Recombinant Mouse Protein FAM203A (Fam203a)

What is FAM203A and how does it compare to FAM203B?

FAM203A is a member of the Family with Sequence Similarity 203 protein family. Unlike its paralog FAM203B which is primarily found in humans and possibly non-human primates, FAM203A is highly conserved across species . This conservation suggests it plays an important biological role maintained throughout evolution. While FAM203B contains two conserved domains of unknown function (DUF383 and DUF384), FAM203A shares these structural features, indicating potential functional similarities despite their different evolutionary distributions .

What expression systems are recommended for recombinant mouse FAM203A?

Multiple expression systems can be utilized for recombinant mouse FAM203A production, each with distinct advantages:

The optimal expression system depends on your specific research requirements, including protein yield needs, downstream applications, and budget constraints .

What is known about the structural domains in mouse FAM203A?

Mouse FAM203A contains two conserved domains of unknown function: DUF383 and DUF384, similar to those found in FAM203B . Structural prediction suggests these domains may be important for protein-protein interactions. The homolog of FAM203A in C. elegans (Y54H5A.2) is thought to help regulate the actin cytoskeleton, suggesting FAM203A might have cytoskeletal regulatory functions . Systematic genetic interaction screens, similar to those performed for cell polarity genes in C. elegans, could help uncover FAM203A's functional networks and potential binding partners .

How can I optimize purification of recombinant mouse FAM203A?

Optimizing purification of recombinant mouse FAM203A involves several strategic decisions:

• Tag selection: Consider His-tag for initial capture via IMAC purification, with C-terminal placement preferable to avoid N-terminal functional domain interference .

• Buffer optimization:

  • Buffers containing 2% sarkosyl have shown higher yield and purity for some recombinant mouse proteins

  • For long-term stability, PBS buffer with stabilizing agents is recommended

• Chromatography strategy:

  • Begin with immobilized metal affinity chromatography for His-tagged protein

  • Follow with size exclusion chromatography to eliminate aggregates

  • Consider ion exchange chromatography as a final polishing step

• Refolding strategy: If the protein forms inclusion bodies, a structured refolding process using glutathione redox system (GSH/GSSG) has proven effective for other recombinant mouse proteins .

Each purification step should be validated by SDS-PAGE and Western blotting to monitor purity and target protein recovery .

What tags are recommended for recombinant mouse FAM203A?

The choice of affinity tag depends on your experimental goals:

For functional studies, including a TEV or PreScission protease cleavage site between the tag and protein is advisable to remove the tag if it interferes with activity . Always validate that the tag doesn't affect protein folding or function through comparative activity assays.

What buffer conditions optimize recombinant mouse FAM203A stability?

Based on established protocols for similar recombinant mouse proteins, the following buffer conditions are recommended:

• Storage buffer composition:

  • Base buffer: PBS (pH 7.4) or Tris-HCl (20-50 mM, pH 7.5-8.0)

  • Salt: 150-300 mM NaCl to prevent aggregation

  • Stabilizing agents: 5-10% glycerol

• Storage recommendations:

  • Short-term (1 month): 4°C

  • Medium-term: -20°C in small aliquots

  • Long-term: -80°C

  • Manual defrost freezer is recommended to avoid repeated freeze-thaw cycles

• Reconstitution guidelines:

  • For lyophilized protein: Reconstitute at 100 μg/mL in sterile buffer

  • For research not sensitive to carrier proteins, consider adding 0.1% BSA or other carrier protein

Conduct accelerated stability studies at different temperatures (4°C, 25°C, 37°C) to establish a stability profile specific to your recombinant FAM203A preparation.

How can I verify the proper folding and activity of recombinant mouse FAM203A?

Verifying proper folding and activity is challenging for proteins like FAM203A with poorly characterized functions. A multi-faceted approach is recommended:

• Biophysical characterization:

  • Circular dichroism spectroscopy to assess secondary structure content

  • Differential scanning fluorimetry to evaluate thermal stability

  • Size exclusion chromatography with multi-angle light scattering to confirm expected oligomerization state

• Limited proteolysis: Properly folded proteins typically show discrete, reproducible fragmentation patterns when subjected to limited proteolysis with enzymes like trypsin or chymotrypsin.

