Recombinant Xenopus laevis Transmembrane protein 214-B (tmem214-b)

What is Xenopus laevis Transmembrane protein 214-B (tmem214-b)?

Transmembrane protein 214-B (tmem214-b) is a 679-amino acid protein expressed in Xenopus laevis. The full-length recombinant version contains an N-terminal His tag and is typically expressed in E. coli expression systems. Structural analysis indicates that the human TMEM214 homolog contains two putative transmembrane domains at amino acids 480-500 and 616-636 . The protein is predominantly localized to the endoplasmic reticulum (ER) membrane, with minimal presence in mitochondria and undetectable levels in cytosol, as demonstrated through cell fractionation and immunoblot analyses .

Based on functional studies of human TMEM214, it likely plays a critical role in ER stress-induced apoptosis pathways, potentially by acting as an anchor for recruitment of procaspase 4 to the ER and facilitating its subsequent activation . The protein's sequence contains specific domains responsible for different functions: the N-terminal cytoplasmic region (amino acids 176-354) is required for binding with procaspase 4, while either of the two transmembrane domains is sufficient for its localization to the ER .

What expression systems are most effective for producing recombinant tmem214-b?

For optimal production of recombinant Xenopus laevis tmem214-b, the E. coli expression system has been successfully employed to generate full-length protein (1-679aa) with N-terminal His tags . When using E. coli systems, researchers should consider the following methodological approach:

• Vector Selection: Use expression vectors containing strong promoters (e.g., T7) with His-tag coding sequences for simplified purification

• Induction Parameters:

  • Optimal IPTG concentration: 0.5-1.0 mM

  • Induction temperature: 16-25°C (lower temperatures may reduce inclusion body formation)

  • Induction duration: 16-20 hours for maximum yield

• Purification Strategy:

  • Initial capture: Ni-NTA affinity chromatography

  • Secondary purification: Size exclusion chromatography

  • Final purity assessment: SDS-PAGE (target >90% purity)

While E. coli systems are effective, mammalian expression systems (HEK293 or CHO cells) may be considered for applications requiring proper glycosylation or when studying protein interactions within a more native-like cellular environment.

How should researchers properly reconstitute and store recombinant tmem214-b?

Proper reconstitution and storage of recombinant tmem214-b is critical for maintaining protein activity and stability. Follow this methodological approach:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to collect all material at the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% (adjustable between 5-50% based on experimental requirements)

• Aliquot the reconstituted protein into smaller volumes to avoid repeated freeze-thaw cycles

Storage Recommendations:

• Short-term storage (up to one week): 4°C

• Long-term storage: -20°C or -80°C in aliquots containing glycerol

• Storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0

Critical Considerations:

• Avoid repeated freeze-thaw cycles as they significantly degrade protein quality

• When thawing, allow the protein to reach room temperature gradually

• For experiments requiring precise concentration, verify protein concentration after reconstitution using standard methods (Bradford or BCA assay)

What experimental approaches can be used to study tmem214-b localization in Xenopus cells or tissues?

To study tmem214-b localization in Xenopus cells or tissues, researchers can employ several complementary methodological approaches:

Immunofluorescence Microscopy:

• Fix cells or tissue sections with 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Block with 5% BSA or normal serum

• Incubate with anti-TMEM214 primary antibody (such as rabbit anti-TMEM214 antiserum raised against recombinant human TMEM214 amino acids 1-110)

• Detect with fluorophore-conjugated secondary antibodies

• Co-stain with organelle markers (e.g., Sec61β for ER, mitochondrial markers)

• Analyze using confocal microscopy

Cell Fractionation and Immunoblotting:

• Isolate distinct cellular fractions (cytosolic, membrane/ER, mitochondrial)

• Perform SDS-PAGE followed by immunoblotting with anti-TMEM214 antibodies

• Compare distribution across fractions using organelle-specific markers as controls

Trypsin Protection Assay:

• Isolate membrane fractions

• Treat with trypsin with or without membrane-disrupting detergents

• Analyze protection patterns by immunoblotting to determine protein orientation in membranes

Expression of Fluorescent Protein Fusions:

• Generate cherry-tagged or GFP-tagged TMEM214 constructs

• Express in Xenopus cells or microinject into embryos

• Analyze localization in live or fixed cells using confocal microscopy

• Co-express with organelle markers to confirm specific localization

How can researchers assess the functional activity of recombinant tmem214-b?

