immunofluorescence localization compared to tagged protein

• Affinity purification of antibodies using immobilized antigen

These antibodies can subsequently be employed for localization studies, co-immunoprecipitation experiments, and functional investigations in S. pombe.

What approaches can address the challenge of functional redundancy when studying SPAC27F1.10?

Functional redundancy presents a significant challenge in characterizing novel proteins. For SPAC27F1.10, researchers should consider these methodological approaches:

• Systematic genetic interactions:

  • Creation of double/triple mutants with related membrane proteins

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

  • Conditional expression systems to study dosage effects

• Stress response profiling:

  • Expose deletion mutants to diverse stressors (oxidative, osmotic, pH, temperature)

  • Quantitative fitness measurements under varied conditions

  • Growth curve analysis with high temporal resolution

• Compensation mechanism identification:

  • Transcriptome analysis of deletion mutants to identify upregulated genes

  • Proteomics to detect altered protein levels

  • Metabolomics to identify pathway rerouting

• Evolutionary analysis:

  • Identification of paralogous genes

  • Cross-species complementation experiments

  • Assessment of conservation patterns across Schizosaccharomyces species

This multi-faceted approach can help overcome the challenges of functional redundancy that often obscure phenotypes in single-gene deletion studies.

How might advanced cell-free expression systems be optimized for structural studies of SPAC27F1.10?

Cell-free expression systems offer advantages for difficult membrane proteins like SPAC27F1.10. A methodological approach to optimization would include:

• Template preparation:

  • Codon optimization for the cell-free system

  • Design of constructs with varying tags and fusion partners

  • Incorporation of stabilizing mutations if structural predictions are available

• Reaction composition optimization:

  • Detergent screening (Brij-58, DDM, digitonin)

  • Lipid nanodisc co-translational incorporation

  • Supplementation with S. pombe membrane fractions

• Expression conditions:

  • Temperature optimization (typically 18-30°C)

  • Incubation time determination (4-24 hours)

  • Feed mechanisms for extended reactions

• Novel lipid sponge droplet integration:

  • Formation of lipid-detergent structures before protein addition

  • Titration of lipid:protein ratios

  • Application of techniques successful with E. coli AcrZ

• Validation of correctly folded protein:

  • Binding assays with predicted partners

  • Limited proteolysis to assess domain structure

  • Circular dichroism for secondary structure assessment

This systematic approach to cell-free expression could overcome many of the challenges associated with traditional recombinant production of this membrane protein.

How might SPAC27F1.10 function in relation to nitrogen starvation response pathways?

Given that many membrane proteins in S. pombe show altered expression during nitrogen starvation, particularly in Δtsc1 and Δtsc2 backgrounds , investigating SPAC27F1.10 in this context represents a promising research direction. Methodological approaches should include:

• Expression analysis:

  • Northern blot comparison of SPAC27F1.10 in wild-type vs. Δtsc1/Δtsc2 strains

  • Time-course expression during nitrogen depletion

  • Reporter gene fusions to monitor real-time expression changes

• Phenotypic characterization:

  • Nitrogen starvation survival in SPAC27F1.10 deletion mutants

  • Sexual differentiation efficiency assessment

  • Microscopic characterization of cellular morphology during starvation

• Genetic interaction mapping:

  • Double mutant analysis with known nitrogen-responsive genes

  • Synthetic genetic array screening under nitrogen limitation

  • Suppressor screens in SPAC27F1.10 deletion backgrounds

• Signaling pathway integration:

  • Analysis of TORC1/TORC2 activity in deletion mutants

  • Assessment of stress-activated MAP kinase pathway activation

  • Investigation of potential interactions with Sck2 or Gpa2 signaling components

These approaches may reveal whether SPAC27F1.10 functions as an upstream sensor, downstream effector, or modulator of nitrogen starvation responses in S. pombe.

What computational approaches can predict SPAC27F1.10 function based on structural features?

Modern computational tools provide valuable insights for proteins lacking experimental characterization. For SPAC27F1.10, a methodological approach using computational prediction would include:

• Structural prediction:

  • AlphaFold2 prediction of tertiary structure

  • Transmembrane topology prediction (TMHMM, Phobius)

  • Identification of conserved domains and motifs

• Functional annotation:

  • Gene Ontology term prediction based on structure

  • Ligand binding site prediction (FTSite, SiteMap)

  • Molecular docking with potential metabolites and signaling molecules

• Network-based prediction:

  • Guilt-by-association analysis using co-expression networks

  • Protein-protein interaction prediction (STRING, DIOPT)

  • Phylogenetic profiling to identify co-evolved genes

• Integration with experimental data:

  • Refinement of predictions using targeted assays

  • Validation through site-directed mutagenesis of predicted functional sites

  • Experimental testing of predicted binding partners

This computational pipeline can generate testable hypotheses about SPAC27F1.10 function that guide experimental design and resource allocation.