Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C1281.03c (SPCC1281.03c)

What cloning methods are most effective for recombinant expression of SPCC1281.03c in S. pombe?

Gap Repair Cloning (GRC) offers significant advantages for cloning SPCC1281.03c in S. pombe. This technique leverages the homologous recombination activity within yeast cells and demonstrates high efficiency with relatively short homology sequences (≥25 bp). The method requires:

• PCR amplification of your gene of interest with primers containing homologous sequences to your target vector

• Linearization of the vector (restriction enzyme digestion is suitable)

• Co-transformation of both fragments into S. pombe

GRC efficiency in S. pombe can reach approximately 70% without specific selection, and remarkably, efficiency increases to >95% in lig4Δ mutant cells where non-homologous end joining is deficient . The technique has been successfully used to construct various marker-containing plasmids (leu1+, ade6+, his5+, lys1+) using stable plasmids like pDblet as backbones .

What expression systems are optimal for functional studies of SPCC1281.03c?

When studying SPCC1281.03c, the nmt1 promoter system in S. pombe provides excellent control over expression levels. This system offers:

• Inducible expression (thiamine-repressible)

• Multiple promoter strength variants (full strength, intermediate, and weak)

• Compatibility with fluorescent tagging approaches

Research has demonstrated successful construction of nmt1 promoter:EGFP fusion plasmids using GRC in S. pombe, which can be applied to SPCC1281.03c for localization and expression studies . The system allows visualization of protein expression through fluorescence imaging, with colonies containing the correctly constructed plasmids showing clear GFP fluorescence when grown on selective media .

What approaches can identify potential alternative splice variants of SPCC1281.03c during different cellular conditions?

Single-molecule real-time (SMRT) sequencing using the Pacific Biosciences (PacBio) platform offers a powerful approach for identifying potential alternative splice variants of SPCC1281.03c. This technique provides:

• Full-length cDNA sequencing capability

• Detection of novel isoforms not previously annotated

• Temporal profiling of expression during cellular processes

To identify potential alternative splice variants:

• Collect RNA samples across relevant time points or conditions (e.g., time course during meiosis)

• Prepare poly(A)+ RNA and generate cDNA libraries

• Sequence using PacBio platform (typically 5 SMRT cells per condition)

• Process data through the Iso-Seq pipeline to generate high-quality, full-length polished consensus sequences

• Analyze using specialized software like SpliceHunter to detect, quantify, and compare splicing patterns

This approach has successfully identified numerous isoforms in S. pombe during meiosis and can reveal condition-specific expression patterns of membrane protein variants .

How can the membrane topology of SPCC1281.03c be experimentally determined?

Determining membrane topology for uncharacterized proteins like SPCC1281.03c requires a multi-faceted experimental approach:

• Computational prediction: Begin with topology prediction algorithms that analyze hydrophobicity patterns, charge distribution, and evolutionary conservation.

• Experimental validation: Employ reporter fusion strategies where:

  • GFP or similar reporters are fused at various positions along the protein sequence

  • Fluorescence microscopy determines which portions face the cytoplasm vs. membrane-enclosed compartments

  • Protease protection assays identify regions accessible to proteolytic digestion

• Site-directed mutagenesis: Create targeted mutations in predicted transmembrane domains to assess their functional importance.

The construction of reporter fusions can be efficiently accomplished using Gap Repair Cloning as demonstrated with the nmt1 promoter:EGFP system, allowing for rapid generation of multiple constructs with different fusion points .

What genetic approaches can reveal the function of SPCC1281.03c in S. pombe?

Multiple genetic approaches can be employed to elucidate the function of uncharacterized membrane proteins like SPCC1281.03c:

• Gene deletion/disruption: Construct knockout strains using GRC-based homologous recombination to integrate selectable markers into the target locus. Analyze the resulting phenotypes under various conditions including:

  • Growth at different temperatures

  • Response to osmotic, oxidative, or cell wall stresses

  • Mating and sporulation efficiency

• Conditional expression systems: Employ the nmt1 promoter system to create strains where SPCC1281.03c expression can be tightly regulated. This allows observation of immediate effects following protein depletion or overexpression .

• Genetic interaction screens: Conduct systematic genetic interaction analyses by crossing SPCC1281.03c mutants with deletion libraries to identify functionally related genes through synthetic lethal or synthetic rescue interactions.

• Fluorescent tagging and localization: Use GRC to create C-terminal or N-terminal fluorescent protein fusions to determine subcellular localization patterns under different conditions or cell cycle stages .

How can the expression profile of SPCC1281.03c be characterized during meiosis and sporulation?

To characterize the expression profile of SPCC1281.03c during meiosis and sporulation:

• Time-course sampling: Collect samples at defined intervals during meiosis (e.g., 0, 2, 4, 6, 8, and 10 hours after induction) to capture the complete process from premeiotic S phase through spore maturation .

• RNA isolation and sequencing: Process samples for:

  • PacBio SMRT sequencing for isoform-level profiling

  • Short-read RNA-seq for quantitative expression analysis

• Data analysis: Analyze the resulting data using specialized software to:

  • Quantify expression changes using the number of full-length circular consensus sequence (FL CCS) reads

  • Identify alternative splice variants that may be meiosis-specific

  • Compare expression patterns with known meiotic regulators

This approach has been successfully used to characterize the dynamic landscape of S. pombe meiosis, revealing substantial transcriptome reshaping with multiple meiosis-specific alternative splicing events .

What expression systems are most suitable for purification of SPCC1281.03c for structural studies?

