Recombinant Bovine Transmembrane protein C10orf57 homolog

How is recombinant bovine C10orf57 homolog typically produced for research applications?

Recombinant bovine transmembrane protein C10orf57 homolog is commonly expressed in E. coli expression systems with an N-terminal histidine tag to facilitate purification . The full-length protein (amino acids 1-124) is encoded by the cDNA sequence corresponding to UniProt ID Q0D2G3.

For optimal expression, researchers should consider the following methodological approach:

• Clone the full coding sequence into an appropriate bacterial expression vector

• Transform into an E. coli strain optimized for protein expression

• Induce protein expression under controlled conditions

• Lyse cells using appropriate detergents that maintain membrane protein structure

• Purify using affinity chromatography (His-tag purification)

• Conduct quality control via SDS-PAGE to ensure >90% purity

The purified protein is typically provided as a lyophilized powder to ensure stability during shipping and storage .

What are the recommended storage and reconstitution procedures for recombinant bovine C10orf57 homolog?

To maintain the functional integrity of recombinant bovine transmembrane protein C10orf57 homolog, specific storage and reconstitution procedures should be followed:

These conditions are optimized to maintain protein stability and function. Prior to experimental use, researchers should verify protein activity using appropriate functional assays specific to transmembrane proteins. The addition of glycerol serves as a cryoprotectant to prevent protein denaturation during freezing and thawing processes .

What are the appropriate experimental controls when using antibodies against bovine C10orf57 homolog in immunological assays?

When conducting immunological assays such as Western blotting, immunohistochemistry, or immunocytochemistry with antibodies against bovine C10orf57 homolog, proper controls are essential for result validation. Based on established protocols for similar proteins:

• Positive Control: Include samples known to express bovine C10orf57 homolog

• Negative Control: Include samples from tissues/cells that do not express the protein

• Antibody Blocking Experiments: Use recombinant protein fragments as blocking controls

For antibody blocking experiments specifically, a recombinant protein control fragment can be used at a 100x molar excess based on the antibody concentration and molecular weight . The antibody-protein control fragment mixture should be pre-incubated for 30 minutes at room temperature before application to the experimental sample .

This methodological approach allows researchers to confirm antibody specificity and eliminate false positive results that may arise from non-specific binding. When interpreting results, any signal that persists despite blocking with the specific peptide fragment should be considered non-specific.

What expression patterns and cellular localization are expected for C10orf57 homolog proteins?

Based on the available data and knowledge of related proteins, C10orf57 homolog/TMEM254 is expected to exhibit specific expression patterns and subcellular localization:

• Tissue Expression: The human ortholog TMEM254 is expressed in multiple tissues, with Gene Ontology annotations indicating it functions as an integral component of membrane structures .

• Subcellular Localization: As a transmembrane protein, C10orf57 homolog is expected to localize to cellular membranes. The protein contains hydrophobic regions typical of transmembrane domains, suggesting insertion into lipid bilayers.

• Topology Prediction: Based on the amino acid sequence, the protein likely has membrane-spanning regions with portions exposed to both the cytoplasmic and extracellular/luminal spaces.

For experimental verification of localization, researchers should consider:

• Immunofluorescence with specific antibodies

• Subcellular fractionation followed by Western blotting

• Epitope tagging (e.g., GFP fusion) for live-cell imaging

When designing such experiments, it's important to ensure that tags do not interfere with the protein's membrane insertion or trafficking.

What experimental approaches can be used to investigate the function of bovine C10orf57 homolog in cellular systems?

To elucidate the function of bovine C10orf57 homolog in cellular systems, several complementary experimental approaches can be employed:

• CRISPR/Cas9-Mediated Gene Editing:

  • Generate knockout cell lines to observe phenotypic changes

  • Create knock-in reporter lines for live visualization of protein expression and localization

  • Introduce specific mutations to identify functional domains

• RNA Interference:

  • Use siRNA or shRNA to achieve transient or stable knockdown

  • Analyze resulting phenotypes and molecular changes

  • Compare with CRISPR knockout to identify potential compensation mechanisms

• Overexpression Studies:

  • Express wildtype or tagged protein to assess effects on cell physiology

  • Perform rescue experiments in knockout backgrounds

  • Test mutant variants to identify critical functional residues

• Interactome Analysis:

  • Conduct BioID or proximity labeling to identify neighboring proteins

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Use yeast two-hybrid screening to identify direct binding partners

• Functional Assays:

  • Assess membrane integrity and transport functions

  • Measure changes in cellular signaling pathways

  • Evaluate effects on cell viability, proliferation, and differentiation

These approaches should be tailored to the specific cellular context in which the protein is naturally expressed. Based on findings from related C10orf proteins, particular attention should be paid to membrane dynamics, cellular stress responses, and potential roles in muscle or cardiac cell function .

