Recombinant Rat Transmembrane protein C10orf57 homolog

What is Transmembrane Protein C10orf57 homolog and what are its key characteristics?

Transmembrane protein C10orf57 homolog, also known as Tmem254, is a 123-amino acid transmembrane protein found in rats (Rattus norvegicus). It features a full-length protein structure with a molecular weight of approximately 26-27 kDa based on structural analysis. The protein contains hydrophobic regions that facilitate its integration into cellular membranes, consistent with its classification as a transmembrane protein . The amino acid sequence of the rat variant is: MGTATGASYFQRGSLFWFTVIAVSFSYYTWVVFWPQSIPYQSLGPLGPFTKYLVDHYHTLLRNGYWLAWLVHVGESLYALVLCRRKGITDSQAQLLWFLQTFLFGVASLSILFAYRPKHQKHN . This sequence is critical for understanding the protein's structure-function relationship in experimental contexts.

How does rat Transmembrane Protein C10orf57 homolog compare to its mouse ortholog?

Comparative analysis between rat and mouse Transmembrane Protein C10orf57 homolog reveals significant sequence conservation with notable species-specific differences. Both proteins have identical length (123 amino acids) and similar structural organization, suggesting conserved functionality across rodent species . The table below highlights key similarities and differences:

These sequence variations may contribute to subtle functional differences that researchers should consider when extrapolating findings between species .

What are the predicted structural domains and topology of Transmembrane Protein C10orf57 homolog?

Transmembrane Protein C10orf57 homolog contains predicted membrane-spanning domains characteristic of integral membrane proteins. Computational analysis using tools similar to those applied for related transmembrane proteins suggests that this protein likely contains 1-4 transmembrane helices with both cytoplasmic and extracellular domains . Topology prediction algorithms would typically identify the N-terminal and C-terminal orientations relative to the membrane, which is crucial for understanding protein function and designing experiments that target specific domains. The hydrophobic regions within the sequence (particularly segments containing sequences like LFWFTVIAVSFSYYTW) likely form the membrane-spanning helices, while charged and polar residues often comprise the soluble domains on either side of the membrane .

What are the optimal conditions for reconstitution and storage of recombinant Transmembrane Protein C10orf57 homolog?

Proper reconstitution and storage of recombinant Transmembrane Protein C10orf57 homolog is critical for maintaining structural integrity and functional activity. The lyophilized protein should be briefly centrifuged prior to opening to ensure all material is at the bottom of the vial. For reconstitution, the protein should be dissolved in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . To maintain stability during storage, the following protocol is recommended:

• Add glycerol to a final concentration of 5-50% (optimally 50%) to the reconstituted protein solution

• Aliquot the solution to minimize freeze-thaw cycles

• Store working aliquots at 4°C for short-term use (up to one week)

• Store long-term aliquots at -20°C/-80°C

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

The storage buffer composition (Tris/PBS-based buffer with 6% Trehalose, pH 8.0) has been optimized to maintain protein stability during freeze-thaw processes and long-term storage .

What expression systems and purification strategies are most effective for producing high-quality recombinant Transmembrane Protein C10orf57 homolog?

The E. coli expression system has been successfully utilized for producing recombinant Transmembrane Protein C10orf57 homolog with high purity (>90% as determined by SDS-PAGE) . For transmembrane proteins, which are typically challenging to express, the following methodology has proven effective:

• Expression System Selection: E. coli provides high yield and cost-effectiveness for this particular protein despite being a transmembrane protein

• Affinity Tag Integration: N-terminal His-tagging facilitates efficient one-step purification using metal affinity chromatography

• Expression Optimization: Parameters including temperature, IPTG concentration, and induction time should be optimized to balance between yield and proper folding

• Solubilization Strategy: Given the protein's transmembrane nature, appropriate detergents are needed during extraction and purification

• Purification Protocol: Typically involves immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography to achieve >90% purity

Researchers should verify protein quality using techniques such as SDS-PAGE, Western blotting, and mass spectrometry to confirm identity, purity, and structural integrity before experimental use.

