Recombinant Human Transmembrane protein 217 (TMEM217)

What is the basic structure and characteristics of human TMEM217?

Transmembrane protein 217 (TMEM217) is encoded by the TMEM217 gene located on chromosome 6 minus strand at position 6p21.2. The gene spans 46,857 base pairs and is flanked by TBC1D22B and PIM1 . The protein's longest isoform consists of 229 amino acids with a predicted molecular weight of 26.6 kDa and an isoelectric point at pH 9.3 . TMEM217 contains four transmembrane domains that primarily consist of uncharged amino acids arranged in predicted alpha helices, with both N-terminus and C-terminus facing the cytosol . A distinctive feature is the C-terminus containing a long predicted coiled tail extending from the final transmembrane domain . The protein contains the domain of unknown function DUF4534 between amino acids 11-171 .

How many isoforms of TMEM217 exist and what are their differences?

TMEM217 has three common isoforms that result from the alternative splicing of three exons. Isoform 1 translates to the longest polypeptide, consisting of 1590 nucleotides. The 5' untranslated region of isoform 1 is relatively short and is predicted to fold into several stem loop domains within conserved areas of the untranslated region . The detailed structural differences between these isoforms and their potential functional implications require further research, as current literature does not fully characterize all variants.

What are the predicted post-translational modifications of TMEM217?

Several predicted phosphorylation and glycosylation sites have been identified on TMEM217 in highly conserved regions of the protein. The phosphorylation sites are located primarily on the C-terminal tail . Additionally, there are two highly conserved cysteine residues that have the potential to form disulfide bonds, which may be critical for maintaining the protein's tertiary structure and function . These post-translational modifications likely play important roles in regulating TMEM217's activity, localization, and interactions with other proteins.

What is the expression pattern of TMEM217 across different tissues?

TMEM217 has been found to have expression correlated with the lymphatic system and endothelial tissues . Gene ontology (GO) annotations for TMEM217 include localization to the fibrillar center, integral component of membrane, and nucleolus . The specific expression patterns across different tissue types, developmental stages, and disease states remain areas requiring more comprehensive investigation.

How can researchers effectively detect endogenous TMEM217 expression in tissue samples?

For detecting endogenous TMEM217 in tissue samples, researchers can employ immunohistochemistry (IHC) or immunofluorescence (IF) using validated antibodies such as TMEM217 Polyclonal Antibody (CAC13969) . This antibody has been validated for ELISA, Western blot (WB), and immunofluorescence applications in human samples . For optimal results when performing IHC/ICC, it is recommended to use proper fixation protocols specific for membrane proteins, consider antigen retrieval methods, and validate antibody specificity using positive and negative controls. Blocking experiments can be conducted using recombinant protein control fragments, such as Human TMEM217 (aa 158-228), at a 100x molar excess compared to the antibody concentration .

What cellular compartments contain TMEM217 and how does this inform its function?

Based on gene ontology annotations, TMEM217 is an integral component of the membrane and has been associated with the fibrillar center and nucleolus . As a transmembrane protein with four predicted transmembrane domains, TMEM217 likely spans cellular membranes with both N-terminus and C-terminus facing the cytosol . This membrane localization suggests potential roles in signal transduction, membrane organization, or transport processes. The specific membrane systems (plasma membrane, endoplasmic reticulum, Golgi, etc.) where TMEM217 predominantly resides require further characterization to better understand its cellular functions.

What are the optimal expression systems for producing recombinant TMEM217?

For recombinant TMEM217 production, both prokaryotic (E. coli) and eukaryotic (HEK293) expression systems have been successfully employed . When selecting an expression system, researchers should consider:

For transmembrane proteins like TMEM217, eukaryotic expression systems generally provide better folding conditions and post-translational modifications that may be essential for proper function and interaction studies.

What purification strategies are most effective for recombinant TMEM217?

Purifying transmembrane proteins presents unique challenges due to their hydrophobic domains. For TMEM217, effective purification strategies include:

• Affinity chromatography: For His-tagged TMEM217, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective . The eluted protein should be immediately buffer-exchanged to remove imidazole.

• Detergent solubilization: Critical for maintaining protein solubility during extraction and purification. Mild detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin are preferable to harsher detergents like SDS that may denature the protein.