For structural biology studies of SPCC1281.03c, consider these expression approaches:

• Homologous expression in S. pombe:

  • Advantages: Native folding environment, appropriate post-translational modifications

  • Method: Use GRC to construct expression vectors with strong promoters (nmt1) and appropriate purification tags

  • Considerations: Yield may be lower than heterologous systems but protein quality often superior

• Heterologous expression optimization:

  • For higher yields, consider expression in specialized systems for membrane proteins

  • Codon optimization may be necessary when switching expression hosts

  • Consider fusion tags that enhance solubility while allowing tag removal during purification

• Construct design considerations:

  • Remove predicted disordered regions that may impede crystallization

  • Consider expressing stable domains individually if full-length protein proves challenging

  • Engineer thermostability through targeted mutations based on computational prediction

The use of GRC allows rapid testing of multiple constructs with different boundaries and tags, enabling efficient optimization of expression conditions for structural studies .

How can potential functions of SPCC1281.03c be predicted through comparative genomics?

To predict potential functions of SPCC1281.03c through comparative genomics:

• Sequence-based analyses:

  • Conduct sensitive homology searches using PSI-BLAST, HHpred, or HMMER

  • Identify remote homologs across different species, particularly focusing on other yeasts and fungi

  • Analyze conserved domains and motifs that may suggest functional roles

• Structural prediction approaches:

  • Generate three-dimensional models using AlphaFold or similar tools

  • Compare predicted structures with known membrane proteins to identify structural similarities

  • Analyze potential ligand-binding pockets or functionally important residues

• Co-expression network analysis:

  • Analyze gene expression datasets across multiple conditions to identify genes with similar expression patterns

  • Build co-expression networks to predict functional associations

  • Integrate with existing protein-protein interaction data

• Phylogenetic profiling:

  • Analyze the presence/absence patterns of SPCC1281.03c across species

  • Identify proteins with similar phylogenetic profiles, suggesting functional relationships

These computational approaches can generate testable hypotheses about SPCC1281.03c function that can then be validated through the experimental methods described in previous sections.

What analytical tools can identify potential post-translational modifications of SPCC1281.03c?

For identification of post-translational modifications (PTMs) in SPCC1281.03c:

• Computational prediction:

  • Use specialized algorithms to predict potential phosphorylation, glycosylation, ubiquitination, and other PTM sites

  • Compare predicted sites across orthologs to identify evolutionarily conserved modification patterns

• Mass spectrometry-based approaches:

  • Purify SPCC1281.03c from cells grown under different conditions

  • Perform proteomic analysis using:

    • Bottom-up proteomics (protein digestion followed by peptide analysis)

    • Middle-down proteomics (partial digestion to create larger peptide fragments)

    • Top-down proteomics (analysis of intact protein)

  • Use specialized fragmentation techniques optimized for PTM identification

• Site-directed mutagenesis validation:

  • Mutate predicted PTM sites to either prevent modification or mimic constitutive modification

  • Assess the functional consequences through localization studies, protein-protein interaction analyses, or phenotypic assays

  • Complement with phospho-specific or other PTM-specific antibodies if available

These approaches can reveal how the function of SPCC1281.03c may be regulated through PTMs during different cellular processes or conditions.

What methods are most effective for identifying protein interaction partners of SPCC1281.03c?

To identify protein interaction partners of membrane proteins like SPCC1281.03c, consider these complementary approaches:

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

  • Generate strains expressing tagged versions of SPCC1281.03c using GRC

  • Optimize membrane protein extraction conditions using appropriate detergents

  • Perform pull-downs followed by mass spectrometry identification

  • Compare results across different conditions to identify condition-specific interactions

• Yeast two-hybrid (Y2H) adaptations:

  • Use split-ubiquitin membrane Y2H systems specifically designed for membrane proteins

  • Screen against genomic or cDNA libraries to identify potential interactors

  • Validate positive hits through reciprocal experiments and secondary assays

• Proximity labeling approaches:

  • Fuse SPCC1281.03c to enzymes like BioID or APEX2

  • Allow in vivo labeling of proximal proteins

  • Purify biotinylated proteins and identify them by mass spectrometry

  • This approach is particularly valuable for capturing transient interactions

• Co-localization studies:

  • Use fluorescently tagged versions of SPCC1281.03c and suspected interaction partners

  • Perform high-resolution microscopy to assess co-localization

  • Consider advanced techniques like Förster resonance energy transfer (FRET) or fluorescence lifetime imaging microscopy (FLIM) to confirm direct interactions

These methods can be implemented using the GRC approaches described earlier to efficiently generate the necessary constructs for interaction studies .

How can CRISPR-Cas9 techniques be adapted for efficient genome editing of SPCC1281.03c in S. pombe?

CRISPR-Cas9 genome editing can be effectively adapted for studying SPCC1281.03c through these approaches:

• CRISPR design considerations for S. pombe:

  • Design guide RNAs with high specificity for the SPCC1281.03c locus

  • Optimize Cas9 expression using appropriate promoters for S. pombe

  • Consider using homology-directed repair templates designed through GRC principles

• Integration with traditional S. pombe techniques:

  • Combine CRISPR-Cas9 with established S. pombe transformation protocols

  • Use selectable markers and reporter systems compatible with S. pombe genetics

  • Apply GRC principles to design repair templates with appropriate homology arms

• Applications for SPCC1281.03c characterization:

  • Generate precise point mutations to test functional hypotheses

  • Create scarless tags for live-cell imaging

  • Implement conditional degradation systems to study protein function

  • Introduce domain swaps to test structure-function relationships

• Validation strategies:

  • Confirm edits through sequencing and functional assays

  • Assess off-target effects through whole-genome sequencing

  • Complement with traditional genetic approaches to validate phenotypes

These advanced genome editing approaches expand the toolkit for studying SPCC1281.03c beyond traditional homologous recombination methods, enabling more precise genetic manipulations.