Recombinant Schizosaccharomyces pombe Uncharacterized protein C27B12.07 (pi071, SPBC27B12.07)

How evolutionarily conserved is protein C27B12.07 across species?

Sequence analysis reveals that C27B12.07 shows significant conservation among fungi, with close homologs identified in:

• Hypsizygus marmoreus (E-value: 2.81722e-111)

• Laccaria amethystina LaAM-08-1 (E-value: 1.52082e-109)

This high degree of conservation among fungi suggests the protein may serve an important biological function despite being uncharacterized. The protein belongs to the DUF1640 family, which consists of sequences derived from hypothetical eukaryotic proteins . The conservation pattern suggests a fungal-specific role rather than a universally conserved function across all eukaryotes.

What is currently known about the cellular localization of this protein?

Based on Gene Ontology annotations, C27B12.07 is predicted to be:

• Located in the mitochondrion (GO:0005739)

• An integral component of membrane (GO:0016021)

• Associated with the mitochondrial membrane (GO:0031966)

This predicted localization suggests a potential role in mitochondrial function, possibly in membrane organization, transport processes, or mitochondrial signaling. Experimental confirmation of this localization would be an essential first step in functional characterization, ideally through fluorescent protein tagging or subcellular fractionation studies.

What are the optimal conditions for recombinant expression and purification of C27B12.07?

For successful recombinant expression and purification of C27B12.07, the following protocol has been established:

Expression System:

• E. coli with N-terminal His-tag fusion

Purification Protocol:

• Express protein in E. coli

• Lyse cells in Tris/PBS-based buffer (pH 8.0)

• Purify using affinity chromatography with Ni-NTA resin

• Elute with imidazole buffer

• Perform buffer exchange to Tris/PBS with 6% Trehalose (pH 8.0)

• Store as lyophilized powder or in solution with 50% glycerol at -20°C/-80°C

Storage Considerations:

• Avoid repeated freeze-thaw cycles

• For long-term storage, aliquot with 50% glycerol and store at -80°C

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

Reconstitution:

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Centrifuge vial briefly before opening to collect contents at the bottom

The purity of the recombinant protein should be greater than 90% as determined by SDS-PAGE .

What analytical techniques are most appropriate for characterizing this uncharacterized protein?

To comprehensively characterize Uncharacterized protein C27B12.07, a multi-technique approach is recommended:

Structural Characterization:

• X-ray crystallography or cryo-EM for 3D structure determination

• Circular dichroism (CD) for secondary structure assessment

• NMR for structural dynamics and ligand binding studies

Functional Analysis:

• Activity-based protein profiling to identify potential enzymatic functions

• Lipid binding assays (given mitochondrial membrane localization)

• Protein-protein interaction screens (pull-downs, crosslinking MS)

Cellular Studies:

• Fluorescent tagging for localization studies in S. pombe

• Fractionation studies to confirm mitochondrial association

• Live-cell imaging to track dynamics

Biophysical Characterization:

• Thermal shift assays to assess stability and ligand binding

• Surface plasmon resonance for interaction studies

• Isothermal titration calorimetry for binding thermodynamics

For membrane proteins like C27B12.07, additional considerations include using appropriate detergents for solubilization and potentially reconstituting the protein in nanodiscs or liposomes for functional studies.

How can I design a gene knockout experiment to study the function of pi071 in S. pombe?

A systematic approach for pi071 knockout in S. pombe would include:

Knockout Strategy Options:

• PCR-based Gene Deletion:

  • Design primers with 80-100 bp homology to regions flanking pi071

  • Amplify a selection marker (e.g., kanamycin resistance)

  • Transform S. pombe with the PCR product

  • Select transformants on appropriate media

• CRISPR-Cas9 Approach:

  • Design guide RNAs targeting the pi071 coding sequence

  • Create a repair template with selection marker

  • Transform S. pombe with Cas9, guide RNA, and repair template

  • Screen for successful editing

Verification Process:

• PCR-verify correct integration at both 5' and 3' junctions

• Confirm absence of pi071 mRNA by RT-PCR

• Verify protein absence by Western blot (if antibodies available)

Phenotypic Analysis Plan:

Controls:

• Wild-type S. pombe strain

• Complementation strain (knockout with reintroduced functional pi071)

• Unrelated gene knockout for stress response comparisons

Previous studies on S. pombe gene deletions have shown that approximately 17.5% of genes are essential for vegetative growth , so it will be important to determine whether pi071 deletion is viable or lethal.

How might C27B12.07 be involved in mitochondrial function based on predicted localization?