To assess the functional activity of recombinant tmem214-b, researchers should implement a multi-faceted approach based on its likely role in ER stress-induced apoptosis (by homology with human TMEM214):

ER Stress-Induced Apoptosis Assay:

• Transfect cells with recombinant tmem214-b or controls

• Treat with ER stress inducers such as thapsigargin (TG) or brefeldin A (BFA)

• Measure apoptosis using:

  • TUNEL assay

  • Annexin V/PI staining and flow cytometry

  • Caspase activity assays

• Compare apoptosis levels between tmem214-b-expressing and control cells

Procaspase 4 Interaction and Activation:

• Perform co-immunoprecipitation assays between tmem214-b and procaspase 4

• Analyze procaspase 4 cleavage/activation by immunoblotting

• Assess downstream markers of apoptosis (e.g., PARP-1 cleavage)

Domain Functionality Assessment:

• Generate truncated variants of tmem214-b lacking specific domains

• Express these variants in cells

• Assess their localization, procaspase 4 binding, and ability to induce apoptosis

• Compare with full-length protein to determine domain-specific functions

Assessment of ER Stress Response:

• Monitor unfolded protein response (UPR) markers including:

  • Bip/GRP78 induction

  • CHOP expression

  • JNK phosphorylation

• Determine whether tmem214-b affects these pathways or functions independently

How does tmem214-b compare between Xenopus laevis and other model organisms, including Xenopus tropicalis?

Comparative analysis of tmem214-b between Xenopus species and other model organisms provides important evolutionary and functional insights:

Sequence Conservation and Evolutionary Analysis:

When working with Xenopus laevis, researchers should note its pseudotetraploid genome, which often results in two copies of genes (S and L homeologs). Experimental design should account for potential functional redundancy between these homeologs . In contrast, Xenopus tropicalis has a diploid genome, potentially simplifying genetic manipulation and analysis .

For comparative studies, researchers can leverage the extensive genomic and genetic resources developed for both Xenopus species, including EST projects, full-length cDNA sequencing, and the Xenopus ORFeome project .

What is the role of tmem214-b in ER stress-induced apoptosis pathways in Xenopus models?

Based on studies of human TMEM214, the Xenopus homolog likely plays a critical role in ER stress-induced apoptotic pathways:

Mechanism of Action:

• TMEM214 localizes to the outer membrane of the ER through its transmembrane domains

• It constitutively associates with procaspase 4 via its N-terminal cytoplasmic region (aa 176-354)

• Upon ER stress, it facilitates the recruitment and activation of procaspase 4

• Activated caspase 4 initiates downstream apoptotic events including PARP-1 cleavage

Pathway-Specific Functions:

• TMEM214-mediated apoptosis appears independent of other ER stress pathways:

  • Does not affect CHOP induction

  • Does not influence JNK phosphorylation

  • Functions independently of Bip/GRP78 upregulation

Experimental Evidence in Cell Models:

• Overexpression of TMEM214 induces apoptosis

• Knockdown inhibits ER stress-induced apoptosis by ~30%

• Does not affect apoptosis induced by external factors (TNFα) or mitochondrial pathway activators (actinomycin D, etoposide)

To study this pathway specifically in Xenopus models, researchers should:

• Establish Xenopus cell lines or primary cultures

• Manipulate tmem214-b expression through overexpression or knockdown approaches

• Induce ER stress with agents like thapsigargin or brefeldin A

• Assess apoptotic markers and pathway components as described in earlier sections

How can researchers address contradictory findings when studying tmem214-b function?

When confronting contradictory findings in tmem214-b research, implement this structured methodological approach:

Systematic Validation Strategy:

• Control for Experimental Variables:

  • Protein preparation: Verify protein integrity through SDS-PAGE and ensure consistent reconstitution methods

  • Expression systems: Test whether E. coli vs. eukaryotic expression affects protein function

  • Cell type specificity: Evaluate tmem214-b function across multiple cell types, as correlation between TMEM214 levels and TG-induced apoptosis varies across cell lines

• Cross-validate with Multiple Techniques:

  • Combine gain-of-function (overexpression) with loss-of-function (knockdown) approaches

  • Employ both in vitro and in vivo models when possible

  • Use complementary assays to measure the same outcome (e.g., multiple apoptosis detection methods)

• Address Isoform-Specific Effects:

  • In X. laevis, distinguish between potential S and L homeologs (similar to dmrt1.S and dmrt1.L described in the context)

  • Generate and test isoform-specific reagents (antibodies, RNAi constructs)

  • Consider potential functional redundancy between homeologs

• Evaluate Pathway Interconnections:

  • Investigate potential crosstalk between tmem214-b-mediated pathways and other ER stress response mechanisms

  • Assess secondary effects on unfolded protein response (UPR) pathways

  • Determine whether contradictory findings might reflect cell-type specific pathway integration

• Standardize Quantification Methods:

  • Establish clear metrics for measuring protein function (e.g., percent apoptotic cells, caspase activation levels)

  • Use appropriate statistical analyses to determine significance

  • Report effect sizes to facilitate cross-study comparisons

How should researchers design experiments to study tmem214-b in Xenopus developmental contexts?

