Recombinant Danio rerio Transmembrane protein 141 (tmem141)

What is Transmembrane protein 141 (tmem141) in Danio rerio?

Transmembrane protein 141 (tmem141) is a protein encoded by the tmem141 gene in zebrafish (Danio rerio). This gene has synonyms including zgc:153677 and is also referred to as si:ch211-268m12.4 in some databases . As a transmembrane protein, it contains domains that span the cell membrane and likely plays roles in cellular signaling, molecular transport, or structural functions. Currently, detailed functional characterization of tmem141 remains limited compared to other transmembrane proteins like Tmem184a, which has been studied in angiogenesis contexts .

Why is zebrafish (Danio rerio) a suitable model for studying tmem141?

Zebrafish serves as an exceptional vertebrate model system for studying gene functions like tmem141 for several compelling reasons. First, the zebrafish genome is fully sequenced and shows significant homology with human genes. Second, the model offers substantial advantages for molecular genetic studies, including transparency during embryonic development, rapid generation time, and established techniques for genetic manipulation . Additionally, zebrafish embryos develop externally, allowing for direct observation of developmental processes. The extensive molecular genetic and genomic data available for zebrafish creates a powerful foundation for investigating protein functions . These characteristics make zebrafish particularly valuable for understanding transmembrane proteins like tmem141 in vertebrate systems.

How is recombinant Danio rerio tmem141 typically produced for research?

Recombinant Danio rerio tmem141 is typically produced using expression systems similar to those used for other zebrafish proteins. The general methodology involves:

• Gene Amplification: The tmem141 gene sequence (or partial sequence) is amplified using RT-PCR from total RNA extracted from zebrafish tissues. Specific primers containing appropriate restriction enzyme sites are designed based on the gene sequence (e.g., similar to the approach used for tnfb with EcoRI and BamHI sites) .

• Vector Construction: The amplified gene is cloned into a suitable expression vector (e.g., pET-32a) after restriction enzyme digestion and ligation .

• Expression System Selection: Depending on the research requirements, the protein can be expressed in prokaryotic systems (E. coli), or eukaryotic systems (yeast, baculovirus, or mammalian cells) . For basic structural studies, E. coli expression systems are commonly used due to their high yield and simplicity.

• Protein Purification: The recombinant protein is purified using techniques such as affinity chromatography or gel extraction. For instance, target proteins can be isolated by cutting the target band from SDS-PAGE gels, similar to the approach used for rZetnfb .

The resulting recombinant protein typically has >90% purity and is stored in a liquid form containing glycerol at -20°C or -80°C for long-term storage .

What are the optimal conditions for expressing and purifying recombinant Danio rerio tmem141?

For optimal expression and purification of recombinant Danio rerio tmem141, researchers should consider:

Expression System Selection:

• E. coli system: Most cost-effective but may have limitations for proper folding of transmembrane proteins

• Yeast or insect cell systems: Better for maintaining protein structure and post-translational modifications

• Mammalian cell systems: Optimal for functional studies requiring mammalian-like modifications

Purification Protocol:

• Include appropriate detergents during cell lysis to solubilize membrane proteins

• Use affinity tags (His-tag or GST-tag) for initial purification

• Consider size exclusion chromatography as a final purification step

• Maintain >90% purity for experimental consistency

Storage Conditions:

• Store in glycerol-containing buffer at -20°C for short-term use

• Store at -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles that can compromise protein integrity

When working with transmembrane proteins like tmem141, maintaining proper folding and stability is particularly challenging due to their hydrophobic domains. Therefore, optimization of expression conditions, detergent selection, and buffer composition is often necessary for each specific application.

How can researchers effectively use tmem141 in functional studies?

To effectively utilize recombinant tmem141 in functional studies, researchers should consider these methodological approaches:

For In Vitro Interaction Studies:

• Develop binding assays to identify potential interaction partners using techniques like pull-down assays or co-immunoprecipitation

• Perform structural analyses through techniques like circular dichroism or protein crystallography to understand protein folding and domain organization

• Study protein-protein interactions through yeast two-hybrid systems or protein arrays, considering the limitations observed in previous two-hybrid studies with other proteins

For In Vivo Functional Studies:

• Genetic Manipulation: Use morpholino-mediated knockdown similar to approaches used for Tmem184a to evaluate developmental phenotypes

• Transgenic Approaches: Create transgenic zebrafish lines expressing modified versions of tmem141 to study structure-function relationships

• Tissue-Specific Expression: Employ constructs with tissue-specific promoters or heat shock-inducible promoters for temporal control of expression

• RNA Microinjection: Inject RNA at the 1-cell or 2-cell stage for transient expression studies, allowing half of the embryo to serve as an internal control when injecting at the 2-cell stage

When designing these studies, researchers should monitor for potential effects on angiogenesis, cell proliferation, or other developmental processes, as transmembrane proteins often play crucial roles in these biological functions.