How can Transmembrane Protein C10orf57 homolog function be assessed in vitro and in cellular systems?

Assessing the function of Transmembrane Protein C10orf57 homolog requires a multi-faceted approach that examines both protein-protein interactions and cellular localization:

• Cellular Localization Studies:

  • Immunofluorescence microscopy using antibodies against the His-tag or the native protein

  • Subcellular fractionation followed by Western blotting

  • Live-cell imaging with fluorescently-tagged protein variants

• Protein-Protein Interaction Analysis:

  • Pull-down assays utilizing the His-tag

  • Co-immunoprecipitation to identify binding partners

  • Yeast two-hybrid or mammalian two-hybrid systems

  • Proximity labeling methods (BioID or APEX)

• Functional Assays:

  • Membrane topology mapping using protease protection assays

  • Ion or metabolite transport assays if channel/transporter function is suspected

  • Site-directed mutagenesis of conserved residues to identify functionally important domains

Similar approaches have been successfully employed for related transmembrane proteins like TMEM225 , providing a methodological framework that can be adapted for C10orf57 homolog functional characterization.

What approaches can be used to investigate the evolutionary conservation and divergence of Transmembrane Protein C10orf57 homolog across species?

Investigating evolutionary patterns of Transmembrane Protein C10orf57 homolog requires comparative genomic and proteomic analyses. Researchers can employ the following methodological approach:

• Sequence Alignment and Phylogenetic Analysis:

  • Multiple sequence alignment of C10orf57 homologs from different species

  • Construction of phylogenetic trees to visualize evolutionary relationships

  • Calculation of sequence conservation scores for individual amino acids

• Structural Conservation Analysis:

  • Homology modeling based on known structures of related proteins

  • Identification of conserved structural motifs across species

  • Mapping of variable regions onto predicted 3D structures

• Functional Domain Conservation:

  • Comparison of transmembrane domain predictions across species

  • Analysis of selection pressure (dN/dS ratios) on different protein regions

  • Identification of species-specific insertions or deletions

Based on available data, rat and mouse C10orf57 homologs show high sequence similarity (approximately 90-95% identity), suggesting important conserved functions . Computational tools similar to those used for TMEM225 analysis (SMART for domain searching, PSORT for subcellular localization prediction, and HMMTOP for transmembrane topology) provide robust methodologies for conducting these evolutionary analyses .

How might differential expression of Transmembrane Protein C10orf57 homolog impact cellular physiology and pathophysiology?

Understanding the physiological and pathophysiological implications of Transmembrane Protein C10orf57 homolog expression requires examining tissue-specific expression patterns and manipulating expression levels. Researchers should consider these methodological approaches:

• Expression Profiling:

  • Quantitative RT-PCR across different tissues and developmental stages

  • RNA-seq analysis to determine expression patterns and potential splice variants

  • Protein-level quantification through Western blotting or mass spectrometry

  • Single-cell transcriptomics to identify cell type-specific expression

• Functional Consequence Analysis:

  • CRISPR/Cas9-mediated knockout or knockdown studies

  • Overexpression models using viral vectors or transgenic approaches

  • Phenotypic characterization of altered expression models

  • Correlation of expression levels with physiological parameters

• Disease Association Studies:

  • Analysis of expression in disease models or tissues

  • Genetic association studies in population cohorts

  • Identification of mutations or variants in patient samples

Similar proteins in this family have shown tissue-specific expression patterns, with some members like TMEM225 showing specific expression in testis , suggesting specialized functions in particular organ systems. The methodological frameworks used to characterize these related proteins provide valuable templates for investigating C10orf57 homolog.

What techniques can be applied to determine the interactome of Transmembrane Protein C10orf57 homolog and identify its functional partners?

