Recombinant Chicken Protein FAM53A (FAM53A)

What is FAM53A protein and what are its known functions in chickens?

FAM53A (family with sequence similarity 53, member A) is also known as dorsal neural-tube nuclear protein (DNTNP) in some literature. The protein is encoded by the FAM53A gene in Gallus gallus (chicken) . Despite being identified and characterized at the sequence level, the specific biological functions of FAM53A in chickens remain largely undefined in current research. The protein is believed to be involved in developmental processes, particularly in neural development, as suggested by its alternative name (dorsal neural-tube nuclear protein) .

The chicken FAM53A protein consists of 418 amino acids and shows sequence conservation with its mammalian orthologs, suggesting evolutionary preservation of its function across vertebrate species . While specific pathway involvement data is limited for the chicken variant, research is ongoing to elucidate its role in cellular signaling and developmental biology.

How is recombinant chicken FAM53A typically produced for research applications?

Recombinant chicken FAM53A protein can be produced using several expression systems, with the choice depending on research requirements for protein folding, post-translational modifications, and yield. Based on available information, successful expression has been achieved in:

• Yeast expression systems, which have yielded chicken FAM53A protein with >90% purity

• Mammalian cell systems, which are particularly useful when native folding and post-translational modifications are critical

For purification and detection purposes, recombinant chicken FAM53A is commonly tagged with affinity tags such as:

• Histidine tag (His-tag) - facilitates purification via metal affinity chromatography

• GST tag - improves solubility and enables purification via glutathione affinity chromatography

• MYC/DDK tags - useful for immunodetection in experimental applications

Standard production typically follows this methodological workflow:

• Cloning of the chicken FAM53A gene into an appropriate expression vector

• Transformation/transfection into the chosen expression system

• Induction of protein expression

• Cell lysis and protein extraction

• Affinity purification using the chosen tag

• Quality control testing for purity (typically >90%) and identity verification

What expression systems are optimal for producing functional recombinant chicken FAM53A?

The choice of expression system for recombinant chicken FAM53A depends significantly on the intended application and required protein characteristics. Based on available research:

For applications requiring the most physiologically relevant form of the protein, mammalian cell expression is preferred. The data indicates that chicken FAM53A has been successfully expressed in mammalian cell systems with preservation of structural integrity . For basic research applications where absolute native conformation is less critical, yeast-expressed protein offers a good balance of quality and yield .

The expression of recombinant adenoviral vectors carrying other chicken proteins has demonstrated successful protein production methodologies that could be adapted for FAM53A expression. These approaches include monitoring expression through fluorescence microscopy and confirming protein production via Western blotting .

What are the recommended storage conditions for maintaining recombinant FAM53A stability?

Proper storage of recombinant chicken FAM53A is crucial for maintaining protein integrity and activity. Based on standard protocols for similar recombinant proteins, the following guidelines are recommended:

For short-term storage (up to 1 month):

• Store at +4°C in PBS buffer

• Avoid repeated freeze-thaw cycles which can lead to protein denaturation

• Consider adding protein stabilizers such as glycerol (5-10%)

For long-term storage:

• Store at -20°C to -80°C

• Aliquot the protein solution to avoid repeated freeze-thaw cycles

• Consider lyophilization for extended shelf-life, particularly for His-tagged variants

What validation methods should researchers employ to confirm recombinant FAM53A identity and activity?

Validation of recombinant chicken FAM53A requires multiple complementary approaches to confirm both identity and functional integrity:

Identity Confirmation Methods:

• SDS-PAGE for molecular weight verification (expected MW ~46-50 kDa, depending on tags)

• Western blotting with anti-FAM53A antibodies or tag-specific antibodies (anti-His, anti-GST)

• Mass spectrometry for peptide mass fingerprinting and sequence verification

• N-terminal sequencing to confirm protein identity

Purity Assessment:

• SDS-PAGE with densitometry analysis (target >80-90% purity)

• Size exclusion chromatography to detect aggregation or degradation

• Endotoxin testing using LAL method (recommended limit: <1.0 EU per μg protein)

• Protein-protein interaction studies with known or predicted binding partners

• Subcellular localization studies (expected to be nuclear based on its alternative name)

• Phosphorylation state analysis, as the protein contains multiple potential phosphorylation sites

When designing validation experiments, it is advisable to include appropriate positive and negative controls and to perform validation under conditions that mimic the intended experimental use.

