Recombinant Schizosaccharomyces pombe UPF0674 endoplasmic reticulum membrane protein C2G5.01 (SPBC2G5.01)

How is recombinant SPBC2G5.01 produced using recombinant DNA technology?

Production of recombinant SPBC2G5.01 involves several key steps using recombinant DNA technology:

• Gene isolation and amplification: The SPBC2G5.01 gene is typically isolated from S. pombe genomic DNA using PCR with specific primers designed to amplify the coding region (nucleotides corresponding to amino acids 1-374) .

• Vector construction: The amplified gene is inserted into an expression vector containing:

  • A strong promoter compatible with the chosen expression system

  • Appropriate selection markers

  • Potentially a fusion tag for detection and purification

  • Sequences that facilitate proper membrane protein expression

• Transformation and expression: The recombinant DNA construct is introduced into a suitable host organism. Due to its membrane protein nature, specialized expression systems may be necessary for proper folding and post-translational modifications .

• Protein production: The recombinant protein is expressed in the host organism under optimized conditions. As SPBC2G5.01 is a membrane protein, expression conditions must be carefully controlled to prevent aggregation and misfolding .

• Purification: The protein is extracted using detergents or other membrane-solubilizing agents and purified using affinity chromatography based on the fusion tag used.

This process leverages the principle that DNA molecules from all organisms share the same chemical structure, making it possible to introduce S. pombe DNA into different host organisms for protein expression .

What expression systems are typically used for producing recombinant Schizosaccharomyces pombe proteins?

Several expression systems can be employed for producing recombinant S. pombe proteins like SPBC2G5.01, each with distinct advantages:

When selecting an expression system for SPBC2G5.01, researchers must consider that expression of foreign proteins requires specialized expression vectors and often necessitates significant restructuring of foreign coding sequences for optimal expression .

What are the primary structural features of SPBC2G5.01 that affect its recombinant expression?

The primary structure of SPBC2G5.01 contains several features that significantly impact recombinant expression strategies:

• Signal sequence: The N-terminal sequence (MINKKLLFLVFALAKGVLADE) likely functions as a signal peptide directing the protein to the endoplasmic reticulum .

• Transmembrane domains: Hydrophobicity analysis indicates multiple transmembrane segments, making it challenging to express in soluble form.

• Charged regions: The protein contains acidic clusters (EEDEYEEDYNM) that may affect folding and stability during recombinant expression.

• Post-translational modification sites: Potential glycosylation and phosphorylation sites that may be essential for proper function but challenging to reproduce in heterologous systems.

• C-terminal basic region: The lysine-rich sequence at the C-terminus (KKVKSSGDISKLSESDQKKRM) may function in membrane targeting or protein-protein interactions .

Understanding these structural elements is essential for designing expression constructs that will produce properly folded and functional protein. Researchers often need to engineer constructs that preserve these features while optimizing for expression in the chosen host system.

What are the challenges in expressing recombinant membrane proteins like SPBC2G5.01?

Expression of membrane proteins like SPBC2G5.01 presents several unique challenges:

• Toxicity to host cells: Overexpression of membrane proteins can disrupt host membrane integrity, leading to growth inhibition or cell death.

• Protein misfolding and aggregation: The hydrophobic nature of transmembrane domains often leads to improper folding and aggregation when overexpressed.

• Insufficient membrane incorporation: Cellular machinery for membrane insertion can become saturated, resulting in cytoplasmic aggregation.

• Post-translational modification differences: Different expression hosts have varying capacities for post-translational modifications necessary for proper function.

• Detergent compatibility: Finding detergents that effectively solubilize the protein while maintaining its native structure is often challenging.

