Recombinant Mouse Transmembrane protein ENSP00000343375 homolog

What is Recombinant Mouse Transmembrane protein ENSP00000343375 homolog?

Recombinant Mouse Transmembrane protein ENSP00000343375 homolog (Q497K7) is a full-length protein (1-211aa) that belongs to the transmembrane protein family. It is commonly expressed with an N-terminal His-tag in E. coli expression systems. The protein is also known as Tmem247 (Transmembrane protein 247) and is derived from Mus musculus (mouse) . This protein represents an important model for studying transmembrane domain structure and function, particularly in the context of cellular signaling and membrane organization.

How should researchers store and reconstitute Recombinant Mouse TMEM247?

For optimal results when working with Recombinant Mouse TMEM247, researchers should follow these methodological guidelines:

Storage Protocol:

• Store the lyophilized protein at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to prevent protein degradation

• Avoid repeated freeze-thaw cycles as this can compromise protein integrity

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquot for long-term storage at -20°C/-80°C

The protein is typically supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain stability during storage and reconstitution processes.

What expression systems are optimal for producing Recombinant Mouse TMEM247?

When designing experiments to express Recombinant Mouse TMEM247, researchers should consider multiple expression systems based on their specific experimental needs:

E. coli Expression System:

• Most commonly used for TMEM247 production

• Advantages: High yield, cost-effective, rapid expression

• Limitations: Lack of post-translational modifications, potential for improper folding of complex transmembrane proteins

• Recommended for: Structural studies, antibody production, protein-protein interaction assays

Mammalian Expression Systems:

• Alternative for more native-like protein production

• Advantages: Proper post-translational modifications, correct folding of mammalian transmembrane proteins

• Limitations: Lower yield, higher cost, longer production time

• Recommended for: Functional studies, cell signaling experiments, subcellular localization studies

For structural biology applications, researchers might consider recent advances in de novo design strategies for transmembrane domains that have been successfully applied to other transmembrane proteins . These approaches could potentially inform experimental design when working with TMEM247.

How can researchers verify the oligomeric state of Recombinant Mouse TMEM247?

Determining the oligomeric state of transmembrane proteins is crucial for understanding their function. Researchers can employ several complementary methods:

Size Exclusion Chromatography (SEC):

• Useful for initial assessment of protein oligomerization

• Sample preparation: Reconstitute protein in appropriate detergent buffer

• Analysis: Compare elution profile with known molecular weight standards

• Limitations: Detergent micelles can affect apparent molecular weight

SDS-PAGE Analysis:

• Standard method for purity assessment and preliminary oligomeric state determination

• Protocol: Both reducing and non-reducing conditions should be tested

• Expected result: TMEM247 shows >90% purity by SDS-PAGE

Advanced Structural Methods:

• X-ray crystallography: Can provide definitive oligomeric state information if crystals can be obtained

• Similar to the approaches used for de novo designed transmembrane domains in recent studies

• Native mass spectrometry: Emerging technique for membrane protein oligomeric state determination

Researchers should note that transmembrane proteins may form different oligomeric states depending on the experimental conditions, including detergent choice, lipid environment, and protein concentration.

How can Recombinant Mouse TMEM247 be utilized in chimeric receptor design studies?

Recent advances in de novo design of transmembrane domains provide insights for researchers interested in using TMEM247 in engineered receptor systems:

Potential Research Applications:

• TMEM247 could be incorporated into chimeric antigen receptors (CARs) similar to other designed transmembrane domains

• The oligomeric state of the transmembrane domain can significantly impact receptor signaling, as demonstrated with other transmembrane proteins

• Researchers could investigate whether TMEM247's transmembrane domain exhibits specific oligomerization properties that might be useful in receptor engineering

Methodological Approach:

• Design fusion constructs incorporating TMEM247 transmembrane regions

• Express in appropriate cell lines (e.g., T cells for CAR applications)

• Assess receptor clustering and downstream signaling

• Compare performance with established transmembrane domains like CD28

The approaches described for de novo design of transmembrane domains could be applied to study or modify TMEM247's transmembrane regions for specific oligomerization states, potentially creating valuable research tools.

What bioinformatic analyses can reveal functional insights about TMEM247?

Bioinformatic approaches can provide valuable insights into potential functions of TMEM247:

Sequence Homology Analysis:

• BLAST searches reveal that TMEM247 shows some sequence similarity to transformation/transcription domain-associated proteins (TRRAP) from various organisms

• Significant alignments with TRRAP_HUMAN (E-value: 1e-24), TRRAP_MOUSE (E-value: 2e-24), and TRA1_DROME (E-value: 5e-24)

• These homologies suggest potential roles in transcriptional regulation or chromatin modification

Structural Prediction Methods:

• Transmembrane helix prediction tools can identify potential membrane-spanning regions

• Ab initio modeling approaches similar to those used for de novo transmembrane domain design could predict structural features

• Molecular dynamics simulations in membrane environments can provide insights into stability and dynamics

Functional Domain Prediction:

• Researchers should analyze the sequence for conserved functional motifs that might indicate specific molecular functions

• Protein-protein interaction prediction algorithms might suggest potential binding partners

How does the mouse TMEM247 compare to human orthologs in structure and function?

