Recombinant Schizosaccharomyces pombe Uncharacterized protein C1071.03c (SPAC1071.03c)

What expression systems are recommended for recombinant production of SPAC1071.03c?

Multiple expression systems have been successfully employed for SPAC1071.03c recombinant production:

For initial characterization studies, the E. coli system typically provides sufficient material, but for functional studies, yeast expression (particularly using S. pombe itself as host) often provides better protein quality with native-like post-translational modifications . When expressing in E. coli, optimization of codon usage for bacterial expression is recommended to improve yield.

What purification strategy works best for SPAC1071.03c?

A multi-step purification protocol is recommended:

• Affinity chromatography using a fusion tag (His-tag is most common)

• Ion exchange chromatography for intermediate purification

• Size exclusion chromatography for final polishing

Researchers report optimal purification results with the following conditions:

• Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail

• Affinity binding: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole (for His-tagged protein)

• Elution: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM imidazole gradient

The protein shows improved stability when stored in Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage. Repeated freeze-thaw cycles should be avoided .

How can I validate the expression and localization of SPAC1071.03c in S. pombe cells?

For comprehensive validation of SPAC1071.03c expression and localization:

• Transcript level analysis:

  • RT-qPCR using gene-specific primers

  • RNA-seq for genome-wide expression context

• Protein level detection:

  • Western blot using antibodies against the native protein or epitope tags

  • Mass spectrometry for protein identification and quantification

• Subcellular localization:

  • Fluorescence microscopy using GFP/mCherry fusion constructs

  • Immunofluorescence with antibodies against the protein or tag

  • Subcellular fractionation followed by Western blot analysis

The membrane-spanning domain prediction suggests this protein may localize to the cell membrane or an organelle membrane. When designing fluorescent fusion proteins, both N-terminal and C-terminal tagging should be tested, as the N-terminal signal sequence might affect proper localization if disrupted by a tag .

What genetic approaches are most effective for studying SPAC1071.03c function?

Several complementary genetic approaches provide insights into SPAC1071.03c function:

• Gene deletion/knockout:

  • Use homologous recombination to create SPAC1071.03c deletion strains

  • Phenotype analysis under various growth conditions and stresses

  • Analyze growth, morphology, cell cycle progression, and mating efficiency

• Conditional expression systems:

  • Implement nmt1 promoter system for thiamine-repressible expression

  • Create temperature-sensitive alleles for rapid inactivation studies

• Genetic interaction mapping:

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

  • Epistasis analysis with known pathway components

  • Suppressor screens to identify functional relationships

• Site-directed mutagenesis:

  • Target conserved residues to assess functional importance

  • Create phospho-mimetic or phospho-deficient mutations at predicted phosphorylation sites

Based on related studies in S. pombe, SGA screens have proven particularly valuable for identifying the functional networks of uncharacterized proteins. When combined with next-generation sequencing approaches, these methods can rapidly generate functional hypotheses for experimental validation .

What omics approaches are useful for characterizing SPAC1071.03c function?

Multi-omics approaches provide complementary data for functional characterization:

For RNA-seq experiments, both polyA-selected and total RNA libraries should be considered to capture possible effects on non-coding RNAs. Differential expression analysis should include annotation of specific genomic regions affected, such as subtelomeric genes that have been shown to be sensitive to some chromatin regulatory factors in S. pombe .

How does SPAC1071.03c relate to mating phenotypes in S. pombe?

S. pombe natural isolates exhibit diverse mating phenotypes that impact various cellular processes. To investigate SPAC1071.03c's potential role in mating:

• Quantitative mating assays:

  • Compare mating efficiency between wild-type and SPAC1071.03c deletion strains

  • Measure inbreeding coefficients using fluorescent markers to track mating partners

  • Analyze in both homothallic (h90) and heterothallic (h+ and h-) backgrounds

• Pheromone response analysis:

  • Monitor pheromone signaling pathway activation

  • Assess morphological changes during mating (shmoo formation)

  • Evaluate SPAC1071.03c expression changes during the mating process

• Cross-strain analysis:

