Recombinant Chicken Protein FAM214A (FAM214A), partial

What is FAM214A and what are its known functional characteristics?

FAM214A (Family With Sequence Similarity 214 Member A) is a protein that has been historically known by other aliases including KIAA1370 and FLJ10980 . Though its precise function remains incompletely characterized, research suggests it plays roles in chromosome segregation during meiosis and purine metabolism regulation . The protein is encoded by the FAM214A gene located on chromosome 15 at position q21.2-q21.3 in humans . Research in model organisms indicates FAM214A is involved in regulating urate levels and inflammatory response programming . Methodologically, researchers investigating this protein should consider utilizing comparative genomics approaches across species, as well as functional studies in model organisms where homologs have been better characterized.

What structural domains characterize the FAM214A protein?

The FAM214A protein contains two conserved domains:

• DUF4210 (Domain of Unknown Function 4210)

• Chromosome_Seg (Chromosome Segregation domain)

While the precise functions of these domains remain uncharacterized, bioinformatic analyses suggest both domains contribute to chromosome segregation during meiosis . Researchers investigating domain function should employ deletion analysis, site-directed mutagenesis, and protein-protein interaction studies to elucidate their specific roles. The combination of structural prediction tools with experimental validation represents the most robust approach to understanding these domains.

What are the recommended methods for expressing recombinant chicken FAM214A protein?

Recombinant chicken FAM214A protein can be expressed using several systems, with the choice depending on specific research needs:

The expression system selection should be guided by the intended application . For structural studies, E. coli-derived protein may be sufficient, while functional studies might require the more complex post-translational modifications achieved in mammalian expression systems. Regardless of the expression system, researchers should verify the purity (≥85% as determined by SDS-PAGE) and confirm activity through appropriate functional assays .

What detection methods are most effective for measuring FAM214A expression?

Several methods can be employed for detecting and measuring FAM214A:

• ELISA: Human FAM214A ELISA kits demonstrate a detection range of 0.78-50 ng/mL with a sensitivity of 0.39 ng/ml, making them suitable for analyzing serum, plasma, tissue homogenates, and cell culture supernatants .

• Western blotting: Can be performed using specific antibodies against FAM214A (e.g., Sigma-Aldrich #HPA039369, 1:1000 dilution), with β-actin typically serving as a loading control. Imaging can be performed using fluorescent secondary antibodies and systems such as the Odyssey® CLx .

• RT-qPCR: For mRNA expression analysis, particularly useful for comparing expression across different tissues or experimental conditions.

• Immunohistochemistry: For tissue localization studies, though the selection of antibodies with appropriate specificity is crucial.

When analyzing FAM214A protein levels, researchers should normalize expression to appropriate housekeeping proteins and include positive and negative controls to ensure assay specificity.

What genome editing approaches are most suitable for studying FAM214A function?

CRISPR-Cas9 approaches have proven effective for targeting FAM214A. Guide RNA sequences designed by the laboratory of Feng Zhang at the Broad Institute have been validated for efficiently targeting the FAM214A gene with minimal off-target effects . When implementing CRISPR-based methodologies:

• Use at least two different guide RNA constructs per target to increase success probability

• Verify guide RNA sequences against your specific target sequence, especially when targeting specific splice variants or exons

• Consider using vectors that include selection markers to facilitate the identification of successfully edited cells

• Validate genomic edits through sequencing and confirm functional effects through appropriate assays

For transcriptional activation studies, the Synergistic Activation Mediator (SAM) system with specific gRNAs for FAM214A has been validated .

How does FAM214A contribute to purine metabolism regulation?

FAM214A appears to play a significant role in purine metabolism regulation, particularly affecting uric acid levels. Research in Drosophila melanogaster demonstrated that knockdown of medusa (mda), the FAM214A homolog, resulted in decreased levels of:

• Phosphoribosyl pyrophosphate (a purine synthesis precursor)

• Uric acid

• Guanosine

The mechanism appears to involve the regulation of the uric acid-induced innate immune inflammatory response downstream of xbp1 . Experimental approaches to investigate this function should include:

• Metabolomic profiling of purine pathway intermediates in FAM214A knockdown/knockout models

• Enzyme activity assays for key purine metabolism enzymes

• Transcriptomic analysis of purine metabolism genes in response to FAM214A modulation

• Isotope tracing experiments to track purine synthesis and degradation rates

These methodologies would provide mechanistic insights into how FAM214A influences purine metabolism pathways.

What is the relationship between FAM214A and inflammatory responses?