What techniques are most effective for studying tmem141 expression patterns in zebrafish?

For comprehensive analysis of tmem141 expression patterns in zebrafish, researchers should employ multiple complementary techniques:

RNA-Based Methods:

• qRT-PCR: Design primers specific for tmem141 (similar to those in Table 1 for tnfb) and use β-actin as a reference gene. Calculate relative expression using the 2^-ΔΔCt method .

• In situ hybridization: Create RNA probes targeting tmem141 to visualize spatial expression patterns in different tissues throughout development.

• RNA-seq: For global transcriptomic analysis to understand tmem141 expression in different developmental stages or experimental conditions.

Protein-Based Methods:

• Antibody Development: Generate polyclonal antibodies against recombinant tmem141 using approaches similar to those employed for tnfb antibody production .

• Western Blotting: Validate antibody specificity through western blot analysis before using for expression studies .

• Immunohistochemistry: Apply validated antibodies to tissue sections to visualize protein localization at cellular and subcellular levels.

Reporter Systems:

• Fluorescent Reporter Constructs: Create transgenic lines with the tmem141 promoter driving fluorescent protein expression to monitor expression patterns in real time.

• Temporal Expression Analysis: Collect samples at multiple developmental time points (similar to the 0.5, 1, 2, 4, 8, and 12 hours post-infection timepoints used for tnfb studies) to create a developmental expression profile .

These methods can be applied to various tissues and developmental stages to create a comprehensive spatiotemporal map of tmem141 expression in zebrafish.

How should researchers analyze protein interaction data for tmem141?

Researchers analyzing protein interaction data for tmem141 should implement a systematic approach:

Data Collection and Quality Control:

• Employ multiple interaction detection methods (yeast two-hybrid, co-immunoprecipitation, proximity labeling)

• Include appropriate positive and negative controls to establish baseline interaction metrics

• Consider the limitations of each method, particularly for transmembrane proteins

Data Integration and Analysis:

• Cross-reference interaction data from multiple sources, recognizing that single approaches may miss significant interactions

• Analyze interactions in context of cellular compartments to identify biologically relevant partners

• Use computational tools to predict interaction interfaces and binding affinities

Interpretation Considerations:

• Be aware of potential discrepancies between different protein interaction datasets, as highlighted in comparative studies of yeast two-hybrid data

• Consider that "differences between the two data sets could be caused by the ability of two-hybrid analysis to detect a very wide range of interactions, and that the sample size, even in genome-scale analyses, may be too small to detect all of the interactions"

• Evaluate interactions in context of gene co-expression patterns, though visual analysis has shown "no clear correlation between co-expression of genes and protein interactions"

By integrating multiple approaches and considering the limitations of each method, researchers can develop a more comprehensive understanding of tmem141's interaction network.

What control experiments are essential when studying recombinant tmem141 function?

When investigating recombinant tmem141 function, the following control experiments are essential:

Protein Quality Controls:

• Purity Verification: Confirm >90% purity through SDS-PAGE and mass spectrometry

• Structural Integrity: Validate proper folding through circular dichroism or limited proteolysis

• Batch Consistency: Test multiple protein batches to ensure reproducibility of results

Experimental Controls:

• Negative Controls: Include:

  • Empty vector expression products

  • Non-induced samples

  • Unrelated transmembrane proteins of similar size

• Positive Controls: If possible, include well-characterized transmembrane proteins with known functions

• Dose-Response Analysis: Test multiple concentrations of recombinant protein to establish physiological relevance

Genetic Controls for In Vivo Studies:

• Rescue Experiments: Test whether phenotypes in knockdown models can be rescued by wild-type tmem141

• Tissue-Specific Controls: When examining tissue-specific effects, include analysis of other tissues as internal controls

• Temporal Controls: Analyze effects at multiple developmental timepoints to distinguish between primary and secondary effects

This comprehensive control strategy ensures that observations are specifically attributable to tmem141 function rather than experimental artifacts.

What are common challenges when working with recombinant tmem141 and how can they be addressed?

Researchers working with recombinant tmem141 frequently encounter several challenges that can be systematically addressed:

Protein Expression Challenges:

• Low Expression Levels:

  • Solution: Optimize codon usage for the expression system

  • Solution: Test multiple expression vectors and host strains

  • Solution: Consider using fusion partners to enhance solubility

• Protein Aggregation:

  • Solution: Express at lower temperatures (16-18°C)

  • Solution: Use solubility enhancers like SUMO or thioredoxin tags

  • Solution: Include appropriate detergents for membrane protein solubilization

Purification Challenges:

• Contaminating Proteins:

  • Solution: Implement multi-step purification strategies

  • Solution: Consider on-column refolding approaches

  • Solution: Optimize wash conditions to maintain >90% purity

• Loss of Function During Purification:

  • Solution: Preserve protein in glycerol-containing buffers

  • Solution: Store at appropriate temperatures (-20°C for short-term, -80°C for long-term)

  • Solution: Avoid repeated freeze-thaw cycles

Functional Assay Challenges:

• Inconsistent Results:

  • Solution: Standardize protein quantification methods

  • Solution: Validate protein activity before each experiment

  • Solution: Establish clear positive and negative controls

Addressing these challenges through systematic optimization is essential for generating reliable data with recombinant tmem141.

