Recombinant Mouse Transmembrane protein 80 (Tmem80)

What is the tissue distribution profile of mouse Tmem80?

Tmem80 exhibits a broad expression pattern across multiple mouse tissues. Based on comprehensive proteomic profiling data, Tmem80 is detected in several brain regions, including the cerebral cortex, cerebellum, hippocampal formation, and midbrain . Additionally, expression data indicates presence in peripheral tissues including kidney, testis, and intestine .

Recent multiregional brain profiling using advanced proteomic approaches has provided greater resolution of Tmem80's distribution pattern:

Importantly, research has highlighted significant discrepancies between mRNA and protein distribution for many transmembrane proteins, including Tmem80, across brain regions . This emphasizes the importance of protein-level validation rather than relying solely on transcriptomic data.

How can recombinant mouse Tmem80 be expressed and purified for functional studies?

Expression and purification of recombinant mouse Tmem80 presents challenges common to transmembrane proteins. Based on established protocols for similar proteins, the following approach is recommended:

Expression Systems:

• Insect Cell Expression: The baculovirus expression system in insect cells (Sf9 or Tnao38) shows superior results for complex transmembrane proteins . For Tmem80, the MultiBac baculovirus expression system has demonstrated efficacy with complex transmembrane proteins and would be appropriate .

• E. coli Expression: While more challenging for transmembrane proteins, E. coli systems can be successful with proper solubilization and refolding strategies . Expression typically results in inclusion bodies requiring specialized solubilization methods.

Purification Protocol:

• For insect cell expression:

  • Infect cells with amplified baculovirus (typically 3 rounds of amplification)

  • Culture for 72 hours post-infection

  • Harvest cells and isolate membrane fractions

  • Solubilize using mild detergents (DDM or LMNG)

  • Purify using affinity chromatography (His-tag strategy commonly used)

• For E. coli expression:

  • Solubilize inclusion bodies with appropriate buffers

  • Employ a dilution method with refolding buffer to obtain native protein structure

  • Confirm proper folding via immunoreactivity testing with specific antibodies

The quality of recombinant Tmem80 should be assessed by SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering) to confirm proper oligomeric state, similar to methods used for other transmembrane proteins .

What experimental design considerations are important when studying Tmem80 function in mouse models?

When designing experiments to investigate Tmem80 function in mouse models, several critical considerations should be addressed:

Sample Size and Statistical Power:

• Implement the "single mouse experimental design" for initial screening, which can efficiently identify phenotypes while using fewer animals

• For validation studies, calculate appropriate sample sizes based on expected effect sizes and variability within the model system

• Consider power analysis to determine minimum sample sizes needed to detect biologically meaningful differences

Control Selection:

• Include both wild-type controls and appropriate technical controls for specific manipulations

• For genetic models, littermate controls are essential to minimize confounding variables

• Consider including positive controls with known phenotypes when available

Randomization Strategy:

• Randomize animal assignment to experimental groups to prevent bias

• For heterogeneous populations, stratified randomization may be necessary

• Document randomization methods in experimental protocols

Phenotypic Characterization:

When analyzing Tmem80 knockout or transgenic models, comprehensive phenotyping should include:

The experimental timeline should account for developmental expression patterns of Tmem80 and potential compensatory mechanisms that may emerge in genetic models .

What methods can be used to deliver recombinant Tmem80 for in vivo functional studies?

For in vivo functional studies, efficient delivery of recombinant Tmem80 protein can be achieved through several approaches:

Electroporation-Based Delivery:

Electroporation has emerged as a robust method for delivering recombinant proteins directly into mammalian cells, offering several advantages for Tmem80 studies:

• Efficiency: Enables delivery into a large cohort of cells simultaneously

• Versatility: Compatible with various cell types including HeLa, RPE, HEK293, and U2OS cells

• Structural integrity: Successfully delivers complex transmembrane proteins without functional impairment

• Biochemical compatibility: Allows for subsequent immunoprecipitation and interaction studies

Protocol implementation involves:

• Preparing recombinant Tmem80 at 5-10 μM concentration

• Using optimized electroporation parameters (typically 1350 V, 30 ms, 1 pulse for mammalian cells)

• Allowing 12-24 hours for proper localization post-electroporation

• Confirming cellular uptake via western blotting or fluorescence microscopy

Alternative Approaches:

• PEGylation: Long-acting PEGylated delivery systems have shown efficacy for controlled release of proteins in vivo

• Viral vector-mediated expression: AAV or lentiviral vectors can be used for sustained expression of Tmem80 in specific tissues

• Nanoparticle encapsulation: Emerging approach for delivering transmembrane proteins across biological barriers

The choice of delivery method should be based on the specific experimental endpoints and temporal requirements of the study.

