Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C6F6.13c (SPAC6F6.13c)

What is SPAC6F6.13c and what organism does it originate from?

SPAC6F6.13c is an uncharacterized membrane protein from the fission yeast Schizosaccharomyces pombe. It is a full-length protein consisting of 778 amino acids (protein identifier O14244). The protein can be expressed recombinantly with an N-terminal His-tag in E. coli systems, as demonstrated in commercial preparations . While classified as "uncharacterized," its membrane localization suggests potential roles in cellular compartmentalization, signaling, or transport functions typical of integral membrane proteins in eukaryotic systems.

What expression systems are optimal for producing recombinant SPAC6F6.13c?

For structural and functional studies requiring properly folded and processed protein, mammalian or insect cell expression systems might offer advantages despite their higher complexity and cost .

What are the recommended storage and handling protocols for SPAC6F6.13c?

Optimal handling of recombinant SPAC6F6.13c requires specific conditions to maintain stability and functionality:

• Upon receipt: Store lyophilized powder at -20°C/-80°C immediately .

• Reconstitution protocol:

  • Briefly centrifuge the vial to collect material at the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • For long-term storage, add glycerol to a final concentration of 5-50%

• Storage recommendations:

  • Aliquot to avoid repeated freeze-thaw cycles

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

  • Long-term storage requires -20°C/-80°C conditions

• Buffer composition: Typically supplied in Tris/PBS-based buffer with 6% Trehalose, pH 8.0 .

As with most membrane proteins, SPAC6F6.13c likely requires detergents or membrane mimetics for long-term stability after purification, though specific detergent recommendations are not provided in the available literature.

What approaches are recommended for functional characterization of SPAC6F6.13c?

As an uncharacterized membrane protein, multiple complementary approaches should be employed to elucidate SPAC6F6.13c function:

• Genomic approaches:

  • CRISPR/Cas9 knockout or knockdown studies

  • Phenotypic screening of deletion mutants

  • Transcriptomic analysis of gene deletion/overexpression

• Proteomic strategies:

  • Identification of interaction partners through co-immunoprecipitation

  • Proximity labeling to identify neighboring proteins

  • Membrane topology mapping

• Localization studies:

  • Fluorescent protein tagging for live-cell imaging

  • Subcellular fractionation and immunoblotting

  • Immunogold electron microscopy for high-resolution localization

• Functional assays:

  • Transport assays if suspected to be a transporter

  • Electrophysiological measurements if suspected to be a channel

  • Enzymatic activity screening

The absence of established functional data for SPAC6F6.13c in database entries suggests this protein remains functionally uncharacterized , presenting opportunities for novel discoveries through systematic investigation.

How might SPAC6F6.13c relate to meiotic processes in Schizosaccharomyces pombe?

While the search results don't directly implicate SPAC6F6.13c in meiosis, research on S. pombe meiosis reveals dynamic gene expression and alternative splicing patterns that could be relevant. Studies on fission yeast meiosis have identified numerous genes with altered expression or alternative splicing during meiotic progression .

To investigate potential meiotic functions of SPAC6F6.13c, researchers could:

• Analyze expression patterns during synchronized meiosis using RT-qPCR or RNA-seq

• Examine alternative splicing events affecting SPAC6F6.13c during meiotic progression

• Create meiosis-specific conditional mutants to observe phenotypic effects

• Investigate protein localization changes during meiotic progression

Research has documented read-through transcripts and alternative splicing events in S. pombe during meiosis that affect protein function and localization . For example, genes like SPAC16E8.02-SPAC16E8.03 show meiosis-specific read-through transcripts that may encode fusion proteins essential for sporulation . Similar mechanisms might affect SPAC6F6.13c expression or function during meiosis.

What structural analysis approaches are most suitable for SPAC6F6.13c?

Membrane protein structural analysis presents unique challenges. For SPAC6F6.13c, consider these approaches:

For initial characterization, computational approaches like transmembrane topology prediction, homology modeling, and evolutionary analysis can provide structural insights before experimental determination . The 778-amino acid sequence suggests multiple transmembrane domains that would require specialized approaches for structural elucidation.

How does SPAC6F6.13c compare to other uncharacterized membrane proteins in S. pombe?

S. pombe contains numerous uncharacterized membrane proteins that present similar research challenges. Comparative analysis approaches include:

• Phylogenetic analysis to identify evolutionary relationships and potential functional conservation

• Co-expression network analysis to identify functionally related gene clusters

• Domain architecture comparison to identify shared structural elements

• Phenotypic profiling of multiple deletion mutants to identify shared or distinct functions

While specific comparative data for SPAC6F6.13c is limited in the search results, researchers investigating this protein should consider the broader context of S. pombe membrane proteome to identify functional relationships and potential research directions .

What purification strategies are most effective for recombinant SPAC6F6.13c?