  • Test SPAC1071.03c function across different natural isolates with varying mating phenotypes

  • Particularly examine isolates with different fractions of ancestral lineages, such as FY29043, FY29022, FY28981, which show varying inbreeding coefficients

• Sporulation and germination analysis:

  • Assess spore formation efficiency

  • Evaluate spore viability and germination rates

Research on S. pombe natural isolates has shown that mating phenotypes vary significantly, with inbreeding coefficients ranging from highly inbred (similar to standard laboratory strain) to more random mating patterns. Mating efficiency also varies substantially (10-50% of cells mating) across different isolates. These differences could interact with the function of SPAC1071.03c .

What is the relationship between SPAC1071.03c and chromatin regulation?

Evidence suggests potential involvement of SPAC1071.03c in chromatin-related processes:

• Histone modification analysis:

  • ChIP-qPCR and ChIP-seq for key histone marks (H3K9me, H3K4me, H3K36me)

  • Focus on heterochromatic regions (centromeres, telomeres, mating loci)

  • Compare wild-type and SPAC1071.03c deletion strains

• Silencing assays:

  • Reporter gene integration at heterochromatic regions

  • Analysis of transcriptional silencing maintenance

• Chromosome organization:

  • Microscopy analysis of nuclear organization

  • 3C/Hi-C mapping of genome topology

• Genetic interactions with chromatin modifiers:

  • Target known chromatin regulators (Clr4, Swi6, Brl1) for genetic interaction studies

  • Test synthetic phenotypes with heterochromatin formation pathways

Studies of related proteins in S. pombe have shown that deletions of some uncharacterized genes can alter H3K9 methylation patterns, particularly at subtelomeric regions, without affecting centromeric heterochromatin. Similar analysis should be performed for SPAC1071.03c to determine if it plays a role in region-specific chromatin regulation .

How does SPAC1071.03c interact with transcriptional machinery?

To investigate potential roles in transcriptional regulation:

• RNA Polymerase II association:

  • Co-immunoprecipitation with RNA Pol II components

  • ChIP-seq for Pol II occupancy comparison between wild-type and deletion strains

  • PRO-seq to measure active transcription and pausing

• Transcription elongation factor interactions:

  • Test interactions with known elongation factors (Ell1, Eaf1)

  • Analyze synthetic phenotypes with elongation factor mutants

  • Determine co-localization at highly transcribed genes

• Mediator complex interaction:

  • Test physical interaction with Mediator components

  • Analyze genetic interactions with Mediator subunits

• Nascent RNA analysis:

  • PRO-seq to quantify transcription rates

  • Analysis of antisense transcription

Related research in S. pombe has identified complexes involving RNA Polymerase II elongation factors (like Ell1/Eaf1) that associate with genes having high RNA Pol II occupancy. Given the prevalence of uncharacterized proteins in these complexes (like SPAC6G9.15c), SPAC1071.03c might function in a similar context .

How can CRISPR-Cas9 be optimized for editing SPAC1071.03c in S. pombe?

CRISPR-Cas9 editing of SPAC1071.03c requires specific optimization for S. pombe:

• Guide RNA design:

  • Select target sites with minimal off-target potential

  • Avoid regions with secondary structure formation

  • Recommended tools: CHOPCHOP, CRISPOR, CRISPRdirect

• Delivery methods:

  • Plasmid-based expression

  • Ribonucleoprotein (RNP) complex transformation

• Repair template design:

  • Homology-directed repair templates with 50-80 bp homology arms

  • Selection marker strategy (antibiotic resistance, auxotrophic markers)

• Validation strategies:

  • PCR-based genotyping

  • Sequencing verification

  • Western blot confirmation of protein modification/elimination

The optimal CRISPR editing protocol for S. pombe includes transformation of cells in early log phase, with a 4-hour recovery in non-selective rich media before plating on selective media. For knock-in applications, longer homology arms (500-1000 bp) significantly improve editing efficiency.

What structural biology approaches are most promising for SPAC1071.03c characterization?

For comprehensive structural characterization of this challenging uncharacterized protein:

• Cryo-electron microscopy (cryo-EM):

  • Advantageous for membrane proteins

  • Can reveal protein complexes in near-native states

  • Resolution increasingly comparable to X-ray crystallography

• X-ray crystallography:

  • Requires high-purity, homogeneous protein preparation

  • Challenging for membrane proteins without proper detergent screening

  • Consider LCP (lipidic cubic phase) crystallization methods

• NMR spectroscopy:

  • Good for dynamic regions and smaller domains

  • Requires isotope labeling (15N, 13C)

  • Can identify binding interfaces with interacting partners

• Integrative structural biology:

  • Combine multiple methods (SAXS, HDX-MS, cross-linking MS)

  • Computational modeling with experimental constraints

  • AlphaFold2 prediction with experimental validation

Researchers should consider domain-based approaches, expressing and characterizing individual domains separately if the full-length protein proves recalcitrant to structural studies.

How can systems biology approaches integrate SPAC1071.03c into cellular network models?

Systems-level analysis can place SPAC1071.03c in the broader cellular context:

• Network integration approaches:

  • Integrate physical interaction data (Co-IP, BioID, Y2H)

  • Combine with genetic interaction networks (SGA screens)

  • Incorporate expression correlation data across conditions

• Pathway enrichment analysis:

  • GO term enrichment of interacting partners

  • KEGG pathway mapping

  • Custom S. pombe-specific pathway analysis

• Dynamic network modeling:

  • Temporal expression/localization changes during cell cycle

  • Response to environmental perturbations

  • Mathematical modeling of potential pathway involvement

• Comparative genomics:

  • Ortholog identification across yeast species

  • Synteny analysis for contextual insights

  • Evolutionary rate analysis for functional constraints

When conducting SGA screens with SPAC1071.03c deletion, prioritize genes identified in multiple replicate screens and validate key interactions with targeted experiments. The integration of diverse data types (genetic, physical, expression) provides the most robust network placement .

What strategies can overcome expression and solubility issues with recombinant SPAC1071.03c?

For challenging expression and solubility:

• Fusion tag optimization:

  • Compare multiple solubility-enhancing tags (MBP, GST, SUMO, TrxA, NusA)

  • Test both N-terminal and C-terminal tag positions

  • Evaluate tag removal efficiency with various proteases

• Expression conditions:

  • Temperature optimization (16°C, 25°C, 30°C, 37°C)

  • Induction strength variation (IPTG concentration or alternative promoters)

  • Media composition (rich vs. minimal, supplementation strategies)

• Solubilization approaches:

  • Screen multiple detergents for membrane protein extraction

  • Test common solubilizing additives (glycerol, arginine, proline)

  • Investigate refolding strategies from inclusion bodies

• Truncation and domain mapping:

  • Express predicted domains individually

  • Create systematic truncation series

  • Remove predicted transmembrane or disordered regions

Data from commercial protein production services indicates that with optimization, production yields of 1-10 mg of purified protein per liter of culture can be achieved for SPAC1071.03c, with purity levels exceeding 90% .

How can I design definitive functional assays for an uncharacterized protein like SPAC1071.03c?

For developing functional assays for an uncharacterized protein:

• Hypothesis generation:

  • Leverage bioinformatic predictions (domains, motifs)

  • Consider genomic context and evolutionary conservation

  • Use preliminary data from interactome studies

• Activity-based assays:

  • Enzymatic activity screens based on predicted domains

  • Binding assays with predicted interaction partners

  • Functional complementation with orthologous genes

• Cellular phenotype assays:

  • Microscopy-based phenotyping (morphology, localization)

  • Growth under various stress conditions

  • Cell cycle progression analysis

• In vivo reporter systems:

  • Develop fluorescent/luminescent readouts for putative activities

  • Split-protein complementation for interaction detection

  • Degron-based stability assays

Successful characterization typically requires an iterative approach, where initial broad assays inform more specific functional tests. For membrane proteins like SPAC1071.03c, assays should consider both membrane integrity and potential transport or signaling functions.