Methodological approaches to address these challenges include:

• Using lower-temperature induction to slow expression and allow proper folding

• Employing fusion partners that enhance solubility or membrane targeting

• Testing multiple detergents and lipid environments for optimal extraction

• Utilizing specialized host strains designed for membrane protein expression

• Implementing directed evolution approaches to identify more expressible variants

Expression of foreign proteins like SPBC2G5.01 requires specialized expression vectors and often necessitates significant restructuring of the coding sequences to overcome these challenges .

How can researchers optimize buffer conditions for SPBC2G5.01 stability during purification?

Optimizing buffer conditions is crucial for maintaining SPBC2G5.01 stability during purification. Based on general principles for membrane proteins and the specific characteristics of SPBC2G5.01, researchers should consider:

• Buffer composition optimization:

• Detergent screening: Systematic testing of multiple detergents (DDM, LMNG, CHAPS) at concentrations slightly above their critical micelle concentration.

• Stability assessment methods:

  • Thermal shift assays to identify stabilizing conditions

  • Size-exclusion chromatography to monitor aggregation

  • Activity assays (if available) to assess functional integrity

• Storage conditions: Based on commercial preparations, SPBC2G5.01 appears to be stable in Tris-based buffer with 50% glycerol at -20°C .

• Additive screening: Testing various additives including specific lipids, cholesterol, or stabilizing compounds that might enhance protein stability.

Methodologically, researchers should implement a systematic approach, testing multiple buffer conditions in parallel using a small-scale purification protocol before scaling up to larger preparations.

What techniques are most effective for validating the proper folding of recombinant SPBC2G5.01?

Validating proper folding of membrane proteins like SPBC2G5.01 is essential before conducting functional studies. Recommended approaches include:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to examine tertiary structure via intrinsic tryptophan fluorescence

  • Differential scanning calorimetry to determine thermal stability profiles

• Functional assays:

  • Lipid binding assays if SPBC2G5.01 interacts with specific lipids

  • Protein-protein interaction studies with known binding partners

  • Transport assays if the protein functions as a transporter

• Structural validation:

  • Limited proteolysis to assess compact, folded domains resistant to digestion

  • Negative-stain electron microscopy to examine protein homogeneity and structure

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to verify monodispersity

• Antibody recognition:

  • Conformation-specific antibodies can be used to distinguish properly folded protein

  • Epitope mapping to verify exposure of key structural elements

• In silico validation:

  • Comparing experimental data with structural predictions

  • Molecular dynamics simulations to assess stability of proposed structures

These methods should be applied systematically, with results compared against controls including denatured protein samples and, when possible, native protein extracted directly from S. pombe.

What approaches can be used to study SPBC2G5.01 localization in cells?

Understanding the subcellular localization of SPBC2G5.01 provides insights into its function. Several complementary approaches can be employed:

• Fluorescence microscopy techniques:

  • GFP fusion constructs: Creating N- or C-terminal GFP fusions of SPBC2G5.01 for live-cell imaging

  • Split-GFP complementation: Using this approach to minimize disruption of targeting signals

  • Super-resolution microscopy: Techniques like STORM or PALM for precise localization within the ER membrane

• Biochemical fractionation:

  • Sequential centrifugation to separate cellular compartments

  • Density gradient fractionation for more refined separation

  • Western blot analysis of fractions using anti-SPBC2G5.01 antibodies

• Immunolocalization:

  • Immunofluorescence using antibodies against SPBC2G5.01

  • Immuno-electron microscopy for ultrastructural localization

  • Co-localization with known ER markers (e.g., calnexin, Sec61)

• Proximity labeling approaches:

  • APEX2 or BioID fusion to label neighboring proteins

  • Analysis of labeled proteins to identify microenvironment

• Functional validation of localization:

  • Mutational analysis of potential targeting sequences

  • Heterologous expression to test conservation of targeting mechanisms

  • Disruption of trafficking machinery to examine effects on localization

These approaches should be conducted in both native S. pombe cells and heterologous expression systems to compare localization patterns and confirm proper trafficking of the recombinant protein.

What strategies can be employed to identify interaction partners of SPBC2G5.01?

Identifying protein interaction partners is crucial for understanding SPBC2G5.01's function in the endoplasmic reticulum. Several complementary strategies can be employed:

• Affinity-based approaches:

  • Co-immunoprecipitation (Co-IP): Using antibodies against SPBC2G5.01 or epitope tags

  • Tandem affinity purification (TAP): Incorporating dual tags for sequential purification

  • Chemical cross-linking: Stabilizing transient interactions prior to purification

• Proximity-based methods:

  • BioID: Fusion with a biotin ligase to biotinylate proximal proteins

  • APEX2: Peroxidase-based proximity labeling

  • Split-protein complementation: Using split reporters like BiFC to visualize interactions

• Genetic approaches:

  • Synthetic genetic arrays (SGAs): Identifying genetic interactions that suggest physical interactions

  • Suppressor screens: Finding mutations that suppress SPBC2G5.01 deletion phenotypes

  • E-MAP (Epistatic Mini Array Profile): Systematic genetic interaction mapping

• Computational prediction:

  • Sequence-based interaction prediction

  • Structural modeling of potential interaction interfaces

  • Network analysis based on co-expression data

• Quantitative proteomics:

  • SILAC: Comparing protein abundances in wild-type vs. SPBC2G5.01 deletion strains

  • TMT/iTRAQ: Multiplex comparison of protein levels across conditions

A systematic workflow combining these approaches could involve:

• Initial computational prediction of potential interactors

• BioID or APEX2 labeling to identify the proximal proteome

• Validation of top candidates using Co-IP or BiFC

• Functional characterization through genetic interaction studies

This multi-faceted approach would help overcome the challenges associated with identifying interaction partners of membrane proteins, which are often difficult to study due to their hydrophobic nature and detergent solubilization requirements.

How can CRISPR-Cas9 technology be applied to study SPBC2G5.01 function?

CRISPR-Cas9 technology offers powerful approaches for investigating SPBC2G5.01 function in S. pombe:

• Gene knockout/knockdown strategies:

  • Complete gene deletion to assess null phenotype

  • Conditional degradation systems (e.g., auxin-inducible degron) for temporal control

  • CRISPRi for tunable repression without modifying the genomic sequence

• Domain-specific modifications:

  • Precise deletion of specific domains to assess their contribution to function

  • Point mutations in key residues identified through sequence conservation

  • Introduction of premature stop codons to create truncated variants

• Tagging approaches:

  • Endogenous tagging with fluorescent proteins or epitope tags

  • Introduction of split reporter tags for interaction studies

  • Insertion of proximity labeling enzymes at the endogenous locus

• Regulatory element modification:

  • Promoter replacements to control expression levels

  • Modification of UTRs to alter translational regulation

  • Installation of inducible systems for temporal control

• High-throughput functional screens:

  • Saturating mutagenesis of the entire gene

  • Library-based screens targeting specific domains

  • Synthetic genetic interaction screens with other genes

Implementation methodology:

• Design multiple sgRNAs targeting different regions of the SPBC2G5.01 gene

• Construct repair templates containing desired modifications flanked by homology arms

• Transform S. pombe with Cas9 and sgRNA expression constructs along with repair templates

• Screen transformants for successful editing

• Validate edits through sequencing and expression analysis

• Phenotypically characterize edited strains under various conditions

This approach allows for precise genetic manipulation to understand SPBC2G5.01 function in its native context, overcoming limitations of heterologous expression systems.

What experimental approaches can resolve contradictions in SPBC2G5.01 functional data?