How can researchers optimize knockdown or knockout strategies for studying tmem141 function in zebrafish?

For effective genetic manipulation of tmem141 in zebrafish, researchers should consider these optimization strategies:

Morpholino Knockdown Approach:

• Design Optimization:

  • Target splice junctions or translation start sites

  • Test multiple morpholinos to confirm specificity

  • Include appropriate controls (standard control morpholinos)

• Delivery Optimization:

  • Inject at the 1-2 cell stage for uniform distribution

  • Titrate concentrations to minimize off-target effects

  • Consider co-injection with p53 morpholino to reduce apoptosis-related artifacts

• Validation Steps:

  • Confirm knockdown efficiency by qRT-PCR or western blot

  • Perform rescue experiments with co-injection of tmem141 mRNA

  • Document developmental timing carefully, as seen in studies of other transmembrane proteins like Tmem184a

CRISPR/Cas9 Knockout Approach:

• Guide RNA Selection:

  • Design multiple guide RNAs targeting early exons

  • Avoid off-target sites through bioinformatic prediction

  • Consider targeting functional domains based on protein prediction

• Efficiency Assessment:

  • Use T7 endonuclease assays or sequencing to verify editing

  • Establish stable lines through careful screening

  • Characterize founders thoroughly before experimental use

• Phenotypic Analysis:

  • Examine vascular development, as transmembrane proteins often affect angiogenesis

  • Assess at multiple developmental stages (similar to time points used in tnfb studies)

  • Consider potential compensatory mechanisms in stable knockout lines

These approaches have been successfully applied to study other zebrafish transmembrane proteins and can be adapted for tmem141 research.

How might tmem141 research contribute to understanding human disease mechanisms?

Research on Danio rerio tmem141 has significant potential to illuminate human disease mechanisms through several avenues:

Comparative Genomics Applications:

• The zebrafish genome shows substantial homology with human genes, making it valuable for understanding conserved functions

• Studies of zebrafish tmem141 can provide insights into the function of human TMEM141, potentially revealing roles in disease processes

• Zebrafish models expressing human TMEM141 variants could help determine pathogenicity of variants identified in clinical sequencing

Developmental Disease Connections:

• As a transmembrane protein, tmem141 may participate in signaling pathways relevant to human developmental disorders

• Similar to studies of Tmem184a in angiogenesis , tmem141 may have roles in vascular development with implications for cardiovascular diseases

• The zebrafish model allows for rapid screening of developmental phenotypes that could be translated to human conditions

Therapeutic Target Potential:

• Understanding tmem141 function could identify new therapeutic targets for diseases involving membrane transport or signaling

• Drug screening in zebrafish tmem141 models could rapidly identify compounds that modulate related pathways

• The "genetic program for sequential specification of pluripotential precursors followed by divergence of distinct lineages... is highly conserved from zebrafish to humans," suggesting findings in zebrafish may translate to human hematopoietic disorders

This translational potential highlights the importance of fundamental research on zebrafish tmem141 for human health applications.

What emerging technologies could enhance tmem141 research in the coming years?

Several cutting-edge technologies are poised to transform research on tmem141 and other transmembrane proteins:

Advanced Imaging Technologies:

• Super-resolution microscopy for visualizing tmem141 localization at the subcellular level with unprecedented detail

• Light-sheet microscopy for real-time imaging of protein dynamics in developing zebrafish embryos

• Correlative light and electron microscopy (CLEM) for connecting protein localization with ultrastructural context

Single-Cell Technologies:

• Single-cell RNA sequencing to map tmem141 expression in rare cell populations and reveal heterogeneity in expression patterns

• Single-cell proteomics to understand protein-level variation across different cell types

• Spatial transcriptomics to preserve spatial information while analyzing gene expression patterns

Genetic Engineering Advances:

• Base editing and prime editing for precise modification of tmem141 sequences without double-strand breaks

• Optogenetic and chemogenetic tools for temporal control of tmem141 expression or activity

• Transient tissue-specific expression using laser-activated promoters, similar to techniques that "can elicit transient expression of injected constructs in precise cell types at exact stages of development"

Structural Biology Innovations:

• Cryo-electron microscopy for high-resolution structural analysis of tmem141 in its native membrane environment

• AlphaFold2 and related AI tools for predicting protein structures and interaction interfaces

• Integrative structural biology approaches combining multiple data types for comprehensive structural models

Implementing these technologies will provide unprecedented insights into tmem141 function and regulation in both normal development and disease states.