• Binding assays:

  • If cytoskeletal regulation is suspected, test binding to actin or actin-regulatory proteins

  • Surface plasmon resonance can detect even weak interactions, similar to binding studies performed with recombinant GDF-3

• Cellular activity assays:

  • Assess ability to rescue phenotypes in FAM203A-depleted cells

  • Monitor changes in cytoskeletal organization based on the suggested role in cytoskeletal regulation

Due to the limited characterization of FAM203A's function, include validated positive controls of proteins with better-characterized folding properties in parallel experiments.

What experimental approaches help investigate FAM203A's potential role in cytoskeletal regulation?

Given the suggested link between the C. elegans homolog and cytoskeletal regulation , several approaches can be implemented:

• Genetic interaction screens: Systematic genetic interaction screens can help position FAM203A within cytoskeletal regulatory networks, similar to approaches used for C. elegans polarity genes . These screens generated a comprehensive polarity network connecting 184 genes, with 72% not previously linked to cell polarity .

• Biochemical assays:

  • Actin polymerization assays using purified components

  • Microtubule co-sedimentation assays

  • In vitro reconstitution of minimal cytoskeletal systems with and without FAM203A

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize potential co-localization with cytoskeletal structures

  • Live-cell imaging in FAM203A knockout/knockdown cells, similar to approaches used in the transgenic mouse model studies

• Proteomic approaches:

  • Proximity labeling to identify proteins in close proximity to FAM203A

  • Quantitative mass spectrometry comparing wildtype and FAM203A-deficient cells

Combining these approaches would provide complementary evidence for FAM203A's role in cytoskeletal regulation.

What controls should be included when using recombinant mouse FAM203A in cellular assays?

Robust experimental design requires appropriate controls:

• Negative controls:

  • Buffer-only treatment to control for buffer components

  • Heat-denatured FAM203A to control for non-specific effects

  • Unrelated protein of similar size and properties

  • If tagged, equivalent concentration of the tag alone

• Positive controls:

  • If investigating cytoskeletal effects, include well-characterized cytoskeletal regulators

  • For binding assays, include proteins with established interactions

• Concentration controls:

  • Test a concentration gradient (e.g., 0.1-10 μg/mL) to establish dose-dependency

  • Determine EC50 values for specific cellular effects

• Specificity controls:

  • Pre-block with blocking peptides or antibodies

  • Use cells with FAM203A knockout/knockdown for rescue experiments, similar to approaches in the 58 kb deletion mouse studies

• System validation:

  • Confirm cellular uptake if necessary (consider fluorescent labeling)

  • Validate antibody specificity between FAM203A and related proteins

Document all control experiments thoroughly, including source, concentration, and treatment conditions for maximum reproducibility.

How can I design experiments to investigate FAM203A's interaction partners?

To identify and characterize FAM203A's interaction partners:

• Affinity purification-mass spectrometry:

  • Express tagged FAM203A in relevant cell lines

  • Perform pulldowns under physiological conditions

  • Analyze by mass spectrometry with appropriate controls

  • Validate top hits by reciprocal co-immunoprecipitation

• Proximity-based labeling:

  • Generate BioID or similar fusion constructs with FAM203A

  • Express in cells and activate labeling

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach can capture even transient interactions

• Genetic interaction screens:

  • Systematic RNAi screens similar to those performed for cell polarity genes in C. elegans can uncover functional relationships

  • The comprehensive polarity network generated from such screens connected 184 genes, demonstrating the power of this approach

• Crosslinking coupled with mass spectrometry:

  • Chemical crosslinking in cells expressing FAM203A

  • Identify crosslinked peptides by mass spectrometry

  • Provides information about interaction interfaces

For all methods, include appropriate controls and validate key findings using orthogonal techniques.