Resolving contradictory experimental data regarding SPBC2G5.01 function requires systematic investigation using complementary approaches:

• Replication with standardized conditions:

  • Establishing consistent experimental protocols across laboratories

  • Controlling for strain background variations

  • Implementing blinded analysis to reduce bias

• Multi-system validation:

  • Testing function in both native S. pombe and heterologous systems

  • Comparing results from different expression systems

  • Assessing function in related species with SPBC2G5.01 homologs

• Domain-specific analysis:

  • Creating chimeric proteins with domains from related proteins

  • Systematic mutational analysis of key residues

  • Deletion analysis to identify functional domains

• Condition-dependent function assessment:

  • Testing under various stress conditions (heat, oxidative, ER stress)

  • Nutrient limitation experiments

  • Cell cycle synchronization to identify phase-specific functions

• Multi-omics integration:

  • Correlating transcriptomic, proteomic, and metabolomic data

  • Network analysis to place contradictory results in broader context

  • Temporal analysis to resolve seemingly contradictory findings

• Experimental design to tackle epistasis:

  • Implementing E-MAP approaches to systematically identify genetic interactions

  • Developing experimental designs to search for epistasis in yeast systems

Methodologically, researchers should implement a decision tree approach:

• Identify specific points of contradiction in the literature

• Design experiments that directly address these contradictions

• Control for all variables that might explain discrepancies

• Perform multiple independent replicates

• Use statistical methods appropriate for the specific type of data

• Publish all data, including negative results, to build a complete understanding

This systematic approach can help resolve contradictions and develop a unified model of SPBC2G5.01 function.

How do post-translational modifications affect SPBC2G5.01 function?

Post-translational modifications (PTMs) likely play critical roles in regulating SPBC2G5.01 function. A comprehensive investigation would include:

• Identification of PTM sites:

  • Mass spectrometry-based proteomics to map modifications

  • Site-specific antibodies for key modifications

  • Comparison of PTMs between native and recombinant protein

• Types of modifications to investigate:

  • Phosphorylation: Particularly on serine/threonine residues in cytoplasmic domains

  • Glycosylation: N-linked modifications on luminal domains

  • Ubiquitination: Particularly relevant for quality control and turnover

  • Palmitoylation: Potentially important for membrane association

• Functional analysis of PTMs:

  • Site-directed mutagenesis of modified residues

  • Inhibition of specific modifying enzymes

  • Expression in systems lacking specific modification capabilities

• Dynamics of modifications:

  • Cell cycle-dependent changes

  • Stress-induced modification patterns

  • Half-life and turnover of modifications

• Structural consequences:

  • How modifications affect protein conformation

  • Impact on protein-protein interactions

  • Effects on membrane topology and integration

Experimental approach workflow:

• Global PTM mapping through mass spectrometry

• Validation of key sites through site-specific antibodies or targeted MS

• Functional characterization through mutagenesis (e.g., phosphomimetic mutations)

• Temporal analysis under various physiological conditions

• Integration of findings into a regulatory model

Since SPBC2G5.01 is an endoplasmic reticulum membrane protein, particular attention should be paid to how PTMs might regulate its trafficking, membrane integration, and protein quality control within the secretory pathway.

What are the best controls to include in experiments using recombinant SPBC2G5.01?

Robust experimental design for recombinant SPBC2G5.01 studies requires carefully selected controls:

• Expression controls:

  • Empty vector control: Cells transformed with expression vector lacking the SPBC2G5.01 gene

  • Housekeeping protein control: Expression of a well-characterized protein using the same system

  • Endogenous expression benchmark: Comparison with native levels in S. pombe

• Protein quality controls:

  • Thermal shift assays: To verify proper folding across preparations

  • Size exclusion profiles: To confirm monodispersity and lack of aggregation

  • Tag-only control: Expressing the tag alone to distinguish tag artifacts

• Functional assay controls:

  • Inactive mutant: A rationally designed non-functional variant

  • Related protein control: A similar membrane protein with distinct function

  • Complementation control: Testing ability to rescue deletion phenotypes

• Localization controls:

  • Known ER markers: Co-localization with established ER proteins

  • Mislocalization mutant: Variant with targeting sequence deleted

  • Other organelle markers: To confirm specificity of localization

• Interaction study controls:

  • Non-specific binding control: Using an unrelated protein with similar properties

  • Detergent-only samples: To identify detergent-specific artifacts

  • Competition controls: With unlabeled protein to verify specificity

A systematic control matrix should be implemented for all experiments, with appropriate positive and negative controls for each experimental condition. This approach helps distinguish genuine biological insights from technical artifacts when working with challenging membrane proteins like SPBC2G5.01.