How can the functionality of recombinant mouse Tmem80 be validated?

Validating the functionality of recombinant Tmem80 is crucial before using it in downstream applications. Recommended validation approaches include:

Structural Integrity Assessment:

• SEC-MALS analysis to confirm proper oligomeric state

• Circular dichroism spectroscopy to verify secondary structural elements

• Native PAGE to evaluate quaternary structure integrity

Biochemical Validation:

• Immunoreactivity testing with specific anti-Tmem80 antibodies via western blotting

• Co-immunoprecipitation assays to confirm interactions with known binding partners

• Functional binding assays if ligands are identified

Cellular Localization:

• Fluorescently tagged Tmem80 should localize to appropriate cellular compartments

• Confirmation of membrane insertion via fractionation studies

• Co-localization with known interacting partners via confocal microscopy

A comprehensive validation approach should include multiple methods to ensure both structural and functional integrity of the recombinant protein.

What gene expression systems are optimal for producing recombinant mouse Tmem80?

The choice of expression system significantly impacts the yield and quality of recombinant Tmem80. Based on data from similar transmembrane proteins, the following systems offer distinct advantages:

Insect Cell Expression Systems:

• Advantages: Superior folding of complex transmembrane proteins, enhanced post-translational modifications, higher yields

• Optimal cell lines: Tnao38 cells show superior expression for many transmembrane proteins compared to Sf9

• Vector systems: MultiBac baculovirus expression vectors allow for multi-subunit protein complex expression

• Culture conditions: Maintain cells at 27°C with appropriate insect cell media (such as SF900-II or ESF921)

Protocol optimization should include:

• Baculovirus amplification through 3 rounds before final infection

• Infection at MOI of 1-3

• Harvest at 72 hours post-infection

• Temperature optimization (typically 27°C is standard, but 21°C may improve folding)

Mammalian Expression Systems:

• Advantages: Native-like post-translational modifications, proper membrane insertion

• Cell lines: HEK293-6E cells demonstrated good expression for transmembrane proteins

• Transfection method: Use of 40 kDa linear PEI instead of 25 kDa PEI significantly improves transient expression

• Considerations: DNA amount, DNA:PEI ratio, cell passage number, and culture medium all impact expression levels

Comparison of Expression Systems:

The passage number of cells significantly impacts transfection efficiency, with a correlation observed between ploidy (DNA content of single cells) and expression levels .

What are the key challenges in studying Tmem80 interactions with other proteins?

Investigating protein-protein interactions involving Tmem80 presents several methodological challenges:

Technical Challenges:

• Maintaining the native membrane environment for authentic interactions

• Distinguishing specific from non-specific interactions due to hydrophobic domains

• Capturing transient or weak interactions that may be physiologically relevant

• Solubilizing membrane proteins without disrupting interaction interfaces

Recommended Approaches:

• Proximity-Based Labeling:

  • BioID or APEX2 fusion proteins to identify proximal interaction partners

  • Allows identification of interactions within the native cellular environment

  • Captures both stable and transient interactions

• Co-Immunoprecipitation Following Protein Delivery:

  • Electroporated recombinant tagged-Tmem80 followed by immunoprecipitation

  • Western blotting for suspected interaction partners

  • Mass spectrometry analysis for unbiased interaction mapping

• Predicted Interaction Networks:

  • Protein coexpression analysis across multiple brain regions to predict endogenous interactions

  • Validation via colocalization studies in neural cell cultures and brain tissue

  • Integration with transcriptomic data to identify context-dependent interactions

For transmembrane proteins like Tmem80, the electroporation approach has demonstrated particular utility, as electroporated proteins establish physiologic interactions with endogenous binding partners that can be detected through both imaging and biochemical methods .

How does gene expression profiling inform Tmem80 function in different mouse tissues?