What insights can be gained from comparative analysis of C10orf57 homologs across different species?

Comparative analysis of C10orf57 homologs across species can provide valuable insights into conserved functions and evolutionary adaptations:

The gene encoding the C10orf57 homolog is conserved across mammals, with the physical arrangement of this gene and surrounding genes being largely maintained across species, including in the dog genome, though with "minor, but striking differences" .

For human TMEM254 (C10orf57), high sequence identity has been documented with mouse and rat orthologs (86% for both), particularly in the region spanning amino acids 33-60 .

Research methodologies for comparative analysis should include:

• Phylogenetic Analysis:

  • Construct evolutionary trees to understand the divergence patterns

  • Identify key evolutionary events that shaped protein function

  • Correlate protein evolution with species-specific adaptations

• Conservation Mapping:

  • Map conserved residues onto predicted structural models

  • Identify potential functional domains based on conservation patterns

  • Target highly conserved regions for mutagenesis studies

• Expression Pattern Comparison:

  • Compare tissue-specific expression across species

  • Identify conserved regulatory elements controlling expression

  • Correlate expression patterns with species-specific physiological traits

• Functional Complementation:

  • Test whether orthologs from different species can rescue defects in model systems

  • Identify species-specific functional differences

  • Create chimeric proteins to map functional domains

This comparative approach can help distinguish fundamental functions that have been preserved throughout evolution from species-specific adaptations, providing insights into both basic biological mechanisms and potential therapeutic targets.

How might post-translational modifications affect the function of bovine C10orf57 homolog?

Although specific post-translational modifications (PTMs) of bovine C10orf57 homolog have not been directly reported in the provided search results, the protein's sequence and membrane localization suggest several potential modification sites that could significantly impact its function:

• Phosphorylation:

  • Analysis of the amino acid sequence reveals multiple serine, threonine, and tyrosine residues that could serve as phosphorylation sites

  • These modifications could regulate protein-protein interactions, subcellular trafficking, or activity

  • Experimental approaches to study phosphorylation include:

    • Phospho-specific antibodies

    • Mass spectrometry with phospho-enrichment

    • Kinase inhibitor studies

    • Site-directed mutagenesis of putative phosphorylation sites

• Glycosylation:

  • As a transmembrane protein, N-linked or O-linked glycosylation could occur on extracellular domains

  • Glycosylation might influence protein stability, trafficking, or interaction with extracellular partners

  • Methods to investigate glycosylation include:

    • Treatment with glycosidases

    • Lectin binding assays

    • Metabolic labeling with modified sugars

    • Mass spectrometry glycoprofiling

• Ubiquitination/SUMOylation:

  • These modifications could regulate protein turnover or change interaction properties

  • Experimental approaches include:

    • Immunoprecipitation under denaturing conditions

    • Expression of tagged ubiquitin/SUMO

    • Proteasome inhibitor studies

    • Identification of modified lysine residues by mass spectrometry

• Palmitoylation/Myristoylation:

  • These lipid modifications often affect membrane association of proteins

  • Could influence the protein's localization to specific membrane domains

  • Can be studied using:

    • Metabolic labeling with modified fatty acids

    • Acyl-biotin exchange chemistry

    • Mass spectrometry with specific enrichment strategies

For each potential modification, researchers should consider both site identification and functional consequences. Comparative analysis with other transmembrane proteins and C10orf family members could provide additional guidance for prioritizing PTM studies.

What are the implications of C10orf57/TMEM254 in cardiac disease models?

Based on genetic mapping studies, C10orf57 (TMEM254) is located within a critical genetic interval associated with both myofibrillar myopathy (MFM) and arrhythmogenic right ventricular cardiomyopathy (ARVC7) . This chromosomal region (10q22.3) has been narrowed down to 4.27 Mbp between markers D10S1645 and D10S1786, containing 18 candidate genes including C10orf57 .

While direct causative mutations in C10orf57 coding regions were not found in the patients studied, regulatory mutations affecting one of the 18 genes in this region (including C10orf57) are hypothesized to be responsible for "a heterogeneous spectrum of clinically distinct myodegenerative disorders, affecting both skeletal and cardiac muscles to variable degrees" .

