Recombinant Schizosaccharomyces pombe Uncharacterized protein C57A10.07 (SPAC57A10.07)

What is SPAC57A10.07 and why is it classified as an uncharacterized protein?

SPAC57A10.07 is a protein encoded in the Schizosaccharomyces pombe genome that remains functionally uncharacterized despite being identified during genome sequencing. It is classified as "uncharacterized" because its biological function, biochemical activity, and structural properties remain largely unknown. The protein lacks significant homology with proteins of known function, making it difficult to predict its role based on sequence comparison alone. Uncharacterized proteins represent significant opportunities for discovering novel biological mechanisms and pathways in this model organism, which shares approximately 70% of its genes as orthologs with human genes .

What are the basic structural features of SPAC57A10.07?

While specific structural information about SPAC57A10.07 is limited, structural features can be predicted using computational tools. These analyses typically include:

These computational predictions should be experimentally validated using techniques such as circular dichroism spectroscopy for secondary structure confirmation or limited proteolysis to identify domain boundaries. S. pombe proteins often contain unique post-translational modifications that may not be accurately predicted by general algorithms .

How is SPAC57A10.07 gene expression regulated in S. pombe?

Understanding the regulation of SPAC57A10.07 requires a multifaceted experimental approach:

Transcriptional Regulation Analysis:

• RNA sequencing (RNA-seq) to measure SPAC57A10.07 mRNA levels under various conditions

• Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors binding to the promoter region

• Promoter analysis using reporter genes to determine critical regulatory elements

Experimental Methodology:

• Replace the native promoter with the thiamine-repressible nmt1 promoter or the rapidly inducible urg1 promoter (which allows induction within 30 minutes compared to the 14-20 hours required for nmt1 induction)

• Create promoter deletion constructs fused to a reporter gene to identify critical regulatory elements

• Perform RNA-seq across different growth conditions and cell cycle stages to create an expression profile

• Utilize the S. pombe deletion library to identify trans-acting factors affecting SPAC57A10.07 expression

A typical RNA-seq experimental design might include:

What experimental approaches are most effective for expressing recombinant SPAC57A10.07?

Successful expression of recombinant SPAC57A10.07 requires careful consideration of expression systems and conditions:

Expression System Selection:

• Homologous expression in S. pombe maintains native folding and post-translational modifications

• Heterologous expression in E. coli offers high yield but may lack proper modifications

• Expression in S. cerevisiae provides a compromise between yield and eukaryotic processing

Optimization Strategies:

• Codon optimization for the chosen expression host

• Use of appropriate promoters (e.g., nmt1 or urg1 for S. pombe)

• Addition of fusion tags for enhanced solubility and purification (His, GST, MBP)

• Inclusion of appropriate signal sequences if secretion is desired

For S. pombe expression, methodology can be adapted from genetic engineering approaches used for other recombinant proteins in this organism:

• Cloning SPAC57A10.07 into an integration vector like pYIplac128

• Transformation into S. pombe using chemical transformation methods

• Induction of expression using the nmt1 promoter (repressed by thiamine) or urg1 promoter for rapid induction

• Verification of expression by Western blot analysis

A comparative expression optimization table might look like:

How can SPAC57A10.07 be purified for structural studies?

Purification of SPAC57A10.07 for structural studies requires a strategic approach to maintain protein integrity:

Cell Lysis Methods:

• For S. pombe, cell disruption by sonication as documented in recombinant protein studies

• Glass bead lysis using a Ribolyser as described in protocols for fission yeast

• French press or homogenization for larger scale preparations

Purification Strategy:

• Affinity Chromatography:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Glutathione-Sepharose for GST fusion proteins

  • Antibody-based purification for epitope-tagged proteins

• Ion Exchange Chromatography:

  • Based on the predicted isoelectric point of SPAC57A10.07

  • Anion exchange (Q-Sepharose) for negatively charged proteins

  • Cation exchange (SP-Sepharose) for positively charged proteins

• Size Exclusion Chromatography:

  • Final polishing step to remove aggregates and achieve high purity

  • Also provides information about the oligomeric state of the protein

A typical purification table should track progress through each step:

For structural studies, buffer optimization for stability and removal of heterogeneity from post-translational modifications may be necessary, especially considering S. pombe's complex glycosylation patterns .

What phenotypes are associated with SPAC57A10.07 deletion or overexpression?

