Recombinant Human UPF0542 protein C5orf43 (C5orf43)

What is UPF0542 protein C5orf43 and what are its key structural features?

Based on available information, UPF0542 protein C5orf43, also known as SMIM15 (Small Integral Membrane Protein 15), is predicted to be an integral component of membrane according to the Alliance of Genome Resources . The "UPF" designation (Uncharacterized Protein Family) indicates that its function was not well characterized when first discovered.

As an integral membrane protein, C5orf43/SMIM15 likely contains transmembrane domains that anchor it within the cell membrane. Structural analysis would typically employ prediction algorithms to identify these domains, signal peptides, post-translational modification sites, and functional domains.

Methodological approaches to confirm these predictions include:

• Protease protection assays to determine membrane topology

• Fluorescence microscopy with tagged protein versions to confirm localization

• Western blotting with domain-specific antibodies

• Mass spectrometry to identify post-translational modifications

How is C5orf43/SMIM15 conserved across species?

Recombinant SMIM15/C5orf43 proteins are commercially available for multiple species including human, cynomolgus/rhesus macaque, rat, mouse, feline, canine, bovine, and equine, suggesting significant conservation across mammals . This conservation often indicates functional importance.

To analyze conservation thoroughly, researchers would typically:

• Perform multiple sequence alignment of C5orf43/SMIM15 orthologs

• Calculate sequence identity and similarity percentages

• Identify conserved domains or motifs

• Use phylogenetic analysis to determine evolutionary relationships

• Compare predicted secondary structures across species

What expression systems are optimal for producing recombinant C5orf43/SMIM15?

The optimal expression system depends on research goals, required protein yield, and downstream applications. For membrane proteins like C5orf43/SMIM15, several expression systems are commonly employed:

For integral membrane proteins like C5orf43/SMIM15, insect cell or mammalian cell expression systems are often preferred to ensure proper folding and membrane insertion. Based on analogous recombinant proteins, baculovirus-infected Sf9 cells might be a suitable system .

Methodological considerations include:

• Addition of appropriate tags (His, FLAG, GST) to facilitate purification

• Optimization of codon usage for the expression host

• Selection of suitable promoters for controlled expression

What purification strategies are most effective for membrane proteins like C5orf43?

Purifying membrane proteins presents unique challenges compared to soluble proteins. A comprehensive purification strategy would include:

• Membrane extraction:

  • Selection of appropriate detergents (e.g., DDM, CHAPS, Triton X-100)

  • Optimization of detergent concentration and extraction conditions

  • Potential use of detergent screens to identify optimal solubilization conditions

• Affinity chromatography:

  • Utilizing engineered tags (His, FLAG, GST) for capture

  • Development of specific antibodies for immunoaffinity purification

  • Careful optimization of binding and elution conditions to maintain protein stability

• Additional purification steps:

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Ion exchange chromatography for further purification

  • Potential detergent exchange during purification for downstream applications

A typical purification workflow might include:

How can expression and purification success be monitored throughout the process?

Monitoring expression and purification of C5orf43/SMIM15 is critical for ensuring experimental success. Methodological approaches include:

• Expression monitoring:

  • Western blotting with tag-specific or protein-specific antibodies

  • qPCR to confirm transcript expression

  • Small-scale test expressions to optimize conditions

  • Fluorescent fusion protein visualization if applicable

• Purification quality control:

  • SDS-PAGE with Coomassie staining to assess purity

  • Western blotting to confirm identity

  • Mass spectrometry for molecular weight confirmation

  • Dynamic light scattering to assess homogeneity

  • Circular dichroism to confirm proper folding

• Functional validation:

  • Binding assays with known or predicted ligands

  • Reconstitution into liposomes to test membrane insertion

  • Activity assays if enzymatic function is known or suspected

Similar to methods used for normalized TCF4 expression analysis, researchers should implement appropriate controls and normalization strategies .

How can functional studies of C5orf43/SMIM15 be designed?

Designing functional studies for a protein with limited known functions requires a multi-faceted approach:

• Gene expression manipulation:

  • RNAi knockdown to assess loss-of-function phenotypes

  • CRISPR-Cas9 knockout to generate complete loss of function

  • Overexpression studies to assess gain-of-function effects

Similar to RNAi transfection methodologies mentioned in the literature , researchers would:

• Select appropriate siRNA targeting sequences

• Optimize transfection conditions

• Assess knockdown efficiency by qPCR and Western blot

• Phenotypic characterization:

  • Cell proliferation assays (similar to those used in published research)

  • Cell migration and adhesion studies

  • Morphological analysis

  • Pathway activation assessment (phosphorylation status of downstream targets)

A systematic experimental approach might include:

What approaches can be used to identify potential interacting partners of C5orf43/SMIM15?

