Recombinant Chicken Transmembrane protein 173 (TMEM173)

How do expression systems affect the quality and functionality of recombinant Chicken TMEM173?

Different expression systems produce recombinant Chicken TMEM173 with varying characteristics that can significantly impact experimental outcomes. Current research supports the use of multiple systems, each with distinct advantages:

For functional studies investigating TMEM173's role in innate immune signaling, mammalian or baculovirus expression systems are generally preferred due to their ability to produce protein with native-like post-translational modifications . When selecting recombinant Chicken TMEM173, researchers should consider the specific requirements of their experimental design and choose an expression system that provides the appropriate balance between yield, cost, and biological relevance.

What are the optimal methods for validating the functional activity of recombinant Chicken TMEM173?

Validating the functional activity of recombinant Chicken TMEM173 requires a multi-faceted approach that confirms both structural integrity and signaling capability. The following methodological workflow is recommended:

Western Blot Analysis

Confirm the identity and integrity of the recombinant protein using validated antibodies. The expected molecular weight of Chicken TMEM173 is approximately 42 kDa (calculated) though the observed weight may be around 37 kDa due to the specific distribution of charged residues .

Cyclic Dinucleotide Binding Assay

Assess binding to cyclic di-GMP or cyclic GMP-AMP using isothermal titration calorimetry or microscale thermophoresis. TMEM173 function depends on its ability to recognize these bacterial second messengers or host-derived cyclic dinucleotides .

Downstream Signaling Activation

Transfect the recombinant protein into chicken cell lines (such as DF-1 fibroblasts or HD11 macrophage-like cells) and measure:

• Phosphorylation of TBK1 and IRF3 by Western blot

• Type I interferon expression by qRT-PCR

• Interferon-stimulated gene expression by RNA-seq or qRT-PCR array

Reconstitution Assay

For the most definitive validation, use CRISPR/Cas9 to generate TMEM173-knockout chicken cell lines, then reconstitute with the recombinant protein and challenge with DNA stimulants such as poly(dA:dT) or cyclic dinucleotides, measuring the restoration of interferon responses.

How does Chicken TMEM173 differ from mammalian STING in structure and function?

Chicken TMEM173 shares fundamental mechanisms with mammalian STING but exhibits species-specific differences that impact experimental design and interpretation. Comparative analysis reveals:

Ligand Specificity

While both chicken and mammalian TMEM173 recognize cyclic dinucleotides, there are subtle differences in binding affinities and specificities. Mammalian STING exhibits a preference for 2'-3' linked cGAMP compared to other linkage isomers, but the exact preference profile of chicken TMEM173 requires specific characterization for each experimental system .

Downstream Signaling Pathway Variations

The core signaling pathway (STING-TBK1-IRF3) is conserved, but chicken TMEM173 activates avian-specific interferon genes and potentially interacts with different regulatory proteins in the signaling cascade. When designing experiments to study chicken TMEM173, researchers should use avian-specific primers and antibodies rather than assuming cross-reactivity with mammalian reagents.

What experimental approaches can identify TMEM173-interacting proteins in avian systems?

Identifying TMEM173-interacting proteins in avian systems requires specialized approaches that account for the unique characteristics of avian cellular components. The following methodological workflow has proven effective:

Co-Immunoprecipitation with Recombinant Tagged Protein

Using recombinant Chicken TMEM173 with affinity tags (such as His, FLAG, or biotinylated Avi-tag), perform co-immunoprecipitation from chicken cell lysates before and after stimulation with cyclic dinucleotides or DNA ligands . This approach can identify both constitutive and stimulus-dependent interactions.

Proximity Labeling Techniques

BioID or TurboID fusion proteins expressed in chicken cell lines can identify proximal proteins through biotinylation, providing insights into the TMEM173 interactome in its native cellular context. This approach is particularly valuable for mapping transient interactions in the STING signaling pathway.

Crosslinking Mass Spectrometry

Chemical crosslinking combined with mass spectrometry analysis can capture both stable and transient interactions, providing structural information about the orientation of interacting proteins within the complex.

