Recombinant Mouse Transmembrane and coiled-coil domain-containing protein 4 (Tmco4)

What is mouse Transmembrane and coiled-coil domain-containing protein 4 (Tmco4)?

Mouse Tmco4, like its human ortholog, is a protein-coding gene that produces a transmembrane protein. It is characterized by multiple transmembrane domains and coiled-coil regions. The protein is predicted to cross the endoplasmic reticulum membrane three times, with the N-terminus likely residing in the cytosol . Mouse Tmco4 shares significant homology with human TMCO4, which consists of 634 amino acids in its most common variant and has a molecular weight of approximately 67.9 kilodaltons with an isoelectric point of 5.48 .

What is the genomic organization of mouse Tmco4?

Based on the human ortholog, mouse Tmco4 likely consists of multiple exons that can produce various transcript variants through alternative splicing. In humans, TMCO4 contains 16 exons and generates 20 different mRNA transcript variants (X1-X20) through different combinations of these exons . The mouse gene likely has a similar structure, though exact details may vary between species. The gene is located on chromosome 1 in humans, but researchers should verify the chromosomal location in mice using genome databases.

What are the key structural features of mouse Tmco4 protein?

Mouse Tmco4 protein contains several important structural domains similar to human TMCO4. These include:

• Three transmembrane regions that anchor the protein in the endoplasmic reticulum membrane

• A large Abhydrolase region that may confer enzymatic activity

• Potential coiled-coil domains that facilitate protein-protein interactions

• Distinct cytosolic and lumenal domains with unique amino acid compositions

These structural features are critical for understanding the protein's potential functions and interactions within cellular environments.

What expression systems are most effective for producing recombinant mouse Tmco4?

For recombinant mouse Tmco4 production, bacterial expression systems using E. coli BL21(DE3) strains are commonly employed. The protocol typically involves:

• PCR amplification of the Tmco4 gene from mouse cDNA

• Cloning into a suitable expression vector (such as pET-28a) with appropriate restriction sites

• Transformation into competent E. coli cells

• Induction of protein expression using IPTG

• Purification using affinity chromatography methods

While bacterial systems are cost-effective, researchers should note that for transmembrane proteins like Tmco4, eukaryotic expression systems (insect or mammalian cells) might yield better results for proper folding and post-translational modifications, though this may increase production complexity and cost.

What purification strategies yield the highest purity of recombinant mouse Tmco4?

Purification of recombinant mouse Tmco4 typically involves multiple steps:

• Expression with a fusion tag (His-tag, GST, etc.) for affinity purification

• Cell lysis using mechanical disruption or detergent-based methods

• Initial purification using affinity chromatography (Ni-NTA for His-tagged proteins)

• Secondary purification using ion exchange or size exclusion chromatography

• Quality assessment using SDS-PAGE and Western blotting

For transmembrane proteins like Tmco4, detergent selection is critical. Mild non-ionic detergents like DDM or CHAPS may help maintain protein structure while solubilizing the membrane-bound protein . Carefully optimized purification protocols are essential to obtain high purity Tmco4 suitable for downstream applications.

How can I assess the quality and proper folding of purified recombinant mouse Tmco4?

Multiple complementary techniques should be employed to assess recombinant Tmco4 quality:

• SDS-PAGE for purity assessment and molecular weight confirmation

• Western blotting with specific antibodies for identity confirmation

• Circular dichroism (CD) spectroscopy to evaluate secondary structure elements

• Thermal shift assays to assess protein stability

• Limited proteolysis to examine folding state

• Functional assays based on predicted protein activity

For transmembrane proteins, additional techniques like detergent screening and size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can provide insights into the protein's oligomeric state and detergent binding .

What are the primary research applications for recombinant mouse Tmco4?

Recombinant mouse Tmco4 has several important research applications:

• Structural studies: Purified protein can be used for crystallography, cryo-EM, or NMR studies to elucidate its three-dimensional structure

• Functional characterization: In vitro assays to determine potential enzymatic activities associated with the Abhydrolase domain

• Interaction studies: Pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens to identify binding partners

• Antibody production: Immunization of animals to generate anti-Tmco4 antibodies for detection and localization studies

• Cancer research: Investigation of potential roles in cancer development based on interactions with cancer-associated proteins

These applications contribute to our understanding of Tmco4's biological functions and potential roles in disease processes.