For studying tmem214-b in Xenopus developmental contexts, researchers should implement this comprehensive experimental design:

Developmental Expression Profiling:

• Extract RNA from multiple developmental stages (oocytes through tadpoles)

• Perform RT-qPCR to quantify tmem214-b expression levels across development

• Use in situ hybridization to determine tissue-specific expression patterns

• Complement with immunohistochemistry using anti-TMEM214 antibodies

Functional Manipulation Strategies:

• mRNA Injection (Gain-of-Function):

  • Synthesize capped mRNA encoding tmem214-b from expression plasmids

  • Microinject into fertilized eggs or early embryos at specified concentrations

  • Include lineage tracers (e.g., GFP mRNA) to track injected cells

  • Analyze phenotypic outcomes at appropriate developmental stages

• CRISPR/Cas9 Genome Editing (Loss-of-Function):

  • Design guide RNAs targeting tmem214-b

  • Inject Cas9 protein/gRNA complexes into fertilized eggs

  • Screen for mutations using Sanger sequencing or T7E1 assay

  • Establish lines with specific mutations for phenotypic analysis

• Dominant Negative Approaches:

  • Design constructs lacking key functional domains (e.g., N-terminal region)

  • Express these constructs to competitively inhibit endogenous protein function

  • Compare phenotypes with complete knockouts

Experimental Controls and Validation:

• Include appropriate controls (uninjected, control mRNA, control gRNA)

• Perform rescue experiments by co-injecting wild-type mRNA with gRNAs

• Validate knockouts/knockdowns at protein level when possible

• Consider potential compensation by related genes or pathways

What common challenges arise when working with recombinant tmem214-b and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant tmem214-b. Here are methodological solutions to address these issues:

Challenge 1: Protein Solubility and Aggregation

• Problem: Transmembrane proteins often aggregate during expression and purification

• Solutions:

  • Optimize buffer composition: Include mild detergents (0.1% Triton X-100 or 0.5% CHAPS)

  • Add stabilizing agents: 6% trehalose, 5-10% glycerol, or 1-2M urea

  • Adjust expression temperature: Lower to 16-18°C during induction

  • Consider fusion partners: MBP or SUMO tags can enhance solubility

  • For reconstitution, use the recommended deionized sterile water with 5-50% glycerol

Challenge 2: Antibody Specificity

• Problem: Cross-reactivity with related proteins or non-specific binding

• Solutions:

  • Validate antibodies using positive and negative controls

  • Consider raising custom antibodies against Xenopus-specific epitopes

  • Perform western blots on samples from knockdown experiments as controls

  • Pre-absorb antibodies with recombinant protein to improve specificity

Challenge 3: Distinguishing Function from Overexpression Artifacts

• Problem: Overexpression can cause non-physiological effects

• Solutions:

  • Use inducible expression systems with titratable promoters

  • Complement overexpression with knockdown/knockout approaches

  • Generate stable cell lines expressing near-endogenous levels

  • Include appropriate controls (inactive mutants, unrelated membrane proteins)

Challenge 4: Protein Degradation During Storage

• Problem: Loss of activity over time

• Solutions:

  • Strictly avoid repeated freeze-thaw cycles

  • Store in small aliquots at -80°C

  • Include protease inhibitors in storage buffers

  • Add stabilizing agents as recommended (50% glycerol, 6% trehalose)

  • Perform activity assays before experiments to verify function

How can advanced techniques be applied to study tmem214-b protein interactions and complex formation?

To characterize tmem214-b protein interactions and complex formation, researchers can employ these advanced methodological approaches:

Co-Immunoprecipitation and Pull-Down Assays:

• Express epitope-tagged tmem214-b (His-tag already present)

• Lyse cells under mild conditions to preserve protein-protein interactions

• Immunoprecipitate using tag-specific antibodies or anti-TMEM214 antibodies

• Analyze co-precipitated proteins by mass spectrometry

• Validate key interactions with reciprocal co-IPs and western blotting

Proximity Labeling Techniques:

• Generate BioID or TurboID fusions with tmem214-b

• Express in Xenopus cells or embryos

• Activate proximity labeling with biotin

• Purify biotinylated proteins and identify by mass spectrometry

• Map the local interactome at the ER membrane

FRET/BRET Interaction Analysis:

• Create fluorescent protein fusions (GFP/CFP and YFP/RFP pairs)

• Co-express potential interaction partners in cells

• Measure resonance energy transfer in live cells

• Quantify interaction strength and dynamics

• Map interaction domains using truncation mutants

Cryo-Electron Microscopy:

• Purify tmem214-b alone or in complex with interaction partners

• Prepare samples for cryo-EM analysis

• Collect and process image data

• Generate structural models of the protein and its complexes

• Identify key structural features mediating interactions

Domain Mapping and Mutagenesis:

• Generate tmem214-b constructs with specific domain deletions or mutations

• Assess the impact on protein interactions and function

• For example, the N-terminal cytoplasmic region (aa 176-354) is critical for procaspase 4 binding

• Map specific residues using alanine-scanning mutagenesis

• Validate functional importance through in vitro and cellular assays

What emerging technologies might advance our understanding of tmem214-b function?

Several cutting-edge methodologies show promise for deepening our understanding of tmem214-b function:

• Single-Cell Transcriptomics and Proteomics:

  • Enable mapping of tmem214-b expression patterns across different cell types during development

  • Reveal cell type-specific co-expression patterns to identify potential functional networks

  • Identify cell populations particularly sensitive to tmem214-b manipulation

• Organoid and Ex Vivo Models:

  • Develop Xenopus organoid systems to study tmem214-b in tissue-specific contexts

  • Apply genome editing in organoids to assess developmental and physiological roles

  • Create reporter lines to monitor ER stress responses in real-time

• Live-Cell Super-Resolution Microscopy:

  • Monitor tmem214-b dynamics and interactions at the ER membrane with nanometer precision

  • Track recruitment of procaspase 4 and other partners during ER stress responses

  • Visualize structural reorganization of ER membranes during apoptosis

• Integrative Multi-Omics Approaches:

  • Combine transcriptomics, proteomics, and metabolomics data from tmem214-b manipulated systems

  • Map the network of pathways affected by tmem214-b activity

  • Identify unexpected connections to other cellular processes

• In Silico Structural Prediction and Molecular Dynamics:

  • Apply AlphaFold or similar AI systems to predict tmem214-b structure

  • Simulate interactions with binding partners

  • Model conformational changes during activation

The continued development of the Xenopus ORFeome project will be particularly valuable, providing libraries designed for multiple applications including expression screening and proteomic analyses, ideally suited to studying transmembrane proteins like tmem214-b .

How might findings from tmem214-b research translate to understanding human disease mechanisms?

Research on Xenopus laevis tmem214-b has significant translational potential for understanding human disease mechanisms:

ER Stress-Related Pathologies:
Given the likely role of tmem214-b in ER stress-induced apoptosis (by homology with human TMEM214), findings may provide insights into diseases characterized by ER dysfunction, including:

• Neurodegenerative Disorders:

  • Alzheimer's, Parkinson's, and ALS feature ER stress and abnormal protein aggregation

  • Understanding tmem214-b's role in mediating ER stress responses could reveal new therapeutic targets

  • Modulation of tmem214-b function might protect neurons from ER stress-induced death

• Metabolic Diseases:

  • Obesity and diabetes involve ER stress in pancreatic β-cells and adipocytes

  • Investigating how tmem214-b regulates ER homeostasis could uncover new disease mechanisms

  • Finding specific small-molecule modulators of tmem214-b function might have therapeutic potential

• Cancer Biology:

  • Cancer cells often display altered ER stress responses to survive under challenging conditions

  • Targeting tmem214-b might selectively enhance apoptosis in cancer cells experiencing ER stress

  • Understanding how tumors modulate this pathway could reveal resistance mechanisms

Comparative Approaches:

• Study differences between Xenopus and human TMEM214 to identify evolutionarily conserved functional elements

• Use Xenopus as a model system for high-throughput screening of compounds targeting tmem214-b/TMEM214

• Validate findings in human cell systems to establish clinical relevance

The availability of genetic tools in Xenopus, including CRISPR/Cas9 genome editing and gynogenetic screens, provides powerful approaches for functional validation of disease-associated variants . These studies could significantly enhance our understanding of how TMEM214 dysfunction contributes to human disease mechanisms.