For membrane proteins with limited characterized interactions, researchers would typically employ both computational predictions and experimental validation:

• Computational prediction approaches:

  • Sequence-based interaction prediction

  • Structural homology modeling to identify potential interaction domains

  • Co-expression analysis across tissues and conditions

  • Phylogenetic profiling to identify functionally related proteins

• Experimental validation methods:

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

  • Proximity-dependent biotinylation (BioID, APEX)

  • Membrane yeast two-hybrid systems

  • FRET/BRET analysis for direct interactions

• Validation and characterization:

  • Co-localization studies

  • Mutagenesis of predicted interaction domains

  • Competition assays

  • Functional assays to determine the significance of interactions

A workflow for identifying and validating protein interactions might include:

• Initial computational predictions to generate candidates

• Primary screening using high-throughput methods like AP-MS

• Secondary validation with orthogonal methods (co-IP, FRET)

• Detailed characterization of confirmed interactions

How might researchers investigate C5orf43/SMIM15 involvement in disease mechanisms?

While specific disease associations for C5orf43/SMIM15 are not detailed in the available information, researchers could employ several approaches to investigate potential disease relevance:

• Genetic association studies:

  • Analysis of variants in C5orf43/SMIM15 in disease cohorts

  • Expression quantitative trait loci (eQTL) analysis

  • Integration with GWAS data for related pathways

  • Sequencing of the gene in patient populations

• Expression analysis in disease states:

  • Differential expression analysis in disease vs. healthy tissues

  • Single-cell RNA-seq to identify cell-type specific changes

  • Protein expression analysis in patient samples

  • Correlation with disease progression or severity

• Functional disease modeling:

  • Generation of disease-associated mutations via site-directed mutagenesis

  • Development of cellular models expressing mutant forms

  • Creation of animal models with corresponding mutations

  • Rescue experiments to confirm causality

This approach resembles methodologies used in investigating genes associated with conditions like schizophrenia, where various molecular techniques are combined to establish disease relevance .

What statistical approaches are recommended for analyzing C5orf43/SMIM15 expression data?

Based on statistical approaches used in similar research, several methods would be appropriate for analyzing C5orf43/SMIM15 expression data:

• For qPCR data:

  • Linear mixed models (similar to those used in the TCF4 expression analysis)

  • ANOVA with appropriate post-hoc tests for multiple conditions

  • t-tests for pairwise comparisons

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) if data is not normally distributed

• For RNA-seq data:

  • DESeq2 or edgeR for differential expression analysis

  • WGCNA for co-expression network analysis

  • GSEA for pathway enrichment analysis

  • Dimension reduction techniques (PCA, t-SNE) for exploratory analysis

• For normalization:

  • Geometric mean of multiple reference genes for qPCR

  • TPM or FPKM methods for RNA-seq

  • TMM (Trimmed Mean of M-values) for RNA-seq count data

  • Batch effect correction using ComBat or similar methods

The approach would be similar to the normalized TCF4 expression analysis in Table 3.9 and Table 3.10, which used statistical analysis applying a linear mixed model .

How can contradictory experimental results about C5orf43/SMIM15 be reconciled?

When faced with contradictory experimental results, systematic analysis and methodological troubleshooting are essential:

• Experimental system differences analysis:

  • Cell type-specific effects (different expression patterns or interaction partners)

  • Species-specific differences in protein function

  • Expression level variations affecting results

  • Different isoforms being studied

• Methodological variations assessment:

  • Different knockdown/knockout strategies targeting different regions

  • Variations in assay sensitivity or specificity

  • Different tags or fusion constructs affecting function

  • Variations in experimental conditions (time points, culture conditions)

• Data analysis approaches:

  • Re-analysis using consistent normalization methods

  • Meta-analysis of multiple datasets

  • Statistical power analysis to assess result reliability

  • Assessment of biological vs. technical variation

Similar to considerations in TCF4 knockdown experiments where data filtering was necessary to remove technical artifacts (e.g., removing plates with inconsistent results), researchers should carefully evaluate experimental conditions and quality control metrics .

A systematic approach to resolving contradictions might include: