Recombinant Bovine Uncharacterized protein C17orf109 homolog

What is Recombinant Bovine Uncharacterized protein C17orf109 homolog?

Recombinant Bovine Uncharacterized protein C17orf109 homolog is a protein of currently unknown function that can be expressed in various host systems for research purposes. According to available resources, it has a UniProt ID of E1BAR0 and consists of 78 amino acids in its full-length form. The protein is classified as "uncharacterized," indicating that its precise biological function has not yet been fully elucidated through experimental validation. Sequence analysis suggests it may contain hydrophobic regions potentially indicative of membrane association. As a recombinant protein, it is typically produced with affinity tags (such as His-tag) to facilitate purification and detection in experimental settings while preserving its native structure as much as possible .

What expression systems are optimal for producing Recombinant Bovine Uncharacterized protein C17orf109 homolog?

What are the optimal storage conditions for Recombinant Bovine Uncharacterized protein C17orf109 homolog?

Proper storage is critical for maintaining the stability and biological activity of Recombinant Bovine Uncharacterized protein C17orf109 homolog. Based on experimental evidence and manufacturer recommendations, the following storage guidelines should be followed:

For long-term storage, the protein should be stored at -20°C to -80°C, with aliquoting necessary to avoid repeated freeze-thaw cycles that can cause protein degradation . Working aliquots can be maintained at 4°C for up to one week for active experiments . The optimal storage buffer typically consists of a Tris/PBS-based buffer containing 50% glycerol at pH 8.0, which helps maintain protein stability during freezing .

When reconstituting lyophilized protein, it is recommended to briefly centrifuge the vial before opening to bring contents to the bottom, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Adding glycerol to a final concentration of 5-50% is recommended for long-term storage, with 50% being the most common concentration used . Repeated freeze-thaw cycles should be strictly avoided as they can significantly reduce protein activity and stability .

How can sequence analysis inform functional predictions for C17orf109 homolog?

Sequence analysis represents a foundational approach to predicting potential functions of uncharacterized proteins like C17orf109 homolog. The analysis should begin with multiple sequence alignment of C17orf109 homologs across species to identify evolutionarily conserved regions, which often correspond to functionally important domains. Comparing the bovine sequence with the mouse homolog reveals conserved motifs, particularly in the hydrophobic region that likely constitutes a transmembrane domain .

Computational prediction tools can identify potential functional motifs, secondary structure elements, and post-translational modification sites. The hydrophobicity profile of C17orf109 homolog strongly suggests a transmembrane region (approximately residues 30-52), while the cysteine-rich C-terminal domain may indicate metal-binding capability or sites for lipid modifications that could anchor the protein to membranes. Transmembrane prediction algorithms like TMHMM or Phobius should be employed to precisely define the boundaries of this domain.

Additionally, tertiary structure prediction using tools like AlphaFold2 can provide insights into potential binding interfaces and functional sites. While the protein is currently uncharacterized, its small size (78 amino acids) and predicted membrane association suggest potential roles in membrane organization, signaling, or protein-protein interactions at membrane interfaces.

What purification strategies are most effective for His-tagged Recombinant Bovine Uncharacterized protein C17orf109 homolog?

Purification of His-tagged Recombinant Bovine Uncharacterized protein C17orf109 homolog should follow a systematic workflow optimized for small proteins with potential membrane-associating properties. The primary purification step typically employs immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins that bind the His-tag with high specificity .

For optimal purification:

• Cell lysis should be performed in buffer containing 20-50 mM Tris-HCl (pH 8.0), 300-500 mM NaCl, and protease inhibitors. For proteins with hydrophobic regions like C17orf109 homolog, addition of mild detergents (0.1% Triton X-100 or DDM) may improve solubility.

• IMAC binding should use 10-20 mM imidazole to reduce non-specific binding, with washing steps at 20-40 mM imidazole and elution at 250-500 mM imidazole.

• Secondary purification via size exclusion chromatography is recommended to separate monomeric protein from aggregates and remove imidazole from the buffer.

• Final purity assessment should be performed using SDS-PAGE, with expected purity greater than 90% as typically determined by gel analysis .

The purified protein can be stored in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 for optimal stability . For proteins with hydrophobic transmembrane domains, addition of glycerol (up to 50%) often enhances stability during storage.

What experimental approaches are recommended for investigating the subcellular localization of C17orf109 homolog?

Determining the subcellular localization of C17orf109 homolog requires multiple complementary approaches due to its uncharacterized nature and predicted membrane association. The following experimental strategy is recommended:

Immunofluorescence microscopy using tagged recombinant protein (GFP, FLAG, or HA) offers visualization of protein distribution patterns within cells. Co-localization studies with established organelle markers (plasma membrane, ER, Golgi, mitochondria) can help identify the specific membrane compartment where C17orf109 homolog resides. For proteins with potential transmembrane domains like C17orf109 homolog, careful consideration of tag position (N- versus C-terminal) is essential to avoid disrupting membrane insertion.

Subcellular fractionation followed by Western blotