Recombinant Human Uncharacterized protein C17orf74 (C17orf74)

What is C17orf74 and what is currently known about its structure?

C17orf74 (chromosome 17 open reading frame 74) is an uncharacterized human protein consisting of 501 amino acids. As suggested by its designation as an "open reading frame," this protein has been identified through genomic sequence analysis but currently has limited functional characterization in the scientific literature . The protein is encoded by a gene located on chromosome 17, which comprises approximately 2.5% of the human genome with about 81 million bases encoding over 1,200 genes .

What is the genomic context of C17orf74?

The C17orf74 gene is located on human chromosome 17, which is notable for containing several clinically significant genes including the tumor suppressor genes p53 and BRCA1 . Chromosome 17 has been associated with various genetic disorders including neurofibromatosis, Alexander disease, Birt-Hogg-Dube syndrome, and Canavan disease . Understanding the genomic neighborhood of C17orf74 may provide context for hypothesizing potential functional roles or disease associations.

What approaches are recommended for initial characterization of an uncharacterized protein like C17orf74?

Initial characterization typically follows a multi-disciplinary approach:

• Bioinformatic analysis: Sequence alignment with known proteins to identify conserved domains

• Expression profiling: Determining tissue-specific or condition-specific expression patterns

• Subcellular localization: Using fluorescently-tagged recombinant protein to determine cellular compartment

• Interactome analysis: Identifying binding partners through techniques like co-immunoprecipitation or yeast two-hybrid screening

• Functional screening: Systematic phenotypic analysis following gene knockdown or overexpression

For C17orf74, researchers should particularly consider its chromosomal context near important tumor suppressor genes when designing functional screens .

What expression systems are optimal for producing recombinant human C17orf74?

When expressing an uncharacterized human protein like C17orf74, researchers should consider multiple expression systems:

For a 501-amino acid protein like C17orf74, mammalian expression systems may be preferable for functional studies to ensure proper folding and post-translational modifications.

How can researchers optimize the purification of recombinant C17orf74?

Purification strategy should be tailored to the specific properties of C17orf74. A general approach includes:

• Tag selection: His-tag, GST, or FLAG tags can facilitate purification, with His-tag being generally less disruptive for a 501-amino acid protein like C17orf74

• Buffer optimization: Test multiple buffer conditions (pH 6.0-8.0, salt concentrations 150-500 mM NaCl)

• Chromatography strategy:

  • Initial capture: Affinity chromatography based on the chosen tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

• Stability assessment: Test thermal stability and time-course degradation in different buffer conditions

Researchers should determine the isoelectric point (pI) of C17orf74 bioinformatically to optimize ion exchange chromatography conditions.

What strategies can researchers employ to identify potential functional domains in C17orf74?

Given the uncharacterized nature of C17orf74, several complementary approaches should be utilized:

• Computational analysis:

  • Sequence homology searches across species

  • Secondary structure prediction

  • Fold recognition algorithms

  • Intrinsically disordered region prediction

• Experimental validation:

  • Limited proteolysis to identify stable domains

  • Hydrogen/deuterium exchange mass spectrometry

  • Domain-specific antibody generation and epitope mapping

  • Systematic truncation library screening for functionality

• Evolutionary analysis:

  • Identification of conserved regions across species

  • Examination of selective pressure across the protein sequence

Since C17orf74 is located on chromosome 17, which houses functionally important genes like p53 and BRCA1, analysis of potential overlapping pathways may provide functional insights .

How can researchers investigate protein-protein interactions involving C17orf74?

Interaction partners often provide crucial insights into protein function. For an uncharacterized protein like C17orf74, a multi-tiered approach is recommended:

• Primary screening methods:

  • Affinity purification-mass spectrometry (AP-MS)

  • Yeast two-hybrid screening

  • Proximity-dependent biotin identification (BioID)

  • Protein microarray screening

• Validation methods:

  • Co-immunoprecipitation with candidate interactors

  • FRET/BRET analysis of direct interactions

  • Surface plasmon resonance for binding kinetics

  • Mammalian two-hybrid assays

• Functional validation:

  • Co-localization studies

  • Mutational analysis of interaction interfaces

  • Competitive binding assays

Given C17orf74's chromosomal location, particular attention should be paid to potential interactions with proteins involved in DNA repair or tumor suppression pathways, given the proximity to p53 and BRCA1 genes on chromosome 17 .

What are the optimal approaches for designing knockout or knockdown experiments for C17orf74?

When designing genetic manipulation experiments for an uncharacterized gene like C17orf74, researchers should consider:

• CRISPR-Cas9 knockout strategy:

  • Design multiple guide RNAs targeting early exons

  • Consider the 501-amino acid structure to ensure complete functional disruption

  • Include control guide RNAs targeting non-essential genes

  • Validate knockout through both DNA sequencing and protein expression analysis

• RNAi knockdown approach:

  • Design siRNAs or shRNAs targeting different regions of the C17orf74 mRNA

  • Validate knockdown efficiency by qRT-PCR and western blotting

  • Include time-course analysis to determine protein half-life

• Inducible systems:

  • Implement doxycycline-inducible shRNA or CRISPR systems

  • Consider degron-based approaches for temporal control of protein depletion

• Rescue experiments:

  • Generate CRISPR-resistant cDNA constructs

  • Create domain-specific deletion mutants for functional mapping

Since chromosome 17 contains genes associated with several disorders, researchers should consider phenotypic assays relevant to these conditions when analyzing C17orf74 knockout effects .

How should researchers analyze potential phenotypes following C17orf74 manipulation?

Given the uncharacterized nature of C17orf74, a broad phenotypic analysis is recommended:

• Cellular phenotypes:

  • Proliferation and cell cycle progression

  • Apoptosis and cell viability

  • Morphological changes

  • Migration and invasion capabilities

• Molecular phenotypes:

  • Transcriptome analysis (RNA-seq)

  • Proteome changes (mass spectrometry)

  • Post-translational modification alterations

  • Signaling pathway activity

• Stress response:

  • DNA damage response (particularly relevant given chromosome 17 context)

  • Oxidative stress sensitivity

  • ER stress response

  • Metabolic stress adaptation

• Model-specific phenotypes:

  • In vivo models: Development, tissue-specific functions

  • 3D culture models: Organoid formation, differentiation capacity

Consider pathway analysis focusing on p53 and BRCA1-related functions, given the chromosomal proximity of C17orf74 to these tumor suppressor genes .

What controls should be included when using recombinant C17orf74 in experiments?

Proper experimental controls are essential when working with an uncharacterized protein:

• Negative controls:

  • Empty vector-transfected cells

  • Irrelevant protein of similar size and properties

  • Heat-denatured C17orf74 protein

  • Tag-only protein (for tagged recombinant versions)

• Positive controls:

  • Known proteins on chromosome 17 for chromosomal localization studies

  • Established proteins in predicted pathways based on bioinformatic analysis

  • Native protein extract for antibody validation

• Technical controls:

  • Multiple independently generated batches of recombinant protein

  • Multiple cell lines to confirm phenotypes

  • Dose-response analysis to establish specificity

• Validation controls:

  • Multiple siRNAs/shRNAs targeting different regions of C17orf74

  • Rescue experiments with siRNA/shRNA-resistant constructs

  • Orthogonal methods to confirm key findings

How can researchers validate the specificity of antibodies against C17orf74?

Antibody validation is particularly crucial for uncharacterized proteins to ensure specific detection:

• Initial validation:

  • Western blot against recombinant protein and endogenous protein

  • Testing in knockout/knockdown systems (signal reduction should be observed)

  • Peptide competition assays

  • Immunoprecipitation followed by mass spectrometry

• Application-specific validation:

  • For immunofluorescence: Co-localization with tagged recombinant protein

  • For ChIP: Comparison with tagged protein ChIP-seq

  • For immunohistochemistry: Comparison with mRNA expression patterns

• Cross-reactivity assessment:

  • Testing against closely related proteins

  • Species cross-reactivity testing

  • Testing in multiple cell types with varying expression levels

• Epitope mapping:

  • Determining the specific region recognized by the antibody

  • Assessing accessibility of the epitope in different applications

What structural biology approaches are suitable for an uncharacterized protein like C17orf74?

Understanding the structure of C17orf74 would provide significant insights into its function. Researchers should consider:

For a 501-amino acid protein like C17orf74, domain-based approaches may be more successful than attempting to solve the entire structure at once.

How can evolutionary analysis contribute to understanding C17orf74 function?

Evolutionary approaches provide valuable context for uncharacterized proteins:

• Ortholog identification:

  • Identify C17orf74 orthologs across species

  • Analyze conservation patterns across phylogenetic trees

  • Identify species-specific adaptations

• Synteny analysis:

  • Examine gene neighborhood conservation

  • Identify co-evolved gene clusters

• Selective pressure analysis:

  • Calculate dN/dS ratios across the protein

  • Identify regions under positive or purifying selection

  • Correlate evolutionary constraints with structural predictions

• Ancestral sequence reconstruction:

  • Determine the evolutionary trajectory of C17orf74

  • Identify key mutations that may have altered function

Given that chromosome 17 contains evolutionarily important genes like p53 and BRCA1, evolutionary analysis may reveal whether C17orf74 has co-evolved with these critical tumor suppressors .