How should I design immunization experiments using recombinant mouse Tmco4?

Based on immunization protocols for other recombinant proteins, an effective design for Tmco4 would include:

• Protein preparation: Use 50-100 μg of highly purified recombinant Tmco4 per immunization

• Adjuvant selection: Mix with Freund's complete adjuvant (primary) and incomplete adjuvant (boosters) in a 1:2 ratio

• Immunization schedule:

  • Primary immunization (day 0)

  • Boost immunizations (days 14, 28, and 42)

  • Blood collection for antibody testing (days 10, 24, 38, and 52)

• Route administration: Subcutaneous injection is recommended

• Antibody evaluation: Monitor antibody production using ELISA, testing both IgM and IgG responses

This protocol can be adapted based on specific research needs and animal model considerations.

What controls should be included when using recombinant mouse Tmco4 in experimental assays?

Rigorous experimental design requires several types of controls:

• Negative controls:

  • Buffer-only controls without Tmco4

  • Irrelevant protein of similar size/structure

  • Denatured Tmco4 (for structure-dependent assays)

• Positive controls:

  • Known binding partners (if established)

  • Related proteins with similar functions

• Validation controls:

  • Multiple detection methods (e.g., different antibodies)

  • Concentration gradients to establish dose-dependence

  • Competition assays with unlabeled protein

• Technical controls:

  • Multiple biological and technical replicates

  • Randomization of sample processing order

  • Blinding in subjective readout assays

These controls help establish specificity and reliability of results involving recombinant Tmco4.

How do post-translational modifications affect mouse Tmco4 function, and how can they be preserved in recombinant protein production?

While specific post-translational modifications (PTMs) of mouse Tmco4 are not well-characterized, transmembrane proteins often undergo glycosylation, phosphorylation, and other modifications critical for function. To study these:

• PTM identification: Use mass spectrometry to map modifications on native Tmco4 isolated from mouse tissues

• Expression system selection:

  • For glycosylation studies: Mammalian or insect cell expression systems

  • For phosphorylation: Co-expression with relevant kinases

• Modification preservation:

  • Use phosphatase inhibitors during purification to maintain phosphorylation states

  • Optimize purification conditions to maintain labile modifications

• Functional comparison: Compare native and recombinant protein activity to assess the impact of PTMs

The choice of expression system significantly influences the PTM profile, with mammalian systems providing the closest match to native modifications .

What approaches can resolve contradictory data regarding mouse Tmco4 protein-protein interactions?

When faced with contradictory interaction data for Tmco4, consider this systematic approach:

• Methodology evaluation: Different techniques (Y2H, co-IP, BioID, FRET) have distinct biases and limitations

• Experimental conditions: Assess buffer composition, detergent selection, and salt concentration effects

• Domain-specific interactions: Test individual domains versus full-length protein

• Cell-type specificity: Validate interactions in multiple cell types and in vivo models

• Quantitative analysis: Use quantitative techniques like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

• Competitive binding studies: Determine if interactions are mutually exclusive

• Structural validation: Use techniques like crosslinking mass spectrometry to identify interaction interfaces

This methodical approach can help reconcile discrepancies and build a coherent model of Tmco4's interactome .

How can I determine the subcellular localization and trafficking dynamics of mouse Tmco4?

A comprehensive approach to studying Tmco4 localization and trafficking includes:

• Fluorescent protein fusion: Generate Tmco4-GFP/RFP fusions (ensuring tag position doesn't disrupt localization signals)

• Live-cell imaging: Track protein movement using confocal microscopy or TIRF microscopy

• Immunofluorescence: Use anti-Tmco4 antibodies with organelle markers in fixed cells

• Biochemical fractionation: Isolate cellular compartments and detect Tmco4 by Western blot

• Electron microscopy: Use immunogold labeling for high-resolution localization

• Trafficking inhibitors: Apply specific inhibitors of cellular trafficking pathways

• Mutagenesis: Identify and mutate putative localization signals in the Tmco4 sequence

Based on its predicted structure, mouse Tmco4 likely localizes primarily to the endoplasmic reticulum membrane, but may also traffic to other compartments depending on cellular context .

