Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C21C3.06 (SPBC21C3.06)

What are the fundamental characteristics of SPBC21C3.06 protein?

SPBC21C3.06 is an uncharacterized membrane protein from the fission yeast Schizosaccharomyces pombe. It consists of 122 amino acids and is available as a recombinant protein with an N-terminal histidine tag when expressed in E. coli . As a membrane protein, it is embedded within cellular membranes, though its precise localization, topology, and function remain to be fully elucidated.

The protein's basic properties are summarized in the following table:

What computational approaches can predict potential functions of SPBC21C3.06?

Several computational approaches can provide insights into the potential functions of uncharacterized proteins like SPBC21C3.06:

• Sequence homology analysis: Comparing the amino acid sequence with characterized proteins using tools like BLAST, HHpred, or HMMER to identify distant homologs.

• Structural prediction: Using tools like AlphaFold2 or RoseTTAFold to predict the 3D structure, which can provide functional insights.

• Domain and motif identification: Scanning for conserved domains using databases like Pfam, PROSITE, or InterPro.

• Transmembrane topology prediction: Using algorithms like TMHMM, Phobius, or TOPCONS to predict membrane-spanning regions.

• Evolutionary analysis: Examining conservation patterns across related species to identify functionally important residues.

When applying these methods, it's essential to integrate multiple lines of evidence rather than relying on a single prediction approach.

What expression systems are recommended for SPBC21C3.06 recombinant production?

While SPBC21C3.06 has been successfully expressed in E. coli as indicated in the available data , membrane proteins often present unique challenges during heterologous expression. The following methodological approaches may be considered:

• E. coli-based expression:

  • BL21(DE3) or C41/C43(DE3) strains specifically designed for membrane proteins

  • Optimization of induction conditions (IPTG concentration, temperature, duration)

  • Fusion with solubility-enhancing tags (MBP, SUMO) in addition to the His-tag

• Yeast expression systems:

  • Pichia pastoris for high-density cultivation and native-like post-translational modifications

  • S. cerevisiae for expression of fungal proteins

  • Native S. pombe expression for authentic processing and folding

• Insect cell expression:

  • Baculovirus expression system for complex eukaryotic proteins

The choice of expression system should be guided by the research objectives and downstream applications.

What purification protocols are most effective for isolating SPBC21C3.06?

Purification of membrane proteins requires specialized approaches:

• Membrane extraction:

  • Efficient cell lysis (sonication, high-pressure homogenization)

  • Membrane isolation via differential centrifugation

  • Solubilization using appropriate detergents (DDM, LMNG, or digitonin)

• Affinity chromatography:

  • Ni-NTA purification utilizing the His-tag

  • Optimization of imidazole concentration in washing and elution buffers

  • Consideration of pH and salt conditions to minimize non-specific binding

• Secondary purification:

  • Size exclusion chromatography to ensure monodispersity

  • Ion exchange chromatography for additional purity

• Quality control:

  • SDS-PAGE and Western blotting for purity assessment

  • Mass spectrometry for identity confirmation

  • Circular dichroism to assess secondary structure integrity

What experimental methods can determine the function of uncharacterized membrane protein SPBC21C3.06?

Determining the function of uncharacterized membrane proteins requires a multi-faceted approach:

• Localization studies:

  • Fluorescent protein tagging for in vivo localization

  • Immunofluorescence microscopy using antibodies against the protein or tag

  • Subcellular fractionation followed by Western blotting

• Interactome analysis:

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

  • Proximity-dependent biotin identification (BioID)

  • Membrane yeast two-hybrid assays

• Phenotypic characterization:

  • Gene deletion/knockout analysis

  • Conditional expression systems

  • Overexpression studies

• Biochemical assays:

  • Transport assays if suspected to be a transporter

  • Enzymatic activity testing based on computational predictions

  • Lipid binding assays

How can researchers design experiments to identify potential interaction partners of SPBC21C3.06?

Identifying interaction partners for membrane proteins requires specialized approaches:

• Affinity-based methods:

  • Pull-down assays using the recombinant His-tagged SPBC21C3.06

  • Co-immunoprecipitation from native cellular contexts

  • Crosslinking mass spectrometry (XL-MS) to capture transient interactions

• Proximity-based methods:

  • BioID or TurboID fusion for proximity labeling

  • APEX2 proximity labeling

  • Split-GFP complementation for binary interactions

• Library screening approaches:

  • Modified membrane yeast two-hybrid screening

  • Phage display against the purified protein

  • Peptide array screening

• Computational prediction and validation:

  • Interactome prediction based on coexpression data

  • Evolutionary coupling analysis

  • Experimental validation of top candidates

How does evolutionary analysis of SPBC21C3.06 inform its potential function?