Evidence suggests FAM214A plays a role in inflammatory regulation:

• FAM214A levels are elevated in inflammatory M1-differentiated mouse bone marrow macrophages

• FAM214A levels are reduced in anti-inflammatory M2-differentiated macrophages

• In Drosophila, mda (FAM214A homolog) activity increases inflammatory response programming

To investigate this relationship, researchers should consider:

• Cytokine profiling in systems with modulated FAM214A expression

• Analysis of inflammatory signaling pathway components (e.g., NF-κB, MAPK pathways)

• Macrophage polarization studies in the presence/absence of FAM214A

• In vivo inflammation models comparing wild-type and FAM214A-deficient contexts

This multi-faceted approach would help clarify how FAM214A influences inflammatory processes and whether it represents a viable therapeutic target for inflammatory conditions.

How does FAM214A influence hyperuricemia-driven pathologies?

Research indicates FAM214A/mda is a potential regulator of hyperuricemia-associated pathologies. In Drosophila models of hyperuricemia and uric acid crystallization, knockdown of mda:

• Limited formation of aggregated uric acid concretions

• Rescued lifespan reduction in hyperuricemia and gout model flies

• Decreased inflammatory response programming

These findings suggest FAM214A may be a promising target for ameliorating negative effects of elevated uric acid burden without the detrimental consequences of targeting upstream genes with pleiotropic effects . For researchers investigating this relationship, approaches should include:

• Crystal formation assays in cell and animal models with modified FAM214A expression

• Histological examination of kidney tissues from hyperuricemic models with altered FAM214A levels

• Inflammatory marker analysis in hyperuricemic conditions with FAM214A modulation

• Survival and phenotypic studies in hyperuricemia animal models with FAM214A genetic manipulation

What are common challenges in purifying recombinant FAM214A protein?

Researchers working with recombinant FAM214A may encounter several challenges:

• Solubility issues: The protein has a molecular weight of 122,472 Da, which can present solubility challenges . Optimization strategies include:

  • Adjusting buffer conditions (pH, salt concentration)

  • Using solubility-enhancing fusion tags (MBP, SUMO)

  • Expressing truncated protein fragments containing functional domains

• Proper folding: Ensuring correct folding is essential for functional studies. Consider:

  • Slow induction at lower temperatures

  • Co-expression with chaperone proteins

  • Refolding protocols if inclusion bodies form

• Maintaining stability: After purification, protein stability should be addressed through:

  • Testing different storage conditions (temperature, buffer compositions)

  • Adding stabilizing agents as appropriate

  • Validating protein activity after storage periods

The format may be lyophilized or liquid, determined during the manufacturing process, which should be selected based on intended applications and stability requirements .

How can researchers address specificity issues when studying FAM214A?

When investigating FAM214A, specificity concerns may arise due to its ubiquitous expression and potential functional overlap with related proteins. To address these challenges:

• Antibody validation: Thoroughly validate antibodies using:

  • Positive and negative control samples

  • Western blotting alongside knockdown/knockout samples

  • Peptide competition assays

  • Multiple antibodies targeting different epitopes

• Genetic manipulation specificity:

  • Design guide RNAs with minimal off-target effects

  • Validate genomic edits through sequencing

  • Rescue experiments to confirm phenotype specificity

  • Use inducible systems to control timing of manipulation

• Cross-reactivity assessment:

  • Compare with related family members (e.g., FAM214B)

  • Perform bioinformatic analyses to identify potential cross-reactive proteins

  • Include appropriate controls in all experiments

Implementing these strategies helps ensure that observed effects are specifically attributable to FAM214A rather than related proteins or off-target effects.

What are the most promising avenues for further FAM214A research?

Based on current knowledge, several research directions appear particularly promising:

• Therapeutic potential in hyperuricemia and gout: Given FAM214A's role in uric acid metabolism and inflammatory responses, investigating its therapeutic potential in these conditions represents a valuable research direction .

• Mechanistic studies of domain functions: Detailed investigation of the DUF4210 and Chromosome_Seg domains would advance understanding of FAM214A's molecular functions .

• Role in immune regulation: The elevated expression in immune tissues suggests important immunological functions that warrant further investigation .

• Chromosome segregation mechanisms: Exploring FAM214A's predicted role in chromosome segregation during meiosis could yield insights into reproductive biology and genetic disorders .

• Interactome mapping: Comprehensive identification of protein-protein interactions would clarify FAM214A's position within cellular signaling networks and metabolic pathways.

Researchers pursuing these directions should employ integrative approaches combining structural biology, functional genomics, and translational research to maximize the impact of their findings.