How does chicken FAM53A compare structurally and functionally to mammalian orthologs?

Comparative analysis of chicken FAM53A with mammalian orthologs provides insights into evolutionary conservation and potential functional significance. While specific comparative data is limited in the available literature, general patterns can be inferred:

The amino acid sequence of chicken FAM53A shows regions of high conservation with mammalian orthologs, particularly in functional domains. Human FAM53A consists of 398 amino acids compared to the 418 amino acids in chicken FAM53A . This slight difference in length suggests potential species-specific adaptations while maintaining core functional domains.

Both chicken and mammalian FAM53A proteins are characterized by:

• Predicted nuclear localization signals

• Multiple phosphorylation sites

• Regions with high density of charged amino acids

Researchers studying chicken FAM53A should consider these evolutionary relationships when designing experiments or interpreting results, particularly when extrapolating findings between species.

What methodological approaches are recommended for studying FAM53A protein-protein interactions?

Investigating protein-protein interactions (PPIs) of chicken FAM53A requires careful selection of methodologies appropriate for nuclear proteins. Based on the limited available data on FAM53A's interactome and standard approaches for nuclear protein interaction studies, the following methods are recommended:

In vitro approaches:

• Pull-down assays using tagged recombinant FAM53A as bait

  • His-tagged or GST-tagged chicken FAM53A can be immobilized on appropriate resins

  • Chicken tissue or cell lysates can be used as prey

  • Interacting proteins can be identified by mass spectrometry

• Surface Plasmon Resonance (SPR) for quantitative binding kinetics

  • Requires highly purified recombinant FAM53A (>90% purity)

  • Can determine binding affinities and kinetics with candidate interacting proteins

Cellular approaches:

• Co-immunoprecipitation (Co-IP) from chicken cell lines

  • Requires specific antibodies against FAM53A or expression of tagged versions

  • Can detect native protein complexes

• Proximity-based labeling methods (BioID, APEX)

  • Expression of FAM53A fused to a biotin ligase or peroxidase

  • Allows identification of proximal proteins in the nuclear compartment

  • Particularly suitable for transient or weak interactions

• Fluorescence-based interaction assays

  • FRET (Förster Resonance Energy Transfer)

  • BiFC (Bimolecular Fluorescence Complementation)

  • Allows visualization of interactions in living cells

When designing PPI experiments for FAM53A, researchers should consider:

• Nuclear localization may require specific lysis conditions to effectively solubilize the protein

• Phosphorylation states may influence interaction partners

• Transient interactions may require crosslinking approaches

• Controls should include tag-only proteins to identify non-specific interactions

What are common challenges in working with recombinant chicken FAM53A and their solutions?

Researchers working with recombinant chicken FAM53A may encounter several technical challenges. Based on experience with similar nuclear proteins and recombinant protein work, the following issues and solutions are relevant:

Researchers have successfully expressed recombinant chicken proteins using adenoviral expression systems, which could be adapted for FAM53A expression if conventional systems prove challenging . The stability of recombinant proteins during temperature fluctuations can be assessed using methods similar to those described for other recombinant proteins, involving exposure to different temperature points followed by functional testing .

How can researchers design meaningful functional studies for a protein with limited characterized activity?

Designing functional studies for chicken FAM53A presents a challenge due to limited information about its specific activities. A systematic approach is recommended:

• Inference-based experimental design

  • Base initial experiments on the protein's alternative name (dorsal neural-tube nuclear protein)

  • Focus on nuclear functions and developmental processes

  • Design experiments that test involvement in neural development pathways

• Domain-based functional analysis

  • Identify conserved domains through bioinformatic analysis

  • Express and test individual domains for specific activities

  • Use truncation mutants to map functional regions

• Localization and temporal expression studies

  • Determine subcellular localization in relevant cell types

  • Map expression patterns during embryonic development

  • Identify tissues and developmental stages with highest expression

• Loss-of-function and gain-of-function studies

  • Use RNA interference or CRISPR/Cas9 to reduce expression

  • Overexpress the protein to identify dose-dependent phenotypes

  • Analyze effects on gene expression profiles using RNA-seq

• Interactome mapping

  • Identify binding partners through approaches discussed in section 3.3

  • Perform functional studies on the most promising interaction partners

  • Use pathway analysis to place FAM53A in biological context

When working with proteins of unknown function like FAM53A, it is essential to use multiple complementary approaches and to design experiments with appropriate controls that can distinguish between specific and non-specific effects.