Determining the protein interactome of Transmembrane Protein C10orf57 homolog requires sophisticated biochemical and cellular approaches. Researchers should consider implementing the following methodological strategies:

• Affinity-Based Approaches:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Utilizing the His-tag for pull-down assays followed by protein identification

  • Cross-linking mass spectrometry (XL-MS) to capture transient interactions

  • Proximity-dependent biotin identification (BioID) or APEX proximity labeling

• Genetic Interaction Screening:

  • Synthetic genetic array (SGA) analysis

  • CRISPR-based genetic screens to identify functional relationships

  • Suppressor/enhancer screens in model organisms

• Computational Prediction and Validation:

  • Protein-protein interaction prediction algorithms

  • Co-expression network analysis across tissues and conditions

  • Structural docking simulations with potential partners

  • Validation of predicted interactions with targeted biochemical assays

The recombinant His-tagged version of the protein provides an excellent tool for affinity-based approaches, as the tag allows specific isolation of the protein complex while minimizing background . Implementing these methods enables researchers to construct a comprehensive interactome map, revealing functional networks and potential regulatory mechanisms.

What analytical methods are most appropriate for assessing the quality and integrity of purified recombinant Transmembrane Protein C10orf57 homolog?

Ensuring the quality and integrity of purified recombinant Transmembrane Protein C10orf57 homolog is essential for reliable experimental outcomes. Researchers should implement a combination of analytical approaches:

• Purity Assessment:

  • SDS-PAGE with Coomassie or silver staining (expected purity >90%)

  • Capillary electrophoresis for high-resolution analysis

  • Reversed-phase HPLC for purity profiling

• Identity Confirmation:

  • Western blotting using anti-His antibodies or specific antibodies against the protein

  • Mass spectrometry for accurate molecular weight determination and peptide mapping

  • N-terminal sequencing to confirm proper translation initiation

• Structural Integrity Evaluation:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Fourier-transform infrared spectroscopy (FTIR) for structural characterization

  • Size-exclusion chromatography to detect aggregation or oligomerization

  • Dynamic light scattering for homogeneity assessment

• Functional Verification:

  • Binding assays with known or predicted interaction partners

  • Activity assays based on predicted function (if known)

  • Thermal shift assays to evaluate protein stability

The commercial preparations typically provide a guarantee of >90% purity as determined by SDS-PAGE , but researchers should independently verify quality parameters before proceeding with critical experiments.

How can researchers troubleshoot common issues encountered when working with recombinant transmembrane proteins like C10orf57 homolog?

Working with transmembrane proteins presents unique challenges. Here are methodological solutions to common problems researchers may encounter:

• Low Solubility or Precipitation:

  • Optimize buffer composition (adjust pH, salt concentration, or add compatible solutes)

  • Evaluate different detergents or lipid nanodisc systems for stabilization

  • Consider using protein stabilizing agents like glycerol, trehalose, or arginine

  • Maintain protein at appropriate temperature to prevent aggregation

• Loss of Activity After Reconstitution:

  • Verify proper folding using spectroscopic methods

  • Test different reconstitution protocols and buffer systems

  • Reconstitute into proteoliposomes or nanodiscs to provide native-like environment

  • Add reducing agents if disulfide bonds might form inappropriately

• Degradation During Storage:

  • Add protease inhibitors to storage buffer

  • Optimize freezing and thawing protocols

  • Store in small aliquots to minimize freeze-thaw cycles

  • Monitor stability using analytical techniques like SDS-PAGE or Western blotting

• Non-specific Binding in Interaction Studies:

  • Optimize washing conditions during pull-down or immunoprecipitation experiments

  • Include appropriate blocking agents to reduce background

  • Use more stringent controls to distinguish specific from non-specific interactions

  • Consider mild crosslinking to stabilize transient interactions

The stability of recombinant Transmembrane Protein C10orf57 homolog can be enhanced by following the recommended storage conditions, including maintaining the protein in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 and adding glycerol to 5-50% final concentration for long-term storage .

What advanced structural characterization techniques can be applied to Transmembrane Protein C10orf57 homolog?