How should researchers design experiments to investigate SPBC2G5.01 in nutrient signaling pathways?

Investigating SPBC2G5.01's potential role in nutrient signaling requires a comprehensive experimental design strategy:

• Growth condition variations:

  • Systematic testing across carbon sources (glucose, glycerol, etc.)

  • Nitrogen limitation experiments

  • Amino acid availability modulation

  • Phosphate and other mineral nutrient restrictions

• Integration with known signaling pathways:

  • TORC1/TORC2 pathway: Examining relationships with TOR kinase components

  • PKA pathway: Testing interactions with cAMP signaling

  • Stress response pathways: Connections with general stress response elements

• Genetic interaction mapping:

  • Synthetic genetic array (SGA) analysis with nutrient signaling components

  • E-MAP approach to systematically identify epistatic interactions

  • Suppressor screens to identify functional relationships

• Phenotypic profiling:

  • Growth rate measurements under various nutrient conditions

  • Cell size and morphology analysis

  • Cell cycle progression monitoring

  • Stress resistance profiling

• Biochemical interaction analysis:

  • Co-immunoprecipitation with known nutrient sensors

  • Phosphorylation status in response to nutrient changes

  • Membrane localization shifts under different conditions

• Multi-omics integration:

  • Transcriptome analysis in wild-type vs. SPBC2G5.01 deletion strains

  • Metabolomic profiling to identify pathway disruptions

  • Phosphoproteomics to map signaling events

Experimental design should follow a systematic approach, starting with phenotypic characterization under various nutrient conditions, followed by genetic interaction mapping to place SPBC2G5.01 within known signaling networks, and finally biochemical studies to define direct interactions and modifications in response to nutrient availability.

What considerations should guide the design of a SPBC2G5.01 structural study?

Designing structural studies for membrane proteins like SPBC2G5.01 requires careful planning and consideration of multiple approaches:

The most prudent approach would be to pursue multiple methods in parallel, with an initial focus on construct optimization and screening across different expression systems, detergents, and stabilization strategies. Subsequently, both crystallography and cryo-EM should be attempted, with computational modeling used to interpret partial structural data and guide further experiments.

How can researchers effectively troubleshoot expression issues with recombinant SPBC2G5.01?

Troubleshooting expression issues with recombinant membrane proteins like SPBC2G5.01 requires a systematic approach:

• Expression construct optimization:

• Host strain selection:

  • Test specialized strains designed for membrane proteins

  • Consider slow-growing strains that may handle toxic proteins better

  • Evaluate eukaryotic hosts for proper post-translational modifications

• Induction parameter optimization:

  • Temperature gradient experiments (15°C to 37°C)

  • Inducer concentration titration

  • Time-course analysis of expression

  • Media composition variations

• Fusion strategy exploration:

  • N-terminal vs. C-terminal tags

  • Inclusion of solubility enhancers (MBP, SUMO)

  • Addition of stabilizing protein partners

  • Incorporation of fluorescent reporters for real-time monitoring

• Codon optimization:

  • Matching codon usage to expression host

  • Avoiding rare codons, especially in clusters

  • Eliminating problematic mRNA secondary structures

  • Optimizing GC content for the expression host

Methodologically, researchers should implement a decision-tree approach, systematically addressing the most common issues first (construct design, host selection) before moving to more specialized optimizations. Each modification should be tested independently to clearly identify beneficial changes, which can then be combined for additive effects.