Recombinant Mouse Protein FAM221A (Fam221a)

What is the basic molecular characterization of mouse Fam221a protein?

Mouse Fam221a, like its human ortholog, is a protein encoded by the Fam221a gene. It belongs to the family with sequence similarity 221 member A. Based on human FAM221A data, the molecular weight is approximately 33.1 kDa with an isoelectric point of 6.01 . The protein structure predominantly consists of random coils (approximately 71%) and alpha helices (21%) with a smaller proportion of extended strands (7%) . These structural characteristics suggest potential flexibility in protein-protein interactions, which may be relevant for experimental design considerations when studying binding partners.

Where is mouse Fam221a predominantly expressed?

Drawing from human expression data, Fam221a likely shows higher expression levels in neural tissues, particularly in the spinal cord and brain, including fetal brain. High expression is also observed in the pancreas and retina . Other tissues with notable expression include the liver, thyroid, and colon . When designing tissue-specific experiments, researchers should consider these expression patterns for optimal detection and functional analysis. RNA-seq or qPCR validation of expression in specific mouse tissues is recommended before proceeding with specialized functional studies.

How evolutionarily conserved is Fam221a across species?

Fam221a has one known paralog, FAM221B, which diverged approximately 1781 million years ago according to evolutionary analyses . Orthologs exist across various mammalian species, suggesting evolutionary conservation of function. When designing cross-species studies or selecting animal models, researchers should consider the degree of homology between human and mouse proteins. Protein alignment analysis would be recommended to identify conserved domains that might be crucial for function and potential targets for antibody development or mutagenesis studies.

What are the recommended methods for detecting mouse Fam221a in tissue samples?

For protein detection, Western blotting using validated antibodies against Fam221a would be the standard approach. Consider using tissues with known high expression levels (spinal cord, brain, pancreas) as positive controls . For mRNA detection, qRT-PCR using primers specific to mouse Fam221a should be employed, with careful primer design to avoid cross-reactivity with the paralog Fam221b. Immunohistochemistry or immunofluorescence can be used for spatial localization studies, particularly in neural tissues where expression appears highest. When performing these analyses, appropriate negative controls (including knockout tissue if available) should be included to confirm antibody specificity.

How can I generate recombinant mouse Fam221a for in vitro studies?

Recombinant mouse Fam221a can be produced using prokaryotic (E. coli) or eukaryotic (mammalian, insect) expression systems. For functional studies, a mammalian expression system is recommended to ensure proper post-translational modifications, as the human ortholog demonstrates phosphorylation, glycosylation, and sulfation sites that may be functionally relevant . The protein can be tagged (His, GST, FLAG) for purification purposes, but researchers should verify that the tag does not interfere with protein folding or function. Optimize expression conditions by testing different cell lines, inducer concentrations, and incubation times. Purification can be achieved through affinity chromatography followed by size exclusion chromatography to ensure homogeneity.

What are effective approaches for studying Fam221a post-translational modifications?

Based on human FAM221A data, researchers should investigate phosphorylation, glycosylation, and sulfation sites in mouse Fam221a . Mass spectrometry is the gold standard for comprehensive PTM profiling. For phosphorylation studies, phospho-specific antibodies (if available) or Phos-tag SDS-PAGE can be employed. For glycosylation analysis, treatment with specific glycosidases followed by mobility shift analysis on Western blots can provide initial insights. When studying PTMs, compare findings across different physiological conditions and disease models to identify functionally relevant modifications. Bioinformatic prediction tools like NetPhos, YinOYang, and The Sulfinator can guide experimental design by identifying potential modification sites .

What experimental approaches are recommended for investigating Fam221a function?

Given the limited understanding of Fam221a function, a multi-pronged approach is advised:

• Gene knockout or knockdown studies using CRISPR-Cas9 or siRNA technologies in relevant cell types (neuronal cells, given high expression in neural tissues)

• Protein interaction studies using co-immunoprecipitation, proximity labeling (BioID, APEX), or yeast two-hybrid screening

• Subcellular localization studies using fractionation and immunofluorescence microscopy

• Transcriptomic and proteomic profiling after Fam221a manipulation

When designing loss-of-function experiments, consider both acute (siRNA) and chronic (stable knockout) approaches, as they may reveal different aspects of protein function. Rescue experiments with wild-type Fam221a should be performed to confirm specificity of observed phenotypes.

Is there evidence connecting mouse Fam221a to neurodegenerative diseases?

While direct evidence for mouse Fam221a in neurodegeneration is limited in the provided search results, the human ortholog FAM221A may have associations with Parkinson's disease . Additionally, the related protein FAM222A has been implicated in Alzheimer's disease and amyloid pathology . Given the high expression of Fam221a in neural tissues , investigation of its role in neurodegeneration models is warranted. Researchers should consider:

• Analyzing Fam221a expression in mouse models of neurodegeneration

• Examining potential interactions with disease-associated proteins

• Evaluating the impact of Fam221a manipulation on disease phenotypes in mouse models

When conducting such studies, include appropriate age-matched controls and consider sex differences in disease manifestation.