Purification of membrane proteins requires specialized approaches. For His-tagged SPAC6F6.13c, consider this optimized workflow:

• Expression optimization:

  • Test multiple E. coli strains (BL21, C41/C43, Rosetta)

  • Optimize induction conditions (temperature, IPTG concentration, duration)

  • Consider membrane-protein-specific expression systems

• Membrane isolation and solubilization:

  • Extract membranes via ultracentrifugation

  • Screen detergents systematically (DDM, LMNG, digitonin)

  • Optimize detergent:protein ratios

• Affinity purification:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA

  • Gradual imidazole elution to separate non-specific binding

  • Buffer optimization to maintain stability

• Secondary purification:

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for additional purity

• Quality assessment:

  • SDS-PAGE and Western blotting

  • Mass spectrometry verification

  • Thermal stability assays

  • Dynamic light scattering for homogeneity

The recombinant protein is typically supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE , indicating successful purification is achievable with optimized protocols.

What analytical techniques can verify proper folding and functionality of purified SPAC6F6.13c?

Without known function, researchers must employ multiple approaches to assess protein quality:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • Differential scanning fluorimetry to measure thermal stability

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

• Functional verification approaches:

  • Liposome reconstitution followed by functional assays

  • Binding studies with predicted interacting partners

  • Complementation of knockout phenotypes

• Structural integrity assessment:

  • Limited proteolysis to identify stable domains

  • Negative-stain electron microscopy

  • Native PAGE to assess homogeneity

These approaches provide complementary information about protein quality before proceeding to more resource-intensive experiments or assays .

What experimental design considerations apply when studying SPAC6F6.13c interactions with other proteins?

Investigating protein-protein interactions involving membrane proteins requires specialized approaches:

• Proximity-based methods:

  • BioID or APEX2 proximity labeling

  • Split-GFP complementation assays

  • Förster resonance energy transfer (FRET)

• Affinity-based methods:

  • Co-immunoprecipitation with membrane-compatible detergents

  • Pull-down assays with recombinant protein

  • Surface plasmon resonance (SPR) for binding kinetics

• Genetic interaction methods:

  • Synthetic genetic arrays

  • Suppressor screens

  • Two-hybrid systems adapted for membrane proteins

• Experimental design considerations:

  • Appropriate controls (non-specific binding, background subtraction)

  • Detergent selection to maintain native interactions

  • Validation through multiple orthogonal techniques

For optimal results, researchers should employ PamChip pairing designs that allow direct comparison of different conditions on the same chip, reducing technical variability . This approach enables more reliable detection of interaction differences across experimental conditions.

How can SPAC6F6.13c be effectively studied in the context of signal peptidase activity?

Search result discusses Spc1, an evolutionarily conserved membrane protein subunit of signal peptidase (SPase) that regulates substrate selection. While not directly linked to SPAC6F6.13c, this research provides methodological insights for membrane protein studies:

• In vivo cleavage assays:

  • Create reporter constructs with modified signal sequences

  • Compare processing efficiency in wild-type vs. mutant backgrounds

  • Use Western blotting to detect cleaved and uncleaved forms

• Co-immunoprecipitation studies:

  • Test if SPAC6F6.13c interacts with components of the signal peptidase complex

  • Examine whether it co-precipitates with proteins carrying uncleaved signal sequences

• Membrane protein processing analysis:

  • Evaluate if SPAC6F6.13c affects processing of model membrane proteins

  • Test effects of overexpression or deletion on signal sequence cleavage

• Targeting sequence analysis:

  • Examine SPAC6F6.13c's own signal sequence or transmembrane segments

  • Investigate whether it undergoes SPase-mediated processing

The study demonstrates that Spc1 protects transmembrane segments from SPase action, sharpening substrate selection . Similar experimental designs could determine if SPAC6F6.13c has related functions or is itself regulated by the signal peptidase system.

How can multi-omics approaches enhance understanding of SPAC6F6.13c function?

Integrating multiple omics datasets provides a comprehensive view of SPAC6F6.13c's cellular context:

• Transcriptomics integration:

  • RNA-seq data across conditions to identify co-regulated genes

  • Analysis of alternative splicing patterns during cellular processes

  • Comparative expression across yeast species

• Proteomics approaches:

  • Quantitative proteomics to measure protein abundance changes

  • Post-translational modification mapping

  • Protein-protein interaction networks

• Metabolomics connections:

  • Metabolite profiling in deletion/overexpression strains

  • Isotope labeling to track metabolic changes

  • GC-MS analysis to identify affected pathways

• Data integration strategies:

  • Network analysis to identify functional modules

  • Machine learning approaches to predict function

  • Visualization tools for multi-dimensional data

The resveratrol inhibition study in search result employed GC-MS metabolomics with PCA and PLS-DA analysis to identify significantly changed metabolites. Similar approaches could reveal metabolic impacts of SPAC6F6.13c perturbation.

What bioinformatic resources are most valuable for analyzing SPAC6F6.13c?

Several specialized resources can aid research on this uncharacterized protein:

These resources provide complementary information for generating testable hypotheses about SPAC6F6.13c function, localization, and interactions. Researchers should regularly check for updates as annotations for uncharacterized proteins frequently evolve as new data becomes available .

What considerations apply when designing experiments to detect alternative splicing of SPAC6F6.13c?