• Size exclusion chromatography: Useful as a polishing step to remove aggregates and obtain homogeneous protein preparations.

• Reconstitution into liposomes or nanodiscs: For functional studies, purified TMEM217 can be reconstituted into artificial membrane systems that mimic the native environment.

When evaluating purification success, researchers should assess both yield and functional integrity through activity assays and structural characterization methods.

How can researchers verify the functionality of recombinant TMEM217?

Verifying the functionality of recombinant TMEM217 requires multiple approaches since its precise function remains to be fully characterized. Recommended verification methods include:

• Structural integrity assessment: Circular dichroism (CD) spectroscopy to confirm proper secondary structure formation, particularly alpha-helical content expected in transmembrane domains.

• Membrane incorporation: Confirming proper membrane insertion using protease protection assays or fluorescence-based membrane incorporation assays.

• Binding partner identification: Pull-down assays or co-immunoprecipitation with predicted interaction partners, followed by mass spectrometry analysis.

• Phosphorylation status: Since TMEM217 has predicted phosphorylation sites , phosphorylation-specific antibodies or mass spectrometry can be used to verify these modifications.

• Functional reconstitution: For membrane proteins with unknown function, reconstitution into liposomes and assessment of various activities (e.g., ion flux, substrate transport) may reveal functional properties.

Since TMEM217 has been predicted to have functions linked to the cytoskeleton , assays examining cytoskeletal organization in cells overexpressing or depleted of TMEM217 could provide functional insights.

What experimental approaches can elucidate TMEM217's interaction with the cytoskeleton?

Given TMEM217's predicted function linked to the cytoskeleton , several experimental approaches can be employed to investigate this relationship:

• Co-immunoprecipitation (Co-IP): Using antibodies against TMEM217 to pull down potential cytoskeletal interaction partners, followed by mass spectrometry identification.

• Proximity labeling: Techniques such as BioID or APEX2 fusion proteins can identify proteins in close proximity to TMEM217 in living cells, potentially revealing transient cytoskeletal interactions.

• Fluorescence microscopy: Co-localization studies using fluorescently-tagged TMEM217 and cytoskeletal components (actin, microtubules, intermediate filaments) can reveal spatial relationships.

• Cytoskeleton disruption experiments: Treating cells with cytoskeleton-disrupting agents (cytochalasin D, nocodazole, etc.) and observing effects on TMEM217 localization and function.

• FRET/BRET assays: To detect direct interactions between TMEM217 and cytoskeletal proteins in living cells.

• Cell migration and adhesion assays: Since cytoskeletal dynamics underlie these processes, examining how TMEM217 overexpression or knockdown affects migration and adhesion could provide functional insights.

How can CRISPR-Cas9 technology be optimized for studying TMEM217 function?

CRISPR-Cas9 technology offers powerful approaches for investigating TMEM217 function through genome editing:

• Knockout generation: Complete deletion of TMEM217 can reveal its necessity for specific cellular processes. Design multiple sgRNAs targeting early exons to maximize knockout efficiency. For TMEM217, targeting the regions encoding the first transmembrane domain (approximately amino acids 11-171 containing the DUF4534 domain) would be effective.

• Knock-in strategies:

  • Endogenous tagging with fluorescent proteins or epitope tags

  • Introduction of specific mutations in predicted functional domains

  • Replacement with orthologous genes to study evolutionary conservation

• CRISPR interference/activation (CRISPRi/CRISPRa): For reversible modulation of TMEM217 expression without permanent genetic changes.

• Experimental design considerations:

  • Include proper controls (non-targeting gRNAs)

  • Verify editing efficiency through sequencing

  • Establish multiple independent clonal cell lines to control for off-target effects

  • Perform rescue experiments by re-expressing TMEM217 to confirm phenotype specificity

• Functional readouts: After successful editing, assess phenotypes related to:

  • Cytoskeletal organization and dynamics

  • Cell morphology and migration

  • Membrane organization and trafficking

  • Tissue-specific functions in endothelial or lymphatic contexts

What are the methodological considerations for restoring full biological activity to recombinant TMEM217?

Restoring full biological activity to recombinant TMEM217 presents challenges similar to those encountered with other membrane proteins. Drawing from approaches used with other transmembrane proteins :

• Membrane reconstitution strategies:

  • Liposome incorporation: The recombinant protein can be reconstituted into phospholipid vesicles that mimic the native membrane environment.