What bioinformatic tools are most useful for predicting mouse Tmco4 function and structure?

A comprehensive bioinformatic analysis of Tmco4 should employ multiple tools:

Bioinformatic analysis suggests that mouse Tmco4, like its human ortholog, likely contains three transmembrane domains and a potential Abhydrolase domain that might confer enzymatic activity. Understanding these structural elements is crucial for designing functional assays .

How can I determine if mouse Tmco4 has enzymatic activity associated with its Abhydrolase domain?

To investigate potential enzymatic activity of the Abhydrolase domain in Tmco4:

• Substrate screening:

  • Test common abhydrolase substrates (p-nitrophenyl esters, thioesters)

  • Screen lipid-based substrates given membrane localization

  • Use substrate libraries with fluorescent or colorimetric readouts

• Activity assays:

  • Measure hydrolytic activity using spectrophotometric methods

  • Monitor product formation using chromatography techniques

  • Employ coupled enzyme assays for indirect detection

• Catalytic residue identification:

  • Identify putative catalytic triad using structural models

  • Generate point mutations in predicted active site residues

  • Compare wild-type and mutant activity levels

• Inhibitor studies:

  • Test effects of general and specific hydrolase inhibitors

  • Perform inhibitor kinetics to determine mechanism

This systematic approach can help characterize any enzymatic function of mouse Tmco4, which remains largely undefined in current literature .

What techniques can differentiate between direct and indirect protein interactions with mouse Tmco4?

Distinguishing direct from indirect interactions requires complementary approaches:

• In vitro binding assays:

  • Purified protein pull-downs with recombinant Tmco4

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI)

  • Isothermal titration calorimetry (ITC) for binding energetics

• Proximity-based methods:

  • FRET/BRET analysis with fluorescently tagged proteins

  • Crosslinking mass spectrometry to identify interaction interfaces

  • Split protein complementation assays (BiFC, PCA)

• Structural studies:

  • X-ray crystallography of protein complexes

  • Cryo-EM of larger assemblies

  • NMR studies of protein-protein interfaces

• In situ validation:

  • Proximity ligation assay (PLA) in fixed cells

  • APEX2-mediated proximity labeling

  • Co-localization with super-resolution microscopy

These approaches provide varying levels of confidence in distinguishing direct binding partners from components of the same complex that don't directly contact Tmco4 .

What evidence connects mouse Tmco4 to disease models, and how can recombinant protein be used to investigate these connections?

Based on the limited information about TMCO4 interactions with cancer-related proteins, researchers can investigate disease relevance through:

• Expression analysis:

  • Compare Tmco4 expression across healthy and diseased tissues

  • Analyze public databases (GEO, TCGA) for expression correlations

  • Perform immunohistochemistry on disease model tissues

• Functional studies:

  • Use recombinant Tmco4 to identify and validate interaction partners implicated in disease

  • Perform knockdown/knockout studies to assess phenotypic effects

  • Rescue experiments with wild-type and mutant recombinant protein

• Structural analysis:

  • Investigate how disease-associated mutations might affect protein structure

  • Use recombinant proteins with introduced mutations for comparative functional studies

• Therapeutic screening:

  • Use purified recombinant Tmco4 in high-throughput screening for modulators

  • Test candidate compounds in cellular and animal models

These approaches can help establish whether Tmco4 plays causal or consequential roles in disease processes .

How should I design experiments to investigate the potential role of mouse Tmco4 in cancer models?