Yeast Two-Hybrid Screening

Using a chicken cDNA library, screen for interactions with different domains of TMEM173. While this approach may yield false positives, it can identify direct binary interactions that might be missed by other methods.

The protein interaction data should be validated using reciprocal co-immunoprecipitation, functional assays measuring interferon production, and microscopy to confirm co-localization in relevant cellular compartments.

What are the best cell models for studying Chicken TMEM173 function?

Selecting appropriate cell models is crucial for studying Chicken TMEM173 function. The following cellular systems offer distinct advantages for different research questions:

Established Chicken Cell Lines

• DF-1 fibroblasts: Immortalized chicken embryo fibroblasts with well-characterized interferon responses

• HD11 cells: Macrophage-like cell line that expresses pattern recognition receptors and mounts robust innate immune responses

• DT40 cells: B-cell derived line amenable to genetic manipulation for creating knockout models

Primary Chicken Cells

• Chicken bone marrow-derived macrophages: Physiologically relevant model for studying innate immune responses

• Chicken embryonic fibroblasts: Primary cells that maintain normal signaling pathways

• Chicken dendritic cells: Critical for understanding TMEM173's role in linking innate and adaptive immunity

Experimental Considerations

When working with recombinant Chicken TMEM173 in these cell models, researchers should:

• Optimize transfection protocols specific to avian cells (electroporation often yields better results than lipid-based methods)

• Use chicken-specific qPCR primers when measuring interferon and ISG responses

• Consider the temperature difference for avian cells (39-40°C rather than 37°C used for mammalian cells)

How can researchers measure the activation of Chicken TMEM173 signaling pathways?

Measuring activation of Chicken TMEM173 signaling pathways requires assays that capture different stages of the signaling cascade. The following comprehensive approach is recommended:

Conformational Changes in TMEM173

• FRET-based assays: Fusion proteins with fluorescent tags can detect the conformational changes that occur upon ligand binding

• Limited proteolysis: Differential sensitivity to proteolytic digestion can reveal structural changes upon activation

Trafficking and Localization

• Immunofluorescence microscopy: Track the movement of TMEM173 from the endoplasmic reticulum to ERGIC and other compartments following stimulation

• Subcellular fractionation: Biochemical approach to quantify the redistribution of TMEM173 between cellular compartments

Phosphorylation Events

• Phospho-specific Western blotting: Detect phosphorylation of TBK1 (Ser172) and IRF3 (Ser396) as indicators of pathway activation

• Phos-tag SDS-PAGE: Enhanced resolution of phosphorylated TMEM173 species

Downstream Gene Expression

• Luciferase reporter assays: Measure interferon promoter activity

• qRT-PCR: Quantify expression of type I interferons and interferon-stimulated genes

• RNA-seq: Comprehensive analysis of the transcriptional response to TMEM173 activation

Functional Readouts

• Antiviral state assessment: Challenge cells with vesicular stomatitis virus expressing GFP (VSV-GFP) and measure viral replication as a functional readout of interferon responses

• Cytokine production: Measure secreted interferons using ELISA or bioassays specific for avian interferons

What are the critical parameters for optimizing recombinant Chicken TMEM173 purification?

Purification of recombinant Chicken TMEM173 presents unique challenges due to its transmembrane domains. The following critical parameters should be considered:

Detergent Selection and Concentration

• Critical micelle concentration (CMC): Use detergents at concentrations above their CMC but not excessively high to avoid protein denaturation

• Detergent type: Mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin better preserve protein structure compared to harsh detergents like SDS

• Detergent exchange: Consider gradually exchanging to amphipols or nanodiscs for long-term stability

Buffer Optimization

• pH range: Test pH 7.0-8.0 to identify optimal conditions for protein stability

• Salt concentration: 150-300 mM NaCl typically provides optimal stability while minimizing aggregation

• Glycerol content: 5-10% glycerol can enhance protein stability during storage

Purification Strategy

For E. coli-expressed protein :