Given the predicted mitochondrial membrane localization of C27B12.07, several potential functional roles can be hypothesized:

Potential Mitochondrial Functions:

• Membrane Organization:

  • Cristae structure maintenance

  • Mitochondrial dynamics (fusion/fission)

  • Membrane lipid composition regulation

• Transport Processes:

  • Ion transport across mitochondrial membranes

  • Metabolite exchange between mitochondrial compartments

  • Protein import into mitochondria

• Signaling Functions:

  • Mitochondrial-nuclear communication

  • Response to cellular stress

  • Metabolic regulation

• Mitochondrial DNA Maintenance:

  • mtDNA organization

  • Nucleoid structure

  • Replication or repair processes

Experimental Approach to Test These Hypotheses:

• Mitochondrial morphology analysis in pi071 deletion strains

• Measurement of mitochondrial membrane potential

• Assessment of respiratory chain complex activities

• Analysis of mitochondrial protein import efficiency

• Examination of mtDNA stability and copy number

S. pombe is an excellent model for studying mitochondrial functions due to its ability to survive with mitochondrial defects through fermentative growth, unlike S. cerevisiae, which has more extensive mitochondrial genome loss during laboratory growth .

What protein-protein interaction studies would be most informative for understanding C27B12.07 function?

To elucidate the function of C27B12.07 through protein interactions, a comprehensive approach should include:

In Vivo Interaction Methods:

• Proximity-Based Labeling:

  • BioID or TurboID fusion with C27B12.07

  • Expression in S. pombe under native promoter

  • MS identification of biotinylated proximity partners

  • Particularly valuable for membrane proteins like C27B12.07

• Co-Immunoprecipitation:

  • Tagged C27B12.07 expression in S. pombe

  • Careful membrane solubilization with appropriate detergents

  • MS analysis of co-purified proteins

  • Reciprocal co-IP validation

• Yeast Two-Hybrid:

  • Split-ubiquitin system for membrane proteins

  • Screening against S. pombe cDNA library

  • Validation of interactions by orthogonal methods

In Vitro Approaches:

• Pull-Down Assays:

  • His-tagged recombinant C27B12.07 as bait

  • Incubation with S. pombe cell lysates

  • MS identification of binding partners

• Crosslinking-Mass Spectrometry:

  • Chemical crosslinking of purified protein complexes

  • Identification of direct binding interfaces

  • Structural insights into interaction modes

Data Analysis Strategy:

• Filter against common contaminants

• Prioritize mitochondrial proteins based on predicted localization

• Network analysis to identify functional clusters

• Integration with genetic interaction data

S. pombe offers excellent tools for studying protein interactions, including established protocols for tagging endogenous genes and sensitive mass spectrometry platforms for protein identification .

How does the study of uncharacterized proteins like C27B12.07 contribute to our understanding of basic cellular processes?

The study of uncharacterized proteins like C27B12.07 is crucial for advancing our understanding of cellular biology for several reasons:

Discovery of Novel Functions:

• Approximately 20-40% of genes in sequenced genomes remain functionally uncharacterized

• These represent opportunities to discover entirely new biological mechanisms

• C27B12.07 belongs to the DUF1640 family, which has no known function

Evolutionary Insights:

• Conservation patterns of uncharacterized proteins provide clues about their importance

• C27B12.07 shows conservation among fungi , suggesting a fungal-specific function

• Studying such proteins helps understand lineage-specific adaptations

Completion of Cellular Networks:

• Uncharacterized proteins represent "missing pieces" in our understanding of cellular pathways

• Their characterization helps complete interaction networks and metabolic maps

• Often reveal unexpected connections between established processes

Methodological Challenges and Innovations:

• Studying proteins without known functions drives development of new research techniques

• Requires integration of computational prediction, structural studies, and functional assays

• Advances in technologies like AlphaFold2 are accelerating characterization of such proteins

Practical Applications:

• Novel proteins often become targets for biotechnological applications

• May reveal new drug targets for antifungal development

• Can lead to discovery of proteins with useful enzymatic activities

A pilot study of gene deletions in S. pombe found that 17.5% of genes were essential for vegetative growth , highlighting the importance of systematic functional characterization of all genes, including uncharacterized ones like pi071.

What are the main challenges in studying proteins with domains of unknown function (DUFs) like C27B12.07?