How might Fam221a function be related to cancer biology?

Human FAM221A has been suggested to play a role in prostate cancer . For mouse studies investigating potential oncogenic or tumor suppressor functions of Fam221a, researchers could:

• Analyze expression in mouse cancer models, particularly prostate cancer

• Perform loss- and gain-of-function studies in cancer cell lines

• Investigate effects on key cancer-related phenotypes (proliferation, migration, invasion, apoptosis)

• Explore potential signaling pathways affected by Fam221a manipulation

In vitro findings should be validated in vivo using appropriate mouse cancer models. Consider using patient-derived xenograft models to bridge mouse studies with human disease relevance.

What are the considerations for developing a Fam221a knockout mouse model?

When generating a Fam221a knockout mouse model, researchers should:

• Design targeting strategies carefully, considering potential overlapping genes or regulatory elements

• Include appropriate reporters (LacZ, GFP) to track expression patterns

• Consider conditional knockout approaches (Cre-loxP) for tissue-specific deletion, particularly in neural tissues where expression is highest

• Plan for comprehensive phenotyping, including neurological, behavioral, and metabolic assessments

How can structural biology approaches enhance understanding of Fam221a function?

Given that human FAM221A consists primarily of random coils (71%) and alpha helices (21%) , structural characterization presents challenges but could provide valuable insights. Researchers might consider:

• X-ray crystallography of purified recombinant protein (though high proportion of random coils may hinder crystallization)

• NMR spectroscopy for solution structure determination, particularly suitable for flexible proteins

• Cryo-EM for larger complexes involving Fam221a

• Computational modeling approaches, including molecular dynamics simulations

Structural studies should focus on identifying functional domains and potential binding interfaces. The protein's reported secondary structure characteristics suggest significant intrinsic disorder, which may indicate a role in dynamic protein-protein interactions or as a scaffold protein.

What are appropriate experimental controls when studying Fam221a in disease models?

When investigating Fam221a in disease contexts, rigorous experimental controls are essential:

• For expression studies: Include multiple reference genes for normalization in qPCR; use multiple antibodies for protein detection when possible

• For disease models: Include age-matched, sex-matched wild-type controls; consider using Fam221a heterozygotes as additional controls

• For therapeutic interventions: Include vehicle-treated groups and consider dose-response relationships

• For behavioral studies: Blind investigators to genotype/treatment; account for circadian variations

How should researchers address species differences when translating mouse Fam221a findings to human applications?

When translating mouse Fam221a findings to human contexts, consider:

• Sequence homology analysis between mouse and human proteins to identify conserved and divergent regions

• Comparative expression pattern analysis across tissues in both species

• Cross-validation of key findings in human cells or samples

• Awareness of potential differences in post-translational modifications and protein interactions

Specific attention should be paid to the conserved post-translational modification sites observed in human FAM221A, including phosphorylation, glycosylation, and sulfation sites . These modifications may be critical for function and could vary between species. Consider using humanized mouse models for studying human-specific aspects of FAM221A function.

What insights can be gained from comparing Fam221a with its paralog Fam221b?

The paralog relationship between Fam221a and Fam221b, with their divergence approximately 1781 million years ago , presents opportunities for comparative functional analysis:

• Sequence and structural comparison to identify conserved domains

• Comparative expression analysis across tissues and developmental stages

• Differential response to experimental perturbations

• Potential compensatory mechanisms in knockout models

When designing experiments to compare paralogs, consider generating double knockout models to address functional redundancy. Comparative biochemical characterization, including interactome analysis, may reveal shared and distinct molecular functions of these related proteins.

What are the most promising research directions for advancing understanding of mouse Fam221a?

Based on current knowledge, future research on Fam221a should prioritize:

• Comprehensive characterization of expression patterns across tissues and developmental stages

• Identification of binding partners and signaling pathways

• Generation and phenotyping of knockout mouse models

• Investigation of roles in neurological disorders, given high expression in neural tissues

• Exploration of potential functions in cancer biology, particularly prostate cancer

The single mouse experimental design approach described in source may facilitate more efficient screening across multiple experimental conditions, potentially accelerating discovery. Integration of molecular, cellular, and in vivo approaches will be essential for developing a comprehensive understanding of Fam221a biology.

How can contradictory findings in Fam221a research be reconciled?

When faced with contradictory findings in Fam221a research:

• Consider differences in experimental systems (cell lines, animal models, experimental conditions)

• Evaluate methodological approaches and their limitations

• Assess potential splice variants or isoforms that might exhibit different functions

• Examine post-translational modifications that could alter protein function in different contexts

• Consider tissue-specific effects, particularly given differential expression patterns