Understanding cross-species conservation of TMEM247 is important for translational research:

Comparative Analysis Approach:

• Perform multiple sequence alignment of mouse TMEM247 with human and other mammalian orthologs

• Identify conserved regions that likely represent functionally important domains

• Analyze species-specific variations that might indicate specialized functions

Evolutionary Considerations:

• Transmembrane proteins often show conservation in membrane-spanning regions while extracellular and intracellular domains may vary

• Comparison with the de novo design principles for transmembrane domains could reveal whether TMEM247's membrane-spanning regions follow similar structural patterns across species

Functional Implications:

• Cross-species conservation patterns can guide mutagenesis studies by highlighting critical residues

• Understanding these differences is crucial when using mouse models to study conditions relevant to human health

What are common challenges in working with Recombinant Mouse TMEM247 and their solutions?

Transmembrane proteins present specific experimental challenges:

Solubility and Aggregation Issues:

• Problem: TMEM247, like many transmembrane proteins, may aggregate during purification or storage

• Solution: Optimize detergent conditions; consider screening different detergents and lipids

• Methodological approach: Test mild detergents like DDM, LMNG, or lipid nanodiscs for protein stabilization

Low Expression Yield:

• Problem: Transmembrane proteins often express poorly in heterologous systems

• Solution: Optimize codon usage for expression host; consider fusion partners that increase expression

• Practical approach: Test different E. coli strains or consider insect cell expression systems for improved yield

Proper Folding Verification:

• Problem: Ensuring correct folding of recombinant transmembrane proteins

• Solution: Employ circular dichroism (CD) spectroscopy to assess secondary structure

• Expected results: Alpha-helical transmembrane domains should show characteristic CD spectra with minima at 208 and 222 nm

How can researchers optimize functional assays for TMEM247?

When designing functional experiments with TMEM247, consider these methodological approaches:

Membrane Incorporation:

• For studying TMEM247 in membrane contexts, researchers can use:

  • Liposome reconstitution

  • Supported lipid bilayers

  • Cell-based expression systems with appropriate controls

Protein-Protein Interaction Studies:

• Proximity-based assays (FRET, BRET, PLA) can detect interactions in cellular contexts

• Pull-down assays with recombinant TMEM247 should include appropriate detergents to maintain protein structure

• Consider crosslinking approaches to capture transient interactions

Functional Readouts:

• Based on potential similarities to TRRAP proteins , consider assays for:

  • Transcriptional regulation

  • Chromatin modification

  • Protein complex formation

What emerging technologies could advance TMEM247 research?

Several cutting-edge approaches could significantly enhance our understanding of TMEM247:

Cryo-electron Microscopy:

• Near-atomic resolution structures of transmembrane proteins in native-like environments

• Could reveal oligomeric states and structural details of TMEM247

• Methodological considerations: Protein stabilization, grid preparation optimization

De Novo Design Approaches:

• Computational design methods for transmembrane domains could provide insights into TMEM247 structure

• These approaches allow programming specific oligomeric interactions

• Potential for designing modified versions of TMEM247 with enhanced stability or novel functions

Single-Molecule Methods:

• Techniques like single-molecule FRET or AFM could probe conformational dynamics

• May reveal functional states not captured in bulk measurements

• Methodological challenge: Labeling strategies that preserve protein function

How might TMEM247 be involved in cellular signaling networks?

Understanding TMEM247's potential role in signaling requires systematic approaches:

Interactome Mapping:

• Proximity labeling methods (BioID, APEX) can identify neighboring proteins in cellular contexts

• Immunoprecipitation combined with mass spectrometry could reveal stable interaction partners

• Yeast two-hybrid screening with cytoplasmic domains might identify signaling mediators

Functional Genomics:

• CRISPR-Cas9 knockout or knockdown studies can reveal phenotypic consequences

• Transcriptional profiling after modulation of TMEM247 expression might uncover regulated pathways

• Phosphoproteomics could determine if TMEM247 affects specific signaling cascades

Relationship to Transcriptional Machinery:

• Given the sequence similarity to transcription-associated proteins , investigate potential roles in gene regulation

• Consider chromatin immunoprecipitation studies if nuclear localization is observed

• Analyze effects on transcription factor activity