Recombinant Human Transmembrane protein FAM155B (FAM155B)

What is the genomic location and structure of the FAM155B gene?

FAM155B (also known as NALF2, CXorf63, TED, or TMEM28) is located on the X chromosome at position Xq13.1 on the positive strand from nucleotides 69504326-69532508. The full gene transcript sequence is 3528 bp with a coding sequence of 1418 bp, containing 3 exons and 2 introns. Genetic neighbors include the EDA (ectodysplasin A) gene downstream and a long intergenic non-protein coding RNA upstream . When designing experiments targeting this gene, researchers should consider its X-chromosomal location, which may affect expression patterns in sex-specific studies and require appropriate controls for X-inactivation in female-derived samples.

What are the structural characteristics of the FAM155B protein?

The FAM155B protein is 472 amino acids long with a predicted molecular weight of 52.5 kDa and an estimated isoelectric point of 8.2. The most abundant amino acids are Leucine (11.4%) and Proline (10%) . The protein contains two transmembrane domains, classifying it as a transmembrane protein. There are two known protein isoforms: isoform 1 is 340 amino acids long, while isoform 2 is 292 amino acids . When working with recombinant FAM155B, researchers should verify which isoform is being used and ensure that the expression system preserves the native structure, particularly the transmembrane domains that may be crucial for function.

How can I effectively express and purify recombinant FAM155B?

For optimal expression and purification of recombinant FAM155B, an E. coli-based expression system with N-terminal GST tagging has been successfully employed . The recommended protocol involves:

• Transformation into an appropriate E. coli strain (BL21 or similar)

• Induction with IPTG at mid-log phase

• Cell lysis in 50 mM Tris-HCl buffer

• Affinity purification using glutathione resin

• Elution with 50 mM Tris-HCl containing 10 mM reduced glutathione, pH 8.0

Post-purification, the protein should be stored at -80°C to maintain stability, with freeze-thaw cycles minimized . When working with transmembrane proteins like FAM155B, researchers should consider that traditional bacterial systems may not provide proper folding of transmembrane domains, potentially necessitating mammalian or insect cell expression systems for functional studies.

What are the validated methods for detecting FAM155B in biological samples?

Several validated methods have been established for detecting FAM155B in various biological samples:

• Western Blotting: Effective for detecting FAM155B in tissue homogenates and cell lysates, typically using antibodies against the N-terminal region .

• ELISA: Commercially available kits can quantitatively measure FAM155B concentrations within a detection range of 0.156-10 ng/ml in tissue homogenates, cell lysates, and biological fluids .

• Immunofluorescence: Useful for visualizing cellular localization of FAM155B, particularly in tissues with known expression like heart, thyroid, and brain.

• RNA-seq/qPCR: For transcriptional analysis of FAM155B expression levels.

When selecting detection methods, researchers should consider the sensitivity requirements of their experiment and the potential for cross-reactivity with related proteins or isoforms.

What sample preparation techniques optimize FAM155B detection in complex biological matrices?

For optimal detection of FAM155B in complex biological matrices, consider these methodological approaches:

• Tissue homogenization: For tissue samples, use a mild detergent buffer (e.g., RIPA) supplemented with protease inhibitors to preserve protein integrity.

• Subcellular fractionation: Since FAM155B is a transmembrane protein, membrane fraction enrichment can significantly improve detection sensitivity.

• Immunoprecipitation: For samples with low FAM155B expression, immunoprecipitation prior to detection can concentrate the target protein.

• Sample dilution: For quantitative ELISA, samples should be properly diluted to fall within the mid-range of the standard curve (0.156-10 ng/ml) for accurate quantification .

When working with brain samples, where FAM155B is highly expressed, particular attention should be paid to reducing background signal from lipid-rich tissues by incorporating additional washing steps in the protocol.

What cellular pathways is FAM155B known to participate in?

Research suggests FAM155B participates in several cellular pathways, though our understanding remains incomplete:

• Endometrial receptivity: FAM155B appears to influence endometrial epithelial cell (EEC) proliferation and may affect the expression of receptivity-related factors like LIF and DKK1 .

• Trophoblast adhesion: Evidence suggests FAM155B can reduce trophoblast adhesion, potentially impacting embryo implantation .

• METTL3 axis interaction: FAM155B functions within the METTL3 pathway, which is involved in RNA methylation and post-transcriptional regulation .

When designing experiments to study FAM155B function, researchers should consider examining these pathways alongside potential novel interactions, employing both gain- and loss-of-function approaches to comprehensively understand its role.

How do I design effective knockdown/knockout experiments for studying FAM155B function?

For robust functional analysis of FAM155B through knockdown or knockout approaches:

• siRNA/shRNA design: Target conserved regions across isoforms for complete knockdown. Recommended target regions include:

  • Exon 2 (present in both major isoforms)

  • Avoid regions with sequence similarity to related family members

• CRISPR-Cas9 knockout: For permanent genetic ablation, design guide RNAs targeting:

  • Early exons to disrupt reading frame

  • Critical functional domains

  • Consider X-chromosome specific targeting strategies

• Validation approaches:

  • Quantify knockdown efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels

  • Include rescue experiments with recombinant FAM155B to confirm specificity

  • Assess potential compensatory upregulation of related family members

When interpreting results, remember that FAM155B knockdown may have different phenotypic consequences depending on the cell type, due to its differential expression across tissues.

What are the challenges in studying FAM155B-protein interactions?