Given the hints that human TMCO4 interacts with cancer-related proteins, a systematic investigation in mouse models would include:

• Expression profiling:

  • Compare Tmco4 levels across normal tissues, precancerous lesions, and tumors

  • Correlate expression with clinical outcomes in animal models

  • Analyze subcellular localization changes during malignant transformation

• Functional modulation:

  • Generate cell lines with Tmco4 knockdown, knockout, or overexpression

  • Assess effects on hallmark cancer phenotypes (proliferation, migration, apoptosis resistance)

  • Test in both in vitro and in vivo cancer models

• Mechanistic studies:

  • Identify cancer-relevant interaction partners using co-IP with recombinant Tmco4

  • Map signaling pathways affected by Tmco4 modulation

  • Investigate effects on specific oncogenic mechanisms

• Therapeutic exploration:

  • Test if recombinant Tmco4 fragments can act as competitive inhibitors

  • Generate antibodies against Tmco4 to block function

  • Screen for small molecules that modulate Tmco4 interactions

This research paradigm can help establish whether Tmco4 represents a potential therapeutic target or biomarker in cancer .

How can CRISPR-Cas9 technology be optimized for studying mouse Tmco4 function?

CRISPR-Cas9 offers powerful approaches for Tmco4 functional studies:

• Gene knockout strategies:

  • Design multiple sgRNAs targeting early exons

  • Create conditional knockout models (floxed alleles)

  • Generate tissue-specific knockouts to bypass potential embryonic lethality

• Knockin applications:

  • Introduce epitope tags for detection without antibodies

  • Create fluorescent protein fusions for live imaging

  • Introduce specific point mutations to test structural hypotheses

• Domain-specific editing:

  • Selectively delete transmembrane domains or the Abhydrolase region

  • Create chimeric proteins to test domain-specific functions

  • Introduce premature stop codons to create truncated proteins

• Transcriptional modulation:

  • Use CRISPRa/CRISPRi to modulate expression without genetic modification

  • Create inducible expression systems to control timing of modification

• Validation strategies:

  • Perform off-target analysis using whole-genome sequencing

  • Rescue experiments with recombinant protein to confirm specificity

  • Create multiple independent cell lines to control for clonal effects

These approaches can provide precise genetic tools for dissecting Tmco4 function in both cellular and animal models.

What emerging technologies offer new insights into mouse Tmco4 structure and interactions?

Several cutting-edge technologies are particularly promising for Tmco4 research:

• Cryo-electron microscopy (Cryo-EM):

  • Allows structural determination without crystallization

  • Particularly valuable for membrane proteins like Tmco4

  • Can capture different conformational states

• Integrative structural biology:

  • Combines multiple data sources (X-ray, NMR, EM, crosslinking)

  • Provides more complete structural models

  • Especially useful for dynamic or flexible regions

• Proximity labeling proteomics:

  • BioID, APEX2, or TurboID fusions to map the Tmco4 microenvironment

  • Identifies transient and stable interaction partners

  • Works in native cellular contexts

• Single-molecule techniques:

  • FRET for studying conformational changes

  • Single-molecule pull-down for stoichiometry determination

  • Optical tweezers for mechanical property analysis

• Artificial intelligence applications:

  • Improved structure prediction with AlphaFold2/RoseTTAFold

  • Network analysis for pathway integration

  • Virtual screening for modulators of Tmco4 function

These technologies can overcome limitations of traditional approaches to membrane protein research and provide unprecedented insights into Tmco4 biology.

What is the current state of mouse Tmco4 research, and what are the critical knowledge gaps?

The current understanding of mouse Tmco4 is limited, with several important knowledge gaps:

• Functional characterization: The precise biological function remains undetermined, particularly regarding the potential enzymatic activity of the Abhydrolase domain

• Physiological role: The importance of Tmco4 in normal development and homeostasis is poorly understood

• Interaction network: A comprehensive map of Tmco4 binding partners is lacking

• Regulation mechanisms: How Tmco4 expression and function are regulated remains unclear

• Disease relevance: Despite hints of cancer connections, specific roles in pathology are not well-established

Addressing these gaps requires a coordinated research approach combining biochemical, structural, genetic, and systems biology methodologies .

How should researchers prioritize experiments to advance understanding of mouse Tmco4?

A strategic research roadmap for advancing Tmco4 knowledge would include: