Recombinant Mouse Uncharacterized protein C17orf74 homolog

What is the current nomenclature and classification of C17orf74 homolog in mice?

The mouse uncharacterized protein C17orf74 homolog is currently classified as a member of the SPEM (Sulphide Production Enhancing Membrane) family member 2, similar to its counterparts in other mammalian species such as horses and primates . The protein remains largely uncharacterized functionally, though its conservation across mammalian species suggests biological significance. Cross-species analysis shows the gene is protein-coding in both horses (C11H17orf74) and western lowland gorillas (C17H17orf74), with identical full name designation as "SPEM family member 2" .

What expression patterns have been observed for C17orf74 homologs across species?

Based on comparative analyses with other mammals, the C17orf74 homolog demonstrates variable expression patterns. In radiation studies with primate models, the human C17orf74 showed persistent differential expression (averaging 0.3 across multiple time points post-radiation exposure) . This suggests potential involvement in radiation response pathways, though expression patterns specifically in mice remain to be comprehensively characterized. Researchers should consider temporal parameters when designing expression studies, given the sustained differential expression observed across the 7-106 day timeframe in primate models .

What structural domains and motifs characterize the C17orf74 protein family?

The C17orf74 protein family, including the mouse homolog, belongs to the V-set protein structural family based on comparative analysis with related genes like VSTM1 (V-set and transmembrane domain containing 1) . The protein likely contains transmembrane domains characteristic of membrane-associated proteins. Structural analysis of gorilla and horse homologs reveals multiple protein isoforms through alternative splicing (e.g., isoforms X1 and X2 in gorilla) , suggesting functional diversity that may also exist in the mouse homolog.

What expression vectors and systems are optimal for producing recombinant mouse C17orf74 homolog?

For efficient expression of recombinant mouse C17orf74 homolog, mammalian expression systems using vectors such as pcDNA3.1 with C-terminal tags (like DYKDDDDK/FLAG) provide reliable results, mirroring successful approaches used with homologs from other species . The CloneEZ™ Seamless cloning technology has proven effective for creating expression-ready ORF clones with this gene family. When designing expression constructs, researchers should consider the full coding sequence length (approximately 1200-1400bp based on horse and gorilla homologs) and optimize codon usage for the selected expression system .

What purification strategies yield highest purity and biological activity for recombinant C17orf74?

Based on structural predictions and data from related proteins, a multi-step purification strategy is recommended. Initial capture using affinity chromatography targeting the C-terminal tag (e.g., anti-FLAG resin for DYKDDDDK-tagged constructs) followed by size exclusion chromatography typically yields high purity. For proteins with predicted transmembrane domains, consider detergent screening (starting with mild detergents like DDM or LMNG) to maintain native conformation. Validation of structural integrity using circular dichroism spectroscopy is advised before functional assays, particularly given the uncharacterized nature of this protein.

What are the critical quality control parameters for validating recombinant mouse C17orf74 homolog preparations?

Quality control for recombinant mouse C17orf74 homolog should include: (1) SDS-PAGE and western blot analysis to confirm molecular weight and immunoreactivity; (2) mass spectrometry for sequence verification; (3) endotoxin testing (<0.1 EU/μg protein) for in vivo applications; (4) aggregation assessment via dynamic light scattering; and (5) functional validation through binding studies with predicted interaction partners. Researchers should establish batch-to-batch consistency metrics and implement storage stability studies (−80°C with 10% glycerol typically maximizes shelf-life for similar proteins).

What experimental approaches can resolve the function of mouse C17orf74 homolog in cellular models?

A multi-faceted approach combining CRISPR-Cas9 gene editing, RNA interference, and overexpression studies provides complementary insights into C17orf74 function. Differential gene expression analysis following radiation exposure, similar to methodologies employed in primate studies, may reveal stress-response functions . Proteomics approaches (BioID or proximity labeling) can identify interaction partners to place C17orf74 within cellular pathways. Cell phenotyping should include assays for membrane integrity, subcellular localization, and response to stressors based on the observation that C17orf74 shows altered expression following radiation exposure in other species .

How can researchers investigate potential radiation response functions of C17orf74 suggested by expression studies?

Given the differential expression of C17orf74 observed in radiation response studies (consistent 0.3-fold change across multiple time points) , researchers should design experiments that examine: (1) protein localization changes following radiation exposure; (2) protein-protein interactions specific to radiation response pathways; (3) transcriptional regulation mechanisms through ChIP-seq of radiation response transcription factors; and (4) phenotypic consequences of C17orf74 knockout/knockdown in radiation sensitivity assays. A time-course design capturing immediate (hours), intermediate (days), and long-term (weeks) responses would align with the persistent expression changes observed in primate models .

What gene knockout/knockdown strategies are most appropriate for studying mouse C17orf74 homolog function?

For studying mouse C17orf74 homolog function, CRISPR-Cas9 gene editing offers the most definitive approach for complete knockout. Design at least four guide RNAs targeting conserved exons present in all potential splice variants, as multiple isoforms have been observed in other species . For temporal control, consider inducible knockdown systems (e.g., doxycycline-inducible shRNA) or conditional knockout models (Cre-loxP) if developmental effects are suspected. Validation should include both genomic verification and protein expression analysis across multiple tissues, particularly those implicated in radiation response based on expression data from related species .

How does the mouse C17orf74 homolog compare structurally and functionally with homologs in other mammalian species?

Comparative analysis reveals significant conservation of C17orf74 across mammals, with the gene designated C11H17orf74 in horses and C17H17orf74 in gorillas . Nucleotide sequence length varies somewhat between species (1383bp in horses, 1218bp in gorilla isoform X2), suggesting potential functional adaptations . All identified homologs encode uncharacterized proteins with the standardized name "SPEM family member 2," indicating conserved but not yet elucidated function . The conservation pattern suggests evolutionary pressure to maintain this gene, particularly within mammals, which reinforces its likely biological importance despite limited functional characterization.

What insights can be gained from analyzing C17orf74 expression in disease models across different species?

Expression analysis from radiation studies shows C17orf74 exhibits persistent differential expression (0.3-fold change) over extended periods (7-106 days post-exposure) . This consistent pattern suggests involvement in long-term adaptive responses rather than acute stress reactions. When designing cross-species comparative studies, researchers should prioritize conditions where stress-response pathways are activated, particularly radiation response, based on existing data. The gene's expression changes should be contextualized within broader pathway alterations, potentially connecting to other differentially expressed genes identified in the same studies, such as VSTM1, ACBD7, and CERCAM .

How might post-translational modifications impact mouse C17orf74 homolog function and interactions?

Analysis should focus on predicted post-translational modification sites conserved across species homologs. Phosphoproteomic studies following cellular stress (particularly radiation exposure) may reveal regulated modification sites given the protein's differential expression in radiation response . Advanced mass spectrometry approaches combining enrichment strategies for phosphorylation, ubiquitination, and acetylation will provide the most comprehensive modification profile. Mutational analysis of identified modification sites combined with functional assays will establish causative relationships between modifications and protein function.

What are the current technical challenges in resolving the crystal structure of mouse C17orf74 homolog?

The primary challenges in crystallizing mouse C17orf74 homolog likely include: (1) potential membrane association making protein purification and crystallization difficult; (2) predicted structural flexibility; and (3) limited protein quantities from recombinant expression systems. Alternative structural biology approaches to consider include cryo-electron microscopy for larger complexes, solution NMR for dynamic regions, and computational modeling based on distant homologs. Expression constructs should be designed with flexible linkers and removable tags, and crystallization trials should include membrane-mimetic environments if transmembrane domains are confirmed.

How can transcriptomic and proteomic approaches be integrated to elucidate the regulatory network of mouse C17orf74 homolog?

An integrated multi-omics approach should combine RNA-seq and proteomics under conditions where C17orf74 displays differential regulation, such as radiation exposure . Time-course designs should capture both immediate regulatory events and prolonged responses, aligning with the sustained expression changes (0.3-fold) observed in primate radiation studies over 7-106 days . Network analysis should integrate transcription factor binding site predictions, epigenetic modifications, and protein-protein interactions to construct a comprehensive regulatory framework. Validation experiments should target predicted key nodes using CRISPR interference for transcriptional regulators and proximity labeling for protein interaction networks.

What tissue distribution patterns and developmental expression timing should researchers expect for mouse C17orf74 homolog?

Based on comparative data from other species, researchers should examine expression across multiple tissue types with particular attention to immune-related tissues given the protein's differential expression in radiation response studies . Developmental timing analysis should include embryonic stages through adulthood, with quantitative PCR using primers designed against conserved regions. Researchers should implement both transcript-level (RNA-seq, qPCR) and protein-level (immunohistochemistry, western blot) detection methods to account for potential post-transcriptional regulation.

What animal models best reflect the physiological contexts where C17orf74 homolog function is critical?

Given the differential expression observed in radiation response studies , mouse models subjected to controlled radiation exposure represent primary systems for investigating physiological relevance. Consider both acute high-dose and chronic low-dose radiation models to capture different response mechanisms. Additionally, stress response models (oxidative stress, DNA damage) may reveal functional importance. For genetic approaches, both conventional knockout models and tissue-specific conditional knockouts should be evaluated, with particular attention to phenotypes in tissues showing highest endogenous expression.