Evolutionary analysis can provide crucial insights into protein function:

• Phylogenetic profiling:

  • Identification of orthologs across fungal species

  • Correlation of presence/absence patterns with specific traits or functions

• Synteny analysis:

  • Examination of gene neighborhood conservation

  • Identification of functionally related genes through genomic context

• Evolutionary rate analysis:

  • Detection of selection signatures (positive or purifying)

  • Identification of functionally constrained regions

• Paralogy relationships:

  • Examination for potential gene duplication events

  • Analysis of concerted evolution patterns observed in fungal gene families

Studies of ribosomal protein genes in fungi have demonstrated that parallel concerted evolution can maintain duplicate copies in many fungal species, suggesting important adaptive roles . Similar evolutionary patterns could provide insights if observed for SPBC21C3.06.

What insights can we gain from comparing SPBC21C3.06 with similar proteins in other fungal species?

Comparative analysis across fungal species can reveal:

• Functional constraints:

  • Highly conserved residues likely critical for function

  • Variable regions potentially involved in species-specific adaptations

• Structural conservation:

  • Transmembrane domain conservation patterns

  • Conservation of potential functional motifs

• Evolutionary history:

  • Whether SPBC21C3.06 has undergone gene conversion events similar to those observed in RPL6 and RPS19 families in budding yeasts

  • Potential lineage-specific adaptations

• Expression pattern correlation:

  • Correlation of expression patterns of orthologs across species

  • Co-expression with functionally related genes

What structural analysis techniques are most suitable for characterizing membrane proteins like SPBC21C3.06?

Structural characterization of membrane proteins presents unique challenges:

• X-ray crystallography:

  • Lipidic cubic phase (LCP) crystallization

  • Fusion with crystallization chaperones (e.g., T4 lysozyme)

  • Antibody fragment co-crystallization

• Cryo-electron microscopy (cryo-EM):

  • Single-particle analysis for larger membrane proteins or complexes

  • Optimization of detergent or nanodisc reconstitution

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Solution NMR for smaller membrane proteins or domains

  • Solid-state NMR for membrane-embedded proteins

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Probing conformational dynamics and solvent accessibility

  • Identifying ligand-binding regions

• Small-angle X-ray scattering (SAXS):

  • Low-resolution envelope determination

  • Conformational ensemble analysis

How can researchers experimentally determine the membrane topology of SPBC21C3.06?

Determining membrane topology is critical for understanding membrane protein function:

• Accessibility mapping:

  • Cysteine substitution combined with membrane-impermeable labeling reagents

  • Protease protection assays

  • Glycosylation mapping using engineered sites

• Fluorescence-based approaches:

  • Green fluorescent protein (GFP) fusion analysis

  • Fluorescence protease protection (FPP) assay

• Antibody-based methods:

  • Epitope insertion and accessibility testing

  • Domain-specific antibody generation and binding analysis

• Chemical crosslinking approaches:

  • Site-specific crosslinking to known membrane landmarks

  • Mass spectrometry analysis of crosslinked peptides

How should researchers approach contradictory experimental data regarding SPBC21C3.06?

When faced with contradictory data, a systematic approach is essential:

• Methodological validation:

  • Verify all experimental controls

  • Assess potential interference from tags or expression systems

  • Evaluate experimental conditions for physiological relevance

• Multi-technique confirmation:

  • Apply orthogonal techniques to verify observations

  • Consider limitations of each experimental approach

  • Integrate data from multiple methodologies

• Cellular context considerations:

  • Evaluate influence of cell type, growth conditions

  • Consider potential post-translational modifications

  • Assess protein-protein or protein-lipid interactions

• Reconciliation strategies:

  • Develop testable hypotheses to explain discrepancies

  • Design experiments to specifically address contradictions

  • Consider dynamic behaviors or multiple functional states

What gene editing strategies are most effective for studying SPBC21C3.06 function in S. pombe?

Several approaches can be employed for genetic manipulation in S. pombe:

• CRISPR-Cas9 methodology:

  • Design of guide RNAs with high specificity

  • Optimization of homology-directed repair templates

  • Strategies for marker-free editing

• Traditional homologous recombination:

  • Long flanking homology targeting

  • Selection marker strategies

  • PCR-based verification methods

• Regulated expression systems:

  • Tetracycline-inducible or repressible systems

  • Thiamine-repressible nmt promoters of varying strengths

  • Estradiol-inducible systems

• Tagging strategies:

  • C-terminal vs. N-terminal tags considering membrane topology

  • Endogenous locus modification vs. ectopic expression

  • Selection of appropriate linker sequences for membrane proteins