Research indicates that S. pombe undergoes extensive alternative splicing, particularly during meiosis . When investigating potential alternative splicing of SPAC6F6.13c:

• Detection methods:

  • RT-PCR with primers spanning potential splice junctions

  • Long-read sequencing (PacBio, Nanopore) to capture full-length isoforms

  • Short-read RNA-seq with splice junction analysis

  • Ribosome profiling to identify translated isoforms

• Experimental conditions:

  • Examine different cell cycle stages

  • Compare vegetative growth vs. meiotic conditions

  • Test stress conditions that might trigger alternative splicing

• Validation approaches:

  • Northern blotting to verify isoform sizes

  • Cloning and sequencing of RT-PCR products

  • Targeted proteomics to detect protein isoforms

As demonstrated in search result , PacBio sequencing effectively detects multiple alternative splicing events in S. pombe, including alternative acceptors, exon inclusions, intron retention, and novel exons. Similar approaches could reveal if SPAC6F6.13c undergoes alternative splicing that affects its structure or function.

How should researchers approach comparative analysis of SPAC6F6.13c across different yeast species?

Evolutionary analysis provides valuable functional insights for uncharacterized proteins:

• Ortholog identification:

  • BLAST searches against fungal genomes

  • OrthoMCL or InParanoid clustering

  • Phylogenetic reconstruction to identify true orthologs

• Sequence conservation analysis:

  • Multiple sequence alignment to identify conserved regions

  • Analysis of selective pressure (dN/dS ratios)

  • Identification of conserved functional motifs

• Functional complementation:

  • Testing if orthologs from other species rescue S. pombe knockout phenotypes

  • Heterologous expression and localization studies

  • Domain swapping experiments

• Comparative genomics:

  • Synteny analysis across fungal genomes

  • Gene neighborhood conservation

  • Co-evolution with interacting partners

Researchers studying SPAC6F6.13c should determine if it represents a conserved or species-specific membrane protein, as this distinction significantly impacts experimental design and interpretation of functional data.

How might cryo-electron microscopy advance structural studies of SPAC6F6.13c?

Cryo-electron microscopy (cryo-EM) has revolutionized membrane protein structural biology and offers several advantages for SPAC6F6.13c research:

• Technical advantages:

  • No crystallization requirement

  • Smaller sample quantities needed compared to X-ray crystallography

  • Visualization of protein in near-native environment

  • Captures multiple conformational states

• Sample preparation strategies:

  • Detergent solubilization followed by vitrification

  • Reconstitution into nanodiscs or amphipols

  • Lipid cubic phase embedding

  • On-grid purification approaches

• Data collection considerations:

  • High-end microscopes with energy filters

  • Direct electron detectors

  • Motion correction software

  • Automated data collection

• Image processing workflows:

  • 2D classification to identify conformational states

  • 3D reconstruction and refinement

  • Focused classification for flexible regions

  • Model building and validation

Recent advances in cryo-EM have enabled structure determination of increasingly smaller membrane proteins, making this approach viable for the 778-amino acid SPAC6F6.13c .

What CRISPR-based approaches are most promising for functional genomics of SPAC6F6.13c?

CRISPR technologies offer powerful tools for investigating SPAC6F6.13c function:

• Knockout strategies:

  • Complete gene deletion

  • Introduction of premature stop codons

  • Frame-shifting indels

• Conditional approaches:

  • Auxin-inducible degron tagging

  • Transcriptional repression (CRISPRi)

  • Conditional promoter replacement

• Tagging applications:

  • Endogenous fluorescent protein fusions

  • Epitope tagging for immunoprecipitation

  • BioID or APEX2 proximity labeling tags

• Base editing applications:

  • Introduction of point mutations

  • Codon optimization

  • Regulatory sequence modification

• Screening approaches:

  • Genome-wide screens for genetic interactions

  • Focused screens targeting membrane protein pathways

  • Combinatorial gene editing

These approaches enable precise manipulation of SPAC6F6.13c to determine its functional importance, localization, and interaction partners without artifacts associated with overexpression systems.

How can single-cell technologies enhance understanding of SPAC6F6.13c expression patterns?

Single-cell approaches reveal cell-to-cell variability that population-based methods obscure:

• Single-cell RNA-seq applications:

  • Cell cycle-dependent expression patterns

  • Identification of rare cell populations with distinct expression

  • Correlation with other genes across single cells

  • Pseudotime analysis during cellular transitions

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF) with specific antibodies

  • Single-cell Western blotting

  • Fluorescent protein reporters in live cells

• Spatial transcriptomics:

  • In situ hybridization techniques

  • Spatial mapping of transcript localization

  • Correlation with cellular structures

• Microfluidic applications:

  • Single-cell isolation and analysis

  • Controlled perturbation studies

  • Time-lapse imaging with reporter systems

These approaches could reveal if SPAC6F6.13c exhibits expression heterogeneity across populations, cell cycle-dependent regulation, or localized expression patterns that suggest specialized functions in subpopulations of S. pombe cells.