Studying FAM155B-protein interactions presents several methodological challenges:

• Transmembrane domain preservation: Standard immunoprecipitation buffers may disrupt membrane protein interactions. Consider:

  • Crosslinking prior to lysis

  • Mild detergents (0.1% digitonin or 1% CHAPS)

  • Membrane-based two-hybrid systems for direct interaction studies

• Tag interference: N-terminal tags (like GST) may interfere with interaction domains. Strategies include:

  • C-terminal tagging alternatives

  • Tag removal via protease cleavage sites

  • Split-tag complementation assays

• Conformational integrity: Ensuring proper folding of recombinant FAM155B for interaction studies requires:

  • Expression in eukaryotic systems for proper post-translational modifications

  • Inclusion of chaperones in expression systems

  • Functional validation of recombinant proteins prior to interaction studies

When designing interaction experiments, combining multiple complementary approaches (co-IP, proximity ligation, FRET) provides stronger evidence for physiologically relevant interactions.

What is known about FAM155B's involvement in reproductive disorders?

Emerging research suggests FAM155B may play a role in reproductive disorders, particularly those related to implantation failures:

• Recurrent Implantation Failure (RIF): CircABCC1 has been shown to reduce endometrial receptivity via the METTL3/FAM155B axis . This pathway appears to impact:

  • Endometrial epithelial cell proliferation

  • Expression of receptivity markers including LIF and DKK1

  • Trophoblast adhesion capabilities

• Potential Mechanisms: FAM155B may influence reproductive processes through:

  • Altering cellular adhesion properties crucial for implantation

  • Modulating cytokine secretion profiles in the endometrium

  • Affecting extracellular matrix composition at the implantation site

For researchers investigating FAM155B in reproductive disorders, examining protein expression in endometrial biopsies from patients with recurrent implantation failure compared to fertile controls may provide valuable insights into its clinical relevance.

How can I effectively analyze FAM155B expression changes in disease states?

For rigorous analysis of FAM155B expression changes in disease contexts:

• Tissue-specific considerations:

  • Brain samples: Compare expression across multiple regions due to heterogeneous expression

  • Heart tissue: Account for chamber-specific and developmental differences

  • Endometrial samples: Control for menstrual cycle phase and hormonal status

• Methodological approach:

  • Combine mRNA and protein quantification to detect post-transcriptional regulation

  • Use in situ hybridization or immunohistochemistry to preserve spatial information

  • Consider single-cell approaches for heterogeneous tissues to avoid dilution effects

• Controls and normalization:

  • Age and sex-matched controls are essential (particularly for X-linked genes)

  • Use multiple reference genes for qPCR normalization

  • Include positive controls with known expression patterns

When interpreting disease-associated changes, consider that altered FAM155B expression may represent either causal factors or compensatory responses to pathological conditions.

How can I develop cell-based assays to study FAM155B function?

Developing robust cell-based assays for FAM155B functional studies requires careful consideration of several factors:

• Cell line selection: Choose cells with either:

  • Endogenous FAM155B expression (cardiac, neural, or thyroid cell lines)

  • Low background expression for overexpression studies

• Reporter system design:

  • For monitoring endometrial receptivity pathway: LIF or DKK1 promoter-driven luciferase reporters

  • For protein-protein interactions: Split complementation assays (BiFC, BRET)

  • For trafficking studies: Fluorescently tagged FAM155B variants

• Functional readouts:

  • Adhesion assays for studying implantation-related functions

  • Calcium imaging for potential channel regulatory functions

  • Proliferation and migration assays for cellular effects

When optimizing these assays, establish dose-response relationships and time-course dynamics to capture the full spectrum of FAM155B's functional impacts.

What are the challenges in reconciling contradictory findings about FAM155B molecular weight?

The literature reports discrepancies in FAM155B molecular weight, with values ranging from 36.41 kDa to 52.5 kDa . Researchers can address these contradictions through:

• Technical considerations:

  • SDS-PAGE migration anomalies due to:

    • Post-translational modifications

    • Hydrophobic transmembrane domains affecting SDS binding

    • Incomplete denaturation

• Isoform variations:

  • The two documented isoforms (340 aa and 292 aa) would have different molecular weights

  • Alternative splicing events may generate tissue-specific variants

• Methodological approach to resolution:

  • Mass spectrometry for precise mass determination

  • Isoform-specific antibodies for discriminating variants

  • Expression of individual isoforms with sequence verification

A comprehensive approach would involve parallel analysis of endogenous and recombinant proteins using multiple detection methods to establish the true molecular characteristics of FAM155B isoforms in different contexts.

What are the most promising directions for future FAM155B research?

Based on current knowledge, several promising research directions for FAM155B include:

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography of FAM155B to resolve transmembrane domain structure

  • Molecular dynamics simulations to understand conformational changes

  • Structure-function studies identifying critical residues for protein interactions

• Disease associations beyond reproductive disorders:

  • Given its expression in brain and heart, investigate potential roles in:

    • Neurodegenerative conditions

    • Cardiac pathologies

    • X-linked disorders

• Signaling pathway integration:

  • Comprehensive interactome mapping using proximity labeling

  • Phosphoproteomics to identify regulatory sites

  • Transcriptional networks controlled by FAM155B modulation

• Therapeutic targeting potential:

  • Development of small molecule modulators of FAM155B function

  • Analysis of FAM155B as a biomarker in endometrial receptivity

  • Investigation of potential roles in embryo implantation failure treatment

Researchers pursuing these directions should employ interdisciplinary approaches combining genomics, proteomics, and functional assays to build a comprehensive understanding of this understudied protein.