• Solubilize inclusion bodies if necessary

• Perform affinity chromatography (IMAC for His-tagged protein)

• Apply ion exchange chromatography to remove contaminating proteins

• Finish with size exclusion chromatography to obtain homogeneous protein

For mammalian or insect cell-expressed protein :

• Optimize cell lysis conditions to extract membrane proteins

• Use affinity purification with appropriate detergent

• Consider on-column detergent exchange to more stable detergents

• Apply size exclusion chromatography as a final polishing step

Quality Control Metrics

• Size exclusion chromatography: Monodisperse peak indicating homogeneous protein

• Dynamic light scattering: Measure particle size distribution to detect aggregation

• Thermal shift assay: Assess protein stability under different buffer conditions

• Functional assays: Confirm cyclic dinucleotide binding capability

How can researchers investigate the role of post-translational modifications in Chicken TMEM173 function?

Post-translational modifications (PTMs) significantly impact TMEM173 function. To investigate their role in Chicken TMEM173, consider the following methodological approaches:

Identification of PTMs

• Mass spectrometry: Perform LC-MS/MS analysis on purified recombinant Chicken TMEM173 from different expression systems to identify phosphorylation, ubiquitination, and other modifications

• Western blotting: Use modification-specific antibodies (phospho, ubiquitin, SUMO) to detect modified forms

• Phos-tag SDS-PAGE: Specifically separate phosphorylated protein species for downstream analysis

Functional Analysis of PTM Sites

• Site-directed mutagenesis: Generate alanine or phosphomimetic (S/T to D/E) mutations at predicted modification sites

• Expression in chicken cells: Compare activity of wild-type vs. mutant proteins using reporter assays

• Domain-specific modifications: Map modifications to specific functional domains (transmembrane, cyclic dinucleotide binding, IRF3 interaction)

Temporal Dynamics of Modifications

• Pulse-chase experiments: Track the kinetics of modification following stimulation

• Inhibitor studies: Use kinase, E3 ligase, or deubiquitinase inhibitors to block specific modification pathways

• Subcellular localization: Correlate modifications with trafficking through cellular compartments

Cross-Species Comparison

Compare modification patterns between chicken, human, and mouse TMEM173 to identify conserved regulatory mechanisms and avian-specific modifications that may account for functional differences.

What strategies can address challenges in structural studies of Chicken TMEM173?

Structural studies of membrane proteins like Chicken TMEM173 present significant challenges. The following strategies can help overcome these obstacles:

Expression Optimization for Structural Studies

• Construct design: Generate truncated constructs removing flexible regions while preserving core functional domains

• Fusion partners: Use thermostabilizing fusion proteins such as T4 lysozyme or BRIL to enhance crystallization properties

• Expression screening: Test multiple expression systems (E. coli, insect cells, mammalian cells) with varying induction conditions

Membrane Protein Stabilization

• Lipid nanodisc reconstitution: Incorporate purified protein into nanodiscs with defined lipid composition mimicking the ER membrane

• Stabilizing ligands: Co-purify with cyclic dinucleotide ligands to stabilize the binding pocket

• Thermostabilizing mutations: Introduce mutations that enhance thermal stability without compromising function

Structural Determination Approaches

• X-ray crystallography: For high-resolution structures of individual domains or stabilized full-length protein

• Cryo-electron microscopy: Particularly valuable for capturing different conformational states of the full-length protein

• Nuclear magnetic resonance (NMR): For dynamic studies of specific domains and ligand interactions

• Cross-linking mass spectrometry: To obtain distance constraints for molecular modeling

Computational Approaches

• Molecular dynamics simulations: Predict conformational changes upon ligand binding

• Homology modeling: Leverage existing structures of human STING to predict chicken TMEM173 structure

• AlphaFold2 prediction: Generate computational models as starting points for experimental validation

Combining these approaches can provide structural insights into Chicken TMEM173 that inform understanding of species-specific differences in innate immune signaling pathways.