Comprehensive gene expression profiling provides valuable insights into potential Tmem80 functions across different tissues:

Multiregional Brain Profiling:

Advanced proteomic approaches have mapped Tmem80 distribution across brain regions, revealing:

• Region-specific expression patterns suggesting specialized functions

• Considerable discrepancy between mRNA and protein distribution, highlighting post-transcriptional regulation

• Co-expression patterns with other transmembrane proteins indicating potential functional networks

Disease-Associated Expression Changes:

Expression profiling in disease models reveals potential involvement of Tmem80 in:

• Left ventricular remodeling in end-stage dilated cardiomyopathy (DCM)

• Participation in metabolic pathways altered in heart failure

• Association with TCA cycle and energy metabolism genes

Chemical Interaction Profiling:

Gene-chemical interaction data indicates differential expression of Tmem80 in response to various compounds:

• Increased expression following exposure to 1,2-dichloroethane and 3,4-methylenedioxymethamphetamine

• Decreased expression with exposure to 2-hydroxypropanoic acid and acrylamide

• Altered methylation patterns in response to arsane

These expression patterns provide foundation hypotheses for functional studies, particularly in metabolic, neurological, and toxicological contexts.

What genetic mouse models are available for studying Tmem80 function?

Several genetic mouse models are available for investigating Tmem80 function:

Targeted Alleles:

• Tmem80<tm1e(EUCOMM)Hmgu>: A targeted mutation mouse model available through the International Mouse Strain Resource (IMSR)

• Generated using EUCOMM targeting strategies for comprehensive functional analysis

• Allows for tissue-specific conditional deletion studies when combined with appropriate Cre recombinase lines

Design Considerations for New Models:

When developing new genetic models to study Tmem80:

• Targeting strategy options:

  • Conventional knockout: Complete gene inactivation

  • Conditional knockout: Tissue-specific or temporally controlled deletion

  • Knockin: Introduction of specific mutations or tagged versions

• Phenotypic analysis framework:

  • Comprehensive tissue expression profiling to identify primary sites for phenotyping

  • Molecular interaction studies to identify affected pathways

  • Physiological assessments based on expression patterns

• Control selection:

  • Littermate controls are essential

  • Consider including heterozygous animals to assess gene dosage effects

  • Age and gender matching critical for interpretable results

When interpreting phenotypes, consider potential compensatory mechanisms that may mask primary functions of Tmem80, particularly in constitutive knockout models.

How can single-cell approaches be applied to study Tmem80 function?

Single-cell methodologies offer powerful approaches to dissect Tmem80 function with unprecedented resolution:

Single-Cell Proteomics:

• Deep learning-assisted transmembrane proteome profiling can identify cell-type specific expression patterns

• Quantification of Tmem80 across diverse cell populations can reveal specialized functions

• Integration with spatial transcriptomics to map expression in tissue context

Functional Analysis in Single Cells:

• Electroporation-based delivery of recombinant Tmem80 variants allows cell-autonomous functional assessment

• Live-cell imaging of fluorescently tagged Tmem80 reveals dynamics and localization patterns

• Patch-clamp electrophysiology can assess functional consequences on membrane properties

Single Mouse Experimental Design:

A novel approach particularly valuable for Tmem80 research:

• Each mouse harbors a different patient-derived xenograft or genetic modification

• Endpoints focus on tumor regression, Event-Free Survival, or specific molecular readouts

• Allows testing across greater genetic diversity with fewer animals

• Particularly useful for initial screening of Tmem80 function across multiple genetic backgrounds

This approach demonstrated strong correlation between results from single-mouse and traditional experimental designs, validating its utility for studying proteins like Tmem80 where function may vary across genetic contexts .

What is known about the structure-function relationship of mouse Tmem80?

Current knowledge about Tmem80 structure-function relationships remains limited, but inferences can be made from available data:

Predicted Structural Features:

• Tmem80 contains multiple transmembrane domains characteristic of the transmembrane protein family

• Likely adopts a multi-pass membrane topology with both cytoplasmic and extracellular domains

• May form homo-oligomeric or hetero-oligomeric complexes based on similar transmembrane proteins

Functional Implications:

• Expression in multiple brain regions suggests potential roles in neuronal signaling or homeostasis

• Co-expression with proteins involved in metabolic pathways indicates possible roles in cellular metabolism

• Chemical interaction data suggests potential involvement in response to environmental toxicants

Further structural studies utilizing techniques such as cryo-electron microscopy would significantly advance understanding of Tmem80's functional mechanisms. Comparative analysis with human TMEM80 could provide evolutionary insights into conserved functional domains.