Studying proteins containing domains of unknown function (DUFs) presents several unique challenges:

Experimental Design Challenges:

• Lack of Functional Hypotheses:

  • No established assays to test function

  • Difficult to design targeted experiments

  • May require unbiased screening approaches

• Expression and Purification Difficulties:

  • Unpredictable solubility and stability properties

  • Unknown cofactor requirements

  • Potential toxicity when overexpressed

• Functional Redundancy:

  • Gene deletion may show no phenotype due to compensatory mechanisms

  • Subtle phenotypes may be difficult to detect

  • May require double/triple knockouts to observe effects

Methodological Solutions:

• Integrated Approaches:

  • Combine computational prediction, structural studies, and phenotypic analysis

  • Use evolutionary information to guide hypothesis generation

  • Apply systems biology techniques to position the protein in cellular networks

• Advanced Structural Techniques:

  • AlphaFold2 or RoseTTAFold for accurate structural prediction

  • Structure-based function prediction

  • Fragment screening to identify potential ligands

• High-Throughput Functional Screening:

  • Metabolite profiling before/after gene deletion

  • Chemical genomics to identify functional interactions

  • Suppressor/enhancer genetic screens

• Specialized Membrane Protein Techniques:

  • For predicted membrane proteins like C27B12.07, use of nanodiscs or liposomes

  • Detergent screening for optimal solubilization

  • Membrane yeast two-hybrid systems for interaction studies

In S. pombe, a pilot gene deletion project revealed methodological challenges, with a region of 18 kb containing multiple genes (including SPBC106.10 and SPBC106.20) being refractory to PCR-based gene deletion , suggesting that technique optimization may be necessary for certain genomic regions.

How can contradictory experimental results about C27B12.07 function be reconciled?

When faced with contradictory results regarding protein function, a systematic approach to reconciliation should be employed:

Sources of Experimental Contradictions:

• Technical Variations:

  • Different expression systems (E. coli vs. yeast vs. baculovirus)

  • Varied purification methods affecting protein conformations

  • Tag positions influencing protein behavior

• Conditional Functions:

  • Protein may perform different functions under different conditions

  • Environmental factors may alter activity

  • Post-translational modifications may switch functions

• Context Dependency:

  • Function may depend on specific interacting partners

  • Subcellular localization might vary in different conditions

  • Genetic background of the model organism can influence results

Reconciliation Strategies:

• Standardized Replication:

  • Reproduce experiments under identical conditions

  • Use multiple technical approaches to verify findings

  • Collaborate with laboratories reporting contradictory results

• Condition Matrix Experiments:

  • Test function across a matrix of different conditions

  • Systematically vary pH, temperature, ionic strength, etc.

  • Examine function in different genetic backgrounds

• Integrative Analysis:

  • Use Bayesian integration of multiple data types

  • Weight evidence based on methodological rigor

  • Create a unified model that accounts for conditional functions

• Resolution Through Structure:

  • Determine if protein can adopt multiple conformations

  • Identify regulatory sites that might explain different behaviors

  • Use mutational analysis to test structural hypotheses

For membrane proteins like C27B12.07, particular attention should be paid to the lipid environment, which can significantly affect protein behavior and might explain seemingly contradictory results obtained under different experimental conditions.

What bioinformatic approaches can predict potential functions for C27B12.07?

Modern bioinformatic approaches offer powerful ways to predict functions for uncharacterized proteins like C27B12.07:

Sequence-Based Methods:

• Sensitive Homology Detection:

  • Profile-based searches (PSI-BLAST, HMMER)

  • Hidden Markov Models for remote homology detection

  • Identification of conserved sequence motifs

• Evolutionary Analysis:

  • Conservation patterns across species

  • Identification of co-evolving residues

  • Phylogenetic profiling to find functionally linked genes

Structure-Based Predictions:

• 3D Structure Prediction:

  • AlphaFold2/RoseTTAFold for accurate structural models

  • Structure comparison with functionally characterized proteins

  • Identification of potential binding pockets

• Function Prediction from Structure:

  • Active site prediction based on structural features

  • Protein-protein docking simulations

  • Virtual screening for potential ligands

Network-Based Approaches:

• Functional Association Networks:

  • Co-expression analysis

  • Predicted protein-protein interactions

  • Genetic interaction profiles

• Pathway Analysis:

  • Placement in known cellular pathways

  • Metabolic reconstruction

  • Signaling pathway prediction

Implementation Workflow:

For mitochondrial membrane proteins like C27B12.07, specialized tools for predicting membrane topology, mitochondrial targeting signals, and lipid interactions should also be employed.

How might high-throughput screening approaches be applied to characterize C27B12.07 function?

High-throughput screening offers powerful approaches to uncover functions of uncharacterized proteins like C27B12.07:

Phenotypic Screening Approaches:

• Chemical-Genetic Profiling:

  • Expose pi071 deletion strain to libraries of bioactive compounds

  • Identify differential sensitivity compared to wild-type

  • Cluster with known gene deletions to predict function

• Synthetic Genetic Array (SGA) Analysis:

  • Cross pi071 deletion with genome-wide deletion collection

  • Identify synthetic lethal/sick interactions

  • Map genetic interaction network to infer function

• High-Content Microscopy Screening:

  • Express fluorescently tagged C27B12.07 in S. pombe

  • Screen for conditions altering localization or abundance

  • Identify phenotypic changes under various perturbations

Biochemical Screening Approaches:

• Ligand and Metabolite Screening:

  • Screen purified C27B12.07 against metabolite libraries

  • Use thermal shift assays to detect binding

  • Identify potential substrates or regulatory molecules

• Protein Interaction Screening:

  • Yeast two-hybrid or split-ubiquitin screens

  • Protein microarray analysis

  • Crosslinking mass spectrometry with various conditions

• Activity-Based Protein Profiling:

  • Screen with activity-based probes

  • Identify potential enzymatic activities

  • Develop targeted biochemical assays based on hits

Data Integration Strategy:

• Cross-reference hits from multiple screens

• Prioritize overlapping functional predictions

• Develop targeted hypotheses for in-depth validation

S. pombe provides excellent resources for high-throughput studies, including genome-wide deletion libraries and established high-throughput genetic screening methods that have been successfully used to study DNA recombination and repair pathways .

What essential control experiments should be included when studying C27B12.07 function?

Robust control experiments are crucial for accurate functional characterization of C27B12.07:

For Genetic Studies:

• Multiple Independent Knockout Lines:

  • Generate at least three independent pi071 deletion strains

  • Ensure consistent phenotypes across all lines

  • Rule out off-target effects or suppressor mutations

• Complementation Controls:

  • Reintroduce wild-type pi071 to knockout strain

  • Verify phenotype rescue

  • Include non-functional mutant versions as negative controls

• Specificity Controls:

  • Compare with deletions of unrelated genes

  • Include deletions of genes with similar predicted functions

  • Test deletions of neighboring genes to rule out positional effects

For Biochemical Studies:

• Protein Quality Controls:

  • Verify protein folding using circular dichroism

  • Assess oligomeric state by size exclusion chromatography

  • Confirm membrane insertion in reconstitution experiments

• Tag Interference Controls:

  • Compare N-terminal versus C-terminal tagged versions

  • Include untagged protein where possible

  • Verify tag does not alter localization or function

• Binding Specificity Controls:

  • Include structurally similar non-binding molecules

  • Test heat-denatured protein as negative control

  • Perform competition assays to confirm specificity

For Localization Studies:

• Co-localization Controls:

  • Include established markers for mitochondria

  • Test multiple mitochondrial compartment markers

  • Use super-resolution microscopy for precise localization

• Fractionation Controls:

  • Verify purity of mitochondrial fractions

  • Include markers for all major cellular compartments

  • Confirm results with orthogonal methods

These control experiments are particularly important for uncharacterized proteins, where the risk of misattributing functions is higher due to the lack of prior knowledge to guide experimental design and interpretation.

How might emerging technologies accelerate the functional characterization of proteins like C27B12.07?

Several cutting-edge technologies are poised to dramatically accelerate the characterization of uncharacterized proteins like C27B12.07:

Structural Biology Advances:

• AI-Powered Structure Prediction:

  • AlphaFold2 and RoseTTAFold for accurate structural models

  • Structure-based function prediction

  • Virtual screening against predicted structures

• Cryo-EM Improvements:

  • Single-particle analysis of smaller proteins

  • Tomography for in situ structural determination

  • Improved membrane protein structure determination

Genetic Engineering Technologies:

• CRISPR-Based Approaches:

  • Base editing for precise mutations without DSBs

  • Prime editing for targeted sequence alterations

  • CRISPRi/CRISPRa for conditional regulation

• High-Throughput Mutagenesis:

  • Deep mutational scanning of entire proteins

  • Multiplexed functional assays

  • Machine learning integration for mutation effect prediction

Single-Cell Technologies:

• Single-Cell Proteomics:

  • High-sensitivity protein detection

  • Spatial proteomics for localization

  • Cell-to-cell variability analysis

• Multi-omics Integration:

  • Correlated transcriptome-proteome-metabolome analysis

  • Single-cell multi-omics for pathway elucidation

  • Systems biology modeling of protein function

Implementation Timeline:

For membrane proteins like C27B12.07, advances in native mass spectrometry, lipid nanodisc technologies, and improved membrane protein crystallization methods will be particularly valuable for functional characterization.