Phenotypic analysis of SPAC57A10.07 deletion or overexpression strains can provide direct insights into its function:

Generation of Deletion and Overexpression Strains:

• Gene deletion using homologous recombination (PCR-based approach) or CRISPR-Cas9

• Overexpression using strong promoters (nmt1) or additional gene copies

• Creation of conditional alleles if deletion is lethal

Phenotypic Analysis:

• Growth Characteristics:

  • Growth rate in rich and minimal media

  • Viability under different temperatures

  • Colony morphology and cell morphology

  • Cell cycle analysis using DAPI staining and FACS analysis

• Stress Sensitivity:

  • Spotting assays on media containing different stressors

  • Growth curve analysis under stress conditions

  • Survival after acute stress exposure (particularly H₂O₂)

• Cell Wall and Morphology Analysis:

  • Microscopic analysis of cell shape and size

  • Aniline blue staining for β-1,3-glucan analysis

  • Calcofluor white staining for cell wall analysis

A pilot study of gene deletion in S. pombe found that approximately 17.5% of genes are essential, compared to 17.8% in budding yeast . The essentiality of SPAC57A10.07 would be a critical piece of information determining the experimental approach.

Phenotypic data should be systematically presented:

What computational approaches can predict the function of SPAC57A10.07?

Computational methods offer powerful approaches to predict functions of uncharacterized proteins like SPAC57A10.07:

Sequence-Based Methods:

• Homology Detection:

  • PSI-BLAST, HHpred, and HMMER for detecting remote homologs

  • Jackhmmer for iterative sequence searches

• Domain and Motif Analysis:

  • InterPro, Pfam, SMART for domain identification

  • ELM for short functional motifs

  • COILS for coiled-coil predictions

• Structural Predictions:

  • AlphaFold2, RoseTTAFold for 3D structure prediction

  • I-TASSER, Phyre2 for fold recognition

  • FoldSeek for structural homology searches

Genomic Context Methods:

• Phylogenetic Profiling:

  • Correlation of presence/absence patterns across species

  • Identification of functionally linked proteins with similar evolutionary profiles

• Gene Expression Correlation:

  • Co-expression analysis using public transcriptomic datasets

  • Identification of genes with similar expression patterns

From genomic analyses, we know that S. pombe shares genes with higher eukaryotes that are not present in S. cerevisiae, such as RNAi machinery genes . If SPAC57A10.07 falls into this category, it may have functions conserved with multicellular organisms rather than with budding yeast.

Results from computational analyses should be systematically evaluated:

How can CRISPR-Cas9 be utilized to study SPAC57A10.07 function in vivo?

CRISPR-Cas9 technology offers powerful tools for studying SPAC57A10.07 function in S. pombe:

CRISPR-Cas9 Applications:

• Gene Knockout:

  • Complete deletion of the SPAC57A10.07 gene

  • Introduction of frameshift mutations or premature stop codons

• Gene Editing:

  • Introduction of point mutations to study specific residues

  • Domain deletions or swaps to assess domain functions

  • Insertion of epitope tags for protein detection and purification

• Gene Regulation:

  • CRISPRi (interference) using catalytically inactive Cas9 (dCas9) to repress transcription

  • CRISPRa (activation) using dCas9 fused to activator domains to enhance transcription

Experimental Design for S. pombe:

• gRNA Design:

  • Selection of target sites with minimal off-target effects

  • Consideration of S. pombe PAM preference

  • Design of multiple gRNAs targeting different regions for efficiency comparison

• Delivery Method:

  • Plasmid-based expression of Cas9 and gRNA

  • Integration of Cas9 into the genome under inducible promoters

  • Direct delivery of Cas9-gRNA ribonucleoprotein complexes

S. pombe researchers have developed techniques to study replication fork arrest and restart , and CRISPR-Cas9 could be used to introduce specific mutations in SPAC57A10.07 to test if it plays a role in these processes.

Design of CRISPR experiments should be carefully documented:

How does post-translational modification affect SPAC57A10.07 activity?

Post-translational modifications (PTMs) can significantly affect protein function, localization, and interactions:

Identification of PTMs:

• Mass Spectrometry-Based Approaches:

  • Enrichment strategies for phosphorylation (TiO₂, IMAC)

  • Enrichment for ubiquitination (K-ε-GG antibodies)

  • Glycan analysis by lectin affinity or hydrazide chemistry

  • Targeted and untargeted PTM discovery using various fragmentation methods

• Biochemical Methods:

  • Western blotting with modification-specific antibodies

  • Mobility shift assays (Phos-tag)

  • ProQ Diamond staining for phosphoproteins

Functional Analysis of PTMs:

• Site-Directed Mutagenesis:

  • Mutation of modification sites to non-modifiable residues (e.g., S→A for phosphorylation)

  • Phosphomimetic mutations (e.g., S→D or S→E)

  • Analysis of mutant phenotypes in vivo

S. pombe proteins undergo various post-translational modifications, including O-mannosylation and N-glycosylation. Research has shown competition between these modifications, where unusual N-glycosylation sites can be masked by O-mannosylation . This interplay could be particularly relevant for SPAC57A10.07 function.

Data on PTMs should be systematically presented:

What high-throughput approaches can be used to identify the molecular function of SPAC57A10.07?

High-throughput approaches offer systematic ways to identify the function of uncharacterized proteins:

Genomic Approaches:

• Synthetic Genetic Array (SGA) Analysis:

  • Systematic creation of double mutants with all viable deletion strains

  • Identification of genetic interactions (synthetic lethality, suppression)

  • Inference of function based on interaction patterns

• Chemical-Genetic Profiling:

  • Testing sensitivity of SPAC57A10.07 deletion or overexpression to a library of compounds

  • Comparison with profiles of known mutants to identify pathway connections

Proteomic Approaches:

• Global Protein-Protein Interaction Mapping:

  • Systematic Y2H or AP-MS screening

  • Comparison of interactome with proteins of known function

• Thermal Proteome Profiling (TPP):

  • Analysis of protein thermal stability changes upon ligand binding

  • Identification of potential substrates or interacting molecules

A pilot gene deletion project in S. pombe assessed the feasibility of a genome-wide deletion project and estimated the percentage of essential genes to be 17.5% . Such systematic deletion projects provide valuable resources for functional genomics studies that can be leveraged to understand SPAC57A10.07.

Results from high-throughput studies should be presented with appropriate statistical analysis:

What are the challenges in crystallizing SPAC57A10.07 for structural determination?

Crystallizing proteins for structural studies presents significant challenges, particularly for uncharacterized proteins:

Common Crystallization Challenges:

• Protein Stability and Homogeneity:

  • Aggregation propensity due to exposed hydrophobic patches

  • Conformational heterogeneity affecting crystal packing

  • Post-translational modifications causing sample heterogeneity

• Intrinsically Disordered Regions (IDRs):

  • Flexible regions hindering crystal formation

  • Strategies include limited proteolysis to remove flexible regions or construct optimization

Optimization Strategies:

• Construct Design:

  • Bioinformatic prediction of domain boundaries

  • Creation of multiple constructs with different N- and C-terminal boundaries

  • Removal of predicted disordered regions

• Crystallization Screening:

  • High-throughput screening of diverse crystallization conditions

  • Variation of protein concentration, temperature, and precipitants

  • Addition of ligands or binding partners to stabilize specific conformations

S. pombe proteins can be successfully expressed and purified for biochemical and structural studies , but the specific challenges for SPAC57A10.07 would depend on its unique properties.

Crystallization optimization results should be systematically tracked:

How can protein-protein interactions of SPAC57A10.07 be systematically mapped?

Identifying protein-protein interactions (PPIs) for SPAC57A10.07 can provide critical insights into its function:

Yeast Two-Hybrid (Y2H) Screening:

• Using SPAC57A10.07 as bait against an S. pombe cDNA library

• Verification of interactions by reverse Y2H and co-immunoprecipitation

• Library screening approach can identify novel interactors

Affinity Purification-Mass Spectrometry (AP-MS):

• Expression of tagged SPAC57A10.07 in S. pombe

• Purification of protein complexes under native conditions

• Mass spectrometric identification of co-purified proteins

• Differentiation between specific interactors and contaminants using quantitative approaches

Proximity-Dependent Biotin Identification (BioID):

• Fusion of SPAC57A10.07 with a biotin ligase (BirA*)

• Biotinylation of proximal proteins in vivo

• Streptavidin purification and mass spectrometry identification

A systematic interaction mapping would be presented as:

S. pombe protein interaction data can reveal connections to specific pathways, as demonstrated in the interaction mapping of other proteins like REM1_SCHPO (meiosis-specific cyclin) .