  • Nanodiscs: These provide a more controlled membrane environment and are amenable to structural studies.

  • Detergent-solubilized micelles: Useful for maintaining solubility while preserving some functional aspects.

• His-tag tethering approach: Similar to methods described for other membrane proteins , recombinant TMEM217 with a His-tag can be tethered to nickel-chelating lipids (NiPCPS) in phospholipid vesicles. This approach has been shown to restore activity to other membrane protein ectodomains by positioning them correctly at the membrane surface.

• Functional assessment: Verify restored activity through:

  • Binding assays with identified interaction partners

  • Structural integrity confirmation via CD spectroscopy or limited proteolysis

  • Functional assays specific to predicted cytoskeletal functions

• Optimization parameters:

  • Lipid composition: The lipid environment significantly affects membrane protein function

  • Protein-to-lipid ratio: Critical for proper incorporation and activity

  • Buffer conditions: pH, ionic strength, and presence of stabilizing agents

These approaches should be tailored to the specific predicted functions of TMEM217 in relation to cytoskeletal dynamics and endothelial/lymphatic tissue functions.

What signaling pathways might involve TMEM217 based on its structure and localization?

While specific signaling pathways involving TMEM217 have not been fully characterized, several possibilities can be inferred from its structural features and expression patterns:

• Cytoskeletal regulation pathways: Given TMEM217's predicted association with the cytoskeleton , it may participate in pathways that regulate cytoskeletal dynamics, potentially involving Rho GTPases, formins, or the Arp2/3 complex.

• Endothelial/lymphatic signaling: Based on its expression correlation with lymphatic and endothelial tissues , TMEM217 might participate in VEGF, Notch, or Tie receptor signaling pathways critical for vascular and lymphatic development and function.

• Membrane trafficking pathways: As a transmembrane protein with potential cytoskeletal links, TMEM217 could function in vesicular trafficking, endocytosis, or exocytosis pathways.

• Cell adhesion and junction formation: Many transmembrane proteins in endothelial tissues contribute to cell-cell adhesion and junction formation, suggesting TMEM217 might participate in these processes.

Research approaches to investigate these pathways include phosphoproteomic analysis following TMEM217 manipulation, transcriptomic profiling to identify affected pathways, and targeted inhibition of specific pathway components to determine epistatic relationships with TMEM217.

What protein-protein interaction studies would be most informative for characterizing TMEM217 function?

To characterize TMEM217 function through protein-protein interactions, the following approaches would be particularly informative:

• Unbiased interaction screening:

  • Yeast two-hybrid screening using the cytoplasmic domains as bait

  • Proximity labeling (BioID, APEX) to identify the interactome in cellular context

  • Affinity purification-mass spectrometry (AP-MS) using full-length or domain-specific constructs

• Candidate-based approaches:

  • Co-immunoprecipitation with potential cytoskeletal partners

  • FRET/BRET assays to detect direct interactions in living cells

  • Protein complementation assays for validation of specific interactions

• Domain-specific interaction mapping:

  • The C-terminal coiled tail domain extending from the final transmembrane domain is particularly interesting for interaction studies

  • The conserved DUF4534 domain (amino acids 11-171) represents another potential interaction interface

• Post-translational modification-dependent interactions:

  • Phosphorylation sites on the C-terminal tail suggest potential interactions with kinases, phosphatases, and phospho-binding proteins

  • Investigation of interaction changes upon phosphorylation/dephosphorylation

• Experimental design considerations:

  • Use both overexpression and endogenous protein approaches

  • Include appropriate controls for non-specific binding

  • Confirm interactions through multiple orthogonal methods

  • Validate functional relevance through mutagenesis of interaction interfaces

How might TMEM217 contribute to immune and inflammatory responses?

While TMEM217 hasn't been directly implicated in immune responses, several transmembrane proteins play crucial roles in these processes. Investigating TMEM217's potential role in immunity would be valuable, particularly given:

• Expression pattern relevance: TMEM217's expression in lymphatic and endothelial tissues places it in contexts critical for immune cell trafficking and inflammatory responses.