A related C10orf family member, C10orf71, has been definitively linked to dilated cardiomyopathy through frameshift variants that result in functional null alleles . Knockout mouse models of C10orf71 showed:

• Abnormal heart morphogenesis during embryonic development

• Cardiac dysfunction in adult mice

• Altered expression and splicing of contractile cardiac genes

• Impaired contractile function while maintaining normal sarcomere structure

Given the positional relationship and potential functional similarities between C10orf family members, C10orf57 warrants investigation in cardiac disease models through:

• Expression analysis in normal and diseased cardiac tissue

• Generation of knockout or knock-in animal models

• Functional studies in cardiomyocyte cell culture systems

• Analysis of patient samples for potential regulatory mutations

These approaches could help clarify whether C10orf57 plays a direct role in cardiac pathology or interacts with other genes in the critical interval to contribute to disease phenotypes.

How can advanced structural biology techniques be applied to study intrinsically disordered transmembrane proteins like C10orf57 homolog?

Studying the structure of transmembrane proteins, particularly those with intrinsically disordered regions, presents unique challenges that require specialized approaches. While C10orf57 specifically has not been characterized as intrinsically disordered in the provided search results, related C10orf proteins like C10orf71 have been identified as intrinsically disordered proteins , suggesting similar structural characteristics might exist in C10orf57.

Advanced structural biology techniques applicable to such proteins include:

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Particularly valuable for characterizing flexible or disordered regions

  • Can provide residue-level dynamics information in solution

  • Requires isotopic labeling (15N, 13C) of the recombinant protein

  • Methodological considerations:

    • Use of membrane mimetics (detergent micelles, bicelles, nanodiscs)

    • Specific pulse sequences optimized for membrane proteins

    • Selective labeling to focus on regions of interest

• Cryo-Electron Microscopy (Cryo-EM):

  • Can visualize membrane proteins in near-native environments

  • May reveal conformational ensembles and structural heterogeneity

  • Methodological approaches:

    • Vitrification in detergent micelles or nanodiscs

    • Use of antibody fragments to increase particle size

    • Classification algorithms to separate different conformational states

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Provides information on protein dynamics and solvent accessibility

  • Can identify structured vs. disordered regions

  • Methodological considerations:

    • Optimization of detergent compatibility with MS

    • Time-resolved measurements to capture dynamics

    • Analysis of peptic digests to map exchange to specific regions

• Molecular Dynamics Simulations:

  • Complement experimental approaches with atomistic models

  • Can predict conformational ensembles and membrane interactions

  • Implementation strategies:

    • Explicit membrane simulations

    • Enhanced sampling techniques

    • Integration with experimental restraints

• Cross-linking Mass Spectrometry (XL-MS):

  • Captures spatial relationships within the protein and with interaction partners

  • Can identify transient interactions and contact regions

  • Methodology includes:

    • Use of membrane-permeable crosslinkers

    • MS/MS analysis to identify cross-linked peptides

    • Integration with structural modeling

By combining these approaches, researchers can develop a comprehensive understanding of the conformational dynamics and functional mechanisms of intrinsically disordered transmembrane proteins like C10orf57 homolog.

What strategies can be employed to develop effective research tools for studying C10orf57 homolog in different experimental systems?

Developing effective research tools is crucial for advancing our understanding of C10orf57 homolog function across different experimental systems. Based on current approaches and technologies, the following strategies are recommended:

• Generation of High-Quality Antibodies:

  • Develop antibodies against multiple epitopes to enable diverse applications

  • Create antibodies specific to post-translationally modified forms

  • Validate antibody specificity using blocking peptides

  • Produce antibodies suitable for various applications (Western blot, immunoprecipitation, immunohistochemistry)

• Expression Constructs:

  • Create mammalian expression vectors with different tags (e.g., fluorescent proteins, epitope tags)

  • Develop inducible expression systems to control expression levels

  • Design constructs for bacterial, insect, and mammalian expression systems

  • Create domain deletion and point mutation variants to map functional regions

• CRISPR-Based Tools:

  • Design guide RNAs with minimal off-target effects

  • Develop conditional knockout systems (e.g., floxed alleles, inducible Cas9)

  • Create knock-in cell lines with endogenous tags

  • Implement CRISPRi/CRISPRa systems for expression modulation

• Reporter Systems:

  • Develop promoter-reporter constructs to study transcriptional regulation

  • Create fusion proteins to visualize subcellular localization

  • Design biosensors to detect protein-protein interactions or conformational changes

  • Implement split-reporter systems for protein interaction studies

• Recombinant Protein Production Optimization:

  • Optimize expression conditions for high yield and proper folding

  • Develop purification protocols that maintain native conformation

  • Create stable storage formulations to preserve activity

  • Produce protein variants with specific labels for biophysical studies

These research tools, when developed with rigorous validation and optimization, will enable comprehensive investigation of C10orf57 homolog function across multiple experimental systems and applications.