What quality control metrics should be established for recombinant FAM53A preparations?

Establishing rigorous quality control (QC) metrics is essential for ensuring reproducible results when working with recombinant chicken FAM53A. Based on standard practices for recombinant proteins and available information, the following QC parameters are recommended:

Physical and Chemical Parameters:

• Purity: >80-90% as determined by SDS-PAGE and densitometry analysis

• Concentration: Accurate protein quantification using BCA or Bradford assay

• Endotoxin levels: <1.0 EU per μg protein using LAL method

• Aggregation state: Assessed by dynamic light scattering or size exclusion chromatography

• pH and buffer composition: Verified to match specifications (typically PBS)

Identity Confirmation:

• Molecular weight: Verification by SDS-PAGE and/or mass spectrometry

• Sequence verification: N-terminal sequencing or peptide mass fingerprinting

• Tag confirmation: Western blot using tag-specific antibodies (His, GST, MYC/DDK)

Functional Parameters:

• Binding activity: If binding partners are identified, measure interaction affinities

• Stability assessment: Thermal shift assay to determine protein stability

• Lot-to-lot consistency: Comparison to reference standards

Documentation Requirements:

• Expression system used and growth conditions

• Detailed purification protocol

• Storage conditions and freeze-thaw history

• Date of production and expiration

• Results of all QC tests performed

Implementing these QC metrics will help ensure that experimental outcomes are attributable to the biological properties of FAM53A rather than variations in protein quality or preparation methods.

What are the most promising avenues for advancing our understanding of chicken FAM53A function?

Based on the current state of knowledge, several research directions appear particularly promising for elucidating the function of chicken FAM53A:

• Developmental biology approaches

  • Detailed expression mapping during chicken embryonic development

  • CRISPR/Cas9-mediated gene editing to create FAM53A knockouts in chicken embryos

  • Analysis of neural tube development in FAM53A-deficient models

• Comparative genomics and evolutionary studies

  • Systematic comparison of FAM53A across vertebrate species

  • Identification of conserved regulatory elements in the FAM53A gene

  • Analysis of selection pressure on different protein domains

• Systems biology approaches

  • Integration of transcriptomics, proteomics, and interactomics data

  • Network analysis to position FAM53A within developmental pathways

  • Computational prediction of function based on structural features

• Structural biology studies

  • Determination of FAM53A three-dimensional structure

  • Identification of functional domains through structural analysis

  • Structure-guided mutagenesis to test function

These research directions would benefit from the availability of high-quality recombinant chicken FAM53A protein and could leverage methodologies that have been successfully applied to other chicken proteins .

How might FAM53A research contribute to broader understanding of avian developmental biology?

Research on chicken FAM53A has potential to contribute significantly to the broader field of avian developmental biology in several ways:

• Neural development insights

  • As a putative dorsal neural-tube nuclear protein , FAM53A studies may illuminate mechanisms of neural tube patterning specific to avian systems

  • Could provide comparative insights between mammalian and avian neural development

  • May help identify avian-specific regulatory networks in neural development

• Evolution of developmental mechanisms

  • Analysis of FAM53A function across species could reveal conserved versus divergent developmental mechanisms

  • Might highlight adaptations specific to avian embryonic development

  • Could provide insights into the evolution of the nuclear proteome in vertebrates

• Model system development

  • Characterization of FAM53A may establish new molecular tools for studying chicken development

  • Could lead to novel reporter systems for developmental processes

  • May provide new markers for specific developmental stages or cell types

• Integration with poultry health research

  • Understanding developmental biology has implications for poultry health and disease resistance

  • May contribute to improved vaccination strategies or genetic selection approaches

  • Could provide molecular targets for enhancing beneficial traits in poultry

The chicken continues to be a valuable model organism for developmental biology, and molecular characterization of proteins like FAM53A contributes to the fundamental knowledge base that supports both basic and applied research in avian biology.