• Experimental approaches to investigate immune functions:

  • Gene expression analysis in immune-activated endothelial cells

  • Effects of TMEM217 modulation on immune cell adhesion and transmigration

  • Analysis of inflammatory cytokine production and response in TMEM217-manipulated cells

  • Assessment of TMEM217 regulation during inflammatory conditions

• Potential connections to inflammatory pathways:

  • Cytokine-induced alterations in TMEM217 expression or localization

  • TMEM217's impact on NF-κB, JAK/STAT, or other inflammatory signaling pathways

  • Effects on endothelial barrier function during inflammation

• Therapeutic implications: If TMEM217 plays roles in inflammatory responses or immune cell interactions, it could represent a novel target for inflammatory disorders or cancer immunotherapy approaches.

Similar to other transmembrane proteins , TMEM217 could potentially influence inflammatory pathways by modulating cytokine receptor signaling, affecting cell adhesion molecules, or participating in immune checkpoint regulation.

What approaches can be used to develop specific inhibitors or modulators of TMEM217?

Developing specific inhibitors or modulators for TMEM217 requires systematic approaches:

• Target validation strategies:

  • Confirm TMEM217's role in specific biological processes through genetic approaches (knockout, knockdown)

  • Identify phenotypes amenable to pharmacological modulation

  • Determine the most druggable domains or interactions

• Small molecule screening approaches:

  • High-throughput screening using cell-based functional assays

  • Fragment-based drug discovery targeting specific domains

  • Structure-based virtual screening if structural data becomes available

• Biologics development:

  • Monoclonal antibodies targeting extracellular loops

  • Peptide inhibitors designed against interaction interfaces

  • Aptamers selected for specific binding to TMEM217

• Assay development for screening:

  • Cell-based reporter assays monitoring downstream signaling

  • Binding assays using purified protein domains

  • Phenotypic screens based on identified TMEM217 functions

• Lead optimization considerations:

  • Specificity profiling against related transmembrane proteins

  • ADME properties optimization

  • Activity assessment in physiologically relevant cell types

The lack of detailed functional information about TMEM217 currently limits targeted drug design, making phenotypic screening approaches particularly valuable in early discovery efforts.

How can researchers design blocking antibodies against TMEM217?

Designing effective blocking antibodies against TMEM217 requires strategic considerations:

• Epitope selection strategy:

  • Target extracellular loops between transmembrane domains

  • Prioritize regions involved in protein-protein interactions

  • Consider conserved regions for broad species reactivity or species-specific regions for selectivity

• Immunization approaches:

  • Recombinant protein fragments corresponding to extracellular domains

  • Synthetic peptides representing specific epitopes

  • DNA immunization encoding the extracellular portion

  • Cell-based immunization with TMEM217-overexpressing cells

• Screening and validation methodology:

  • ELISA-based binding screens

  • Flow cytometry to confirm binding to native protein

  • Functional assays to identify antibodies with blocking activity

  • Epitope mapping to characterize binding sites

• Engineering considerations:

  • Humanization for potential therapeutic applications

  • Affinity maturation to enhance potency

  • Format selection (IgG, Fab, scFv) based on application needs

• Quality control and validation:

  • Specificity testing using TMEM217 knockout cells as negative controls

  • Cross-reactivity assessment with related proteins

  • Stability and binding kinetics characterization

Commercial antibodies such as TMEM217 Polyclonal Antibody (CAC13969) can serve as useful tools for initial studies, while custom antibody development may be necessary for specific blocking applications.

How conserved is TMEM217 across species and what does this suggest about its function?

Evolutionary conservation analysis of TMEM217 provides important functional insights:

Researchers studying TMEM217 should consider these evolutionary patterns when designing experiments and interpreting results across model organisms.

What can structural predictions and modeling reveal about TMEM217 function?