Structural characterization of transmembrane proteins requires specialized approaches due to their hydrophobic nature and membrane integration. Researchers can employ these methodological strategies:

• X-ray Crystallography:

  • Develop crystallization conditions using detergent-solubilized protein

  • Employ lipidic cubic phase (LCP) crystallization for membrane proteins

  • Use antibody fragments or fusion partners to facilitate crystallization

  • Implement surface entropy reduction mutations to improve crystal packing

• Cryo-Electron Microscopy:

  • Single-particle analysis for purified protein or protein complexes

  • Prepare samples in detergent micelles, nanodiscs, or amphipols

  • Implement focused classification for flexible regions

  • Use subtomogram averaging for in-membrane structural analysis

• NMR Spectroscopy:

  • Solution NMR for smaller domains or fragments

  • Solid-state NMR for full-length protein in membrane mimetics

  • Use selective isotope labeling strategies to reduce spectral complexity

  • Employ paramagnetic relaxation enhancement (PRE) for distance constraints

• Computational Modeling:

  • Homology modeling based on related proteins with known structures

  • Ab initio modeling for unique structural elements

  • Molecular dynamics simulations in explicit membrane environments

  • Integrate experimental constraints from limited resolution methods

While no high-resolution structure has been reported for Transmembrane Protein C10orf57 homolog based on the available search results, these methodological approaches provide a roadmap for researchers aiming to elucidate its structure-function relationships.

How might high-throughput screening approaches be used to identify modulators of Transmembrane Protein C10orf57 homolog function?

Developing high-throughput screening (HTS) methodologies for identifying modulators of Transmembrane Protein C10orf57 homolog requires establishing robust functional readouts. Researchers should consider these approaches:

• Assay Development and Optimization:

  • Establish cell-based reporter systems linked to protein function

  • Develop binding assays using fluorescence or FRET technologies

  • Create phenotypic screens based on known or predicted functions

  • Optimize assay conditions for Z-factor >0.5 to ensure statistical reliability

• Screening Implementation:

  • Screen chemical libraries (small molecules, peptides, or biologics)

  • Perform CRISPR/RNAi screens to identify genetic modulators

  • Use fragment-based approaches for initial hit identification

  • Implement parallel screening with orthogonal assays to reduce false positives

• Hit Validation and Characterization:

  • Confirm activity using dose-response curves

  • Evaluate specificity using related proteins as counterscreens

  • Determine mechanism of action through biochemical and cellular assays

  • Assess structure-activity relationships for chemical modulators

• Target Engagement Verification:

  • Cellular thermal shift assay (CETSA) to confirm binding in cells

  • Microscale thermophoresis or surface plasmon resonance for binding kinetics

  • Photoaffinity labeling to identify binding sites

  • Competition assays with known ligands or substrates

These methodological approaches provide a framework for discovering and developing modulators that could serve as both research tools and potential therapeutic leads for conditions involving this protein.

What potential applications exist for using recombinant Transmembrane Protein C10orf57 homolog in biotechnology and therapeutic development?

Recombinant Transmembrane Protein C10orf57 homolog has several potential biotechnological and therapeutic applications that researchers can explore through these methodological approaches:

• Antibody Development:

  • Generate and validate monoclonal antibodies for research and diagnostic applications

  • Develop function-blocking or function-enhancing antibodies

  • Create antibody-drug conjugates for targeted therapeutic delivery

  • Implement phage display to identify high-affinity antibody fragments

• Diagnostic Applications:

  • Develop immunoassays for protein detection in biological samples

  • Create protein-based biosensors for relevant analytes

  • Establish reference standards for clinical laboratory tests

  • Design point-of-care diagnostic platforms

• Therapeutic Development:

  • Evaluate as a drug target for specific diseases based on function

  • Develop recombinant protein therapeutics for replacement therapy

  • Design peptide mimetics targeting key functional domains

  • Explore gene therapy approaches to modulate expression

• Biotechnological Tools:

  • Utilize as a membrane protein expression tag or fusion partner

  • Develop as a scaffold for membrane protein engineering

  • Create protein-based biomaterials with unique properties

  • Employ as an affinity tag for membrane protein purification

The availability of high-purity recombinant protein provides a valuable starting point for these applications, though further characterization of the protein's native function is essential for developing targeted approaches.