How can I address solubility issues when expressing recombinant mouse FAM203A?

Solubility challenges with recombinant mouse FAM203A can be addressed through multiple strategies:

• Expression optimization:

  • Lower induction temperature (15-18°C)

  • Reduce IPTG concentration (0.1-0.25 mM)

  • Use rich media like TB instead of LB

• Construct design:

  • Try different expression constructs (full-length vs. specific domains)

  • Test different affinity tags known to enhance solubility (MBP, SUMO, GST)

  • Optimize codon usage for the expression host

• Solubilization strategies:

  • If inclusion bodies form, mild detergents like sarkosyl (2%) can help

  • Consider buffer additives: arginine (50-200 mM), low concentrations of urea (1-2 M)

  • Test different pH conditions within the 6.0-8.5 range

• Co-expression approaches:

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Consider co-expression with known binding partners if identified

The optimal expression parameters for several mouse recombinant proteins have been found to be BL21(DE3) strain in TB culture medium with 0.25 mM IPTG induction at 15°C for 24 hours .

What approaches can be used to study potential post-translational modifications of FAM203A?

To investigate post-translational modifications (PTMs) of FAM203A:

• Mass spectrometry-based approaches:

  • Enrich for specific PTMs (phosphopeptides, glycopeptides)

  • Use both bottom-up (tryptic peptides) and top-down (intact protein) MS

  • Compare PTM landscapes across different tissues/conditions

• Specific PTM detection methods:

  • Phosphorylation: Phos-tag gels, phospho-specific antibodies

  • Glycosylation: Lectin blotting, PNGase F treatment

  • Ubiquitination: Ubiquitin pulldowns

• Comparative expression systems:

  • Compare PTM patterns between proteins expressed in E. coli, insect cells, and mammalian cells

  • Use mammalian cell expression for complex PTMs

• Prediction and validation:

  • Use bioinformatic tools to predict potential PTM sites

  • Generate site-specific mutants

  • Compare functional properties of wild-type and mutant proteins

For each identified modification, investigate its functional significance through mutation studies and correlation with biological activities.

How can I validate antibody specificity for mouse FAM203A?

Ensuring antibody specificity between mouse FAM203A and related proteins requires rigorous validation:

• Sequence analysis:

  • Identify unique epitopes specific to FAM203A not present in FAM203B

  • Generate peptide-specific antibodies targeting these regions

• Knockout/knockdown controls:

  • Test antibodies in cells with FAM203A knockout

  • Use siRNA knockdown for comparative Western blotting

• Overexpression validation:

  • Express tagged versions of FAM203A and FAM203B

  • Confirm antibody recognizes only the target protein

• Cross-reactivity testing:

  • Perform Western blots with purified recombinant FAM203A and FAM203B

  • Establish detection limits and cross-reactivity thresholds, similar to approaches used in ELISA analyses for other recombinant proteins

• Multiple antibody approach:

  • Use antibodies targeting different epitopes

  • Consistent results across antibodies increase confidence

Document all validation steps thoroughly, including positive and negative controls, to establish clear evidence of specificity.

What quality control measures should be implemented for recombinant mouse FAM203A?

Comprehensive quality control for recombinant mouse FAM203A should include:

• Purity assessment:

  • SDS-PAGE analysis with Coomassie staining (aim for >95% purity)

  • Western blotting with specific antibodies

  • Mass spectrometry to identify contaminants

• Quantification methods:

  • Absorbance at 280nm using protein-specific extinction coefficient

  • BCA or Bradford assay for concentration determination

  • Amino acid analysis for absolute quantification

• Endotoxin testing:

  • LAL assay to ensure levels below 0.1 EU per 1 μg protein

  • Critical for cell culture applications

• Functional validation:

  • Binding assays with potential interaction partners

  • Activity assays based on predicted function

  • Bioactivity testing in appropriate cell lines

• Batch consistency:

  • Lot-to-lot comparison using standardized assays

  • Retention of reference standards