In the absence of experimental structures, computational modeling can provide valuable insights into TMEM217:

• Transmembrane domain prediction and modeling:

  • Hydrophobicity analysis confirms four transmembrane domains

  • Alpha-helical wheel projections can reveal amphipathic characteristics

  • Molecular dynamics simulations of transmembrane domains in lipid bilayers

• Structure prediction approaches:

  • Template-based modeling using structurally characterized transmembrane proteins

  • Ab initio modeling for unique domains like DUF4534

  • Integration of co-evolutionary information to constrain models

• Functional site prediction:

  • Identification of potential binding pockets

  • Mapping of conserved residues onto structural models

  • Electrostatic surface analysis to identify potential interaction sites

• Post-translational modification site analysis:

  • Structural context of predicted phosphorylation sites on the C-terminal tail

  • Accessibility assessment of potential glycosylation sites

  • Modeling effects of modifications on protein conformation

• Protein-protein interaction interface prediction:

  • Docking simulations with predicted interaction partners

  • Identifying surface patches with characteristics of protein interfaces

These computational approaches can guide experimental design by generating testable hypotheses about TMEM217 structure-function relationships.

What disease associations have been reported for TMEM217 and how can they be further investigated?

While specific disease associations for TMEM217 are not explicitly reported in the provided search results, investigating potential disease connections requires systematic approaches:

• Bioinformatic mining of disease databases:

  • GWAS catalog analysis for TMEM217 locus associations

  • Analysis of somatic mutations in cancer genomics databases

  • Gene expression changes in transcriptomic disease datasets

  • Pathway enrichment analysis to identify disease-relevant processes

• Experimental approaches to investigate disease relevance:

  • Expression analysis in disease versus normal tissues

  • Functional studies in disease-relevant cell types

  • Patient-derived sample analysis (mutations, expression changes)

  • Animal models with TMEM217 manipulation in disease contexts

• Potential disease areas for investigation based on TMEM217 characteristics:

  • Vascular and lymphatic disorders (based on expression pattern)

  • Cytoskeletal-related diseases (based on predicted function)

  • Inflammatory conditions (based on tissue expression)

  • Cancer progression (many transmembrane proteins affect cell adhesion and migration)

• Translational research considerations:

  • Biomarker potential assessment

  • Therapeutic target validation

  • Patient stratification based on TMEM217 status

How might TMEM217 be leveraged for therapeutic applications?

Exploring TMEM217's therapeutic potential requires systematic evaluation:

• Target validation approaches:

  • Genetic manipulation in disease models

  • Antibody-mediated blocking studies

  • Expression correlation with disease severity or prognosis

• Potential therapeutic modalities:

  • Monoclonal antibodies targeting extracellular regions

  • Small molecule inhibitors of specific functions

  • Antisense oligonucleotides or siRNAs for expression modulation

  • Gene therapy approaches for loss-of-function contexts

• Application-specific considerations:

  • For vascular/lymphatic disorders: targeting endothelial TMEM217

  • For inflammatory conditions: modulating immune cell-endothelial interactions

  • For cytoskeletal disorders: affecting specific structure-function relationships

• Drug development pathway planning:

  • Assay development for high-throughput screening

  • Lead optimization strategies

  • Translational models for preclinical validation

  • Biomarker strategy for patient selection

• Delivery challenges and solutions:

  • Tissue-specific targeting approaches

  • Membrane protein accessibility considerations

  • Pharmacokinetic optimization

While specific therapeutic applications for TMEM217 remain to be determined, its transmembrane nature and tissue expression pattern suggest potential as a drug target if disease relevance is established.

What are the key resources available for TMEM217 research?

Researchers investigating TMEM217 can access several key resources:

• Commercial reagents:

  • Antibodies: TMEM217 Polyclonal Antibody (CAC13969) validated for ELISA, WB, and IF applications

  • Recombinant proteins: Human TMEM217 (aa 158-228) Control Fragment , Full Length Human TMEM217 protein with His-tag

  • cDNA clones: Rattus norvegicus TMEM217 gene cDNA ORF clone

• Database resources:

  • UniProt ID: Q8N7C4 (Human TMEM217)

  • Entrez Gene ID: 221468 (Human) , 687483 (Rat)

  • Previous identifiers: C6orf128, dJ355M6.2

• Bioinformatic tools relevant for transmembrane protein analysis:

  • TMHMM/Phobius for transmembrane domain prediction

  • NetPhos for phosphorylation site prediction

  • NetNGlyc/NetOGlyc for glycosylation site prediction

  • PSIPRED for secondary structure prediction

• Experimental protocols adapted for transmembrane proteins:

  • Detergent solubilization methods

  • Membrane protein expression systems

  • Reconstitution approaches

• Genetic tools:

  • CRISPR-Cas9 reagents for gene editing

  • siRNA/shRNA resources for knockdown studies

Researchers should note that while some resources are commercially available, comprehensive tool development specifically for TMEM217 remains an active area for development.

What considerations are important when selecting antibodies for TMEM217 detection?

Selecting appropriate antibodies for TMEM217 detection requires careful consideration:

• Epitope location and accessibility:

  • Antibodies targeting extracellular loops are ideal for flow cytometry and live-cell applications

  • C-terminal epitopes are accessible in fixed and permeabilized samples

  • N-terminal epitopes may be less accessible due to protein topology

• Validation status assessment:

  • Verify antibody validation for specific applications (WB, IF, IP, ELISA)

  • Evaluate validation methods (knockout controls, overexpression, peptide blocking)

  • Consider validation in relevant tissue/cell contexts

• Application-specific selection criteria:

  • For Western blotting: Ability to recognize denatured epitopes

  • For immunoprecipitation: Affinity and specificity under native conditions

  • For immunohistochemistry: Compatibility with fixation methods

  • For flow cytometry: Recognition of native conformation

• Controls and validation approaches:

  • Use recombinant protein controls like Human TMEM217 (aa 158-228) Control Fragment

  • Perform blocking experiments with a 100x molar excess of protein fragment control

  • Include TMEM217 knockout or knockdown samples as negative controls

• Available options:

  • TMEM217 Polyclonal Antibody (CAC13969) has been validated for ELISA, WB, and IF applications in human samples

  • Consider species reactivity needs based on research model (human antibodies show ~30% sequence identity with mouse/rat orthologs)

Proper antibody selection and validation are critical for generating reliable TMEM217 research data.

What are the most pressing questions about TMEM217 that remain to be answered?

Several critical knowledge gaps regarding TMEM217 present opportunities for high-impact research:

• Fundamental function determination:

  • What is the precise molecular function of TMEM217?

  • How does its predicted cytoskeletal association manifest functionally?

  • What are the binding partners and interaction network of TMEM217?

• Physiological role clarification:

  • What is TMEM217's role in lymphatic and endothelial tissues?

  • How is TMEM217 expression regulated during development and in response to stimuli?

  • What phenotypes result from TMEM217 deficiency in model organisms?

• Structural biology questions:

  • What is the three-dimensional structure of TMEM217?

  • How do the four transmembrane domains organize in the membrane?

  • What structural changes occur upon post-translational modification?

• Disease relevance exploration:

  • Is TMEM217 dysregulated in specific diseases?

  • Do TMEM217 variants contribute to disease susceptibility?

  • Could TMEM217 serve as a biomarker or therapeutic target?

• Experimental technology development:

  • What are optimal expression and purification systems for functional TMEM217?

  • How can TMEM217 activity be measured in high-throughput formats?

  • What model systems best recapitulate TMEM217 physiology?

Addressing these questions will require interdisciplinary approaches combining molecular biology, structural biology, cell biology, and systems biology methods.

What novel techniques might advance our understanding of TMEM217 function?

Emerging technologies that could significantly advance TMEM217 research include:

• Advanced structural biology approaches:

  • Cryo-electron microscopy for membrane protein structure determination

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Single-particle tracking for membrane organization and dynamics

  • Native mass spectrometry for intact complex analysis

• Genome editing innovations:

  • Base editing for precise point mutations

  • Prime editing for targeted sequence replacements

  • Multiplexed CRISPR screening for functional genomics

  • CRISPRi/CRISPRa for reversible expression modulation

• Protein interaction technologies:

  • Proximity labeling advances (TurboID, Split-TurboID)

  • Optical control of protein interactions (optogenetics)

  • Single-molecule fluorescence methodologies

  • Protein complementation assays with improved sensitivity

• Imaging innovations:

  • Super-resolution microscopy techniques (STORM, PALM, STED)

  • Live-cell imaging with improved spatiotemporal resolution

  • Correlative light and electron microscopy

  • Expansion microscopy for detailed subcellular localization

• Systems biology approaches:

  • Multi-omics integration for network-level understanding

  • Machine learning for pattern recognition in large datasets

  • Spatial transcriptomics/proteomics for tissue context

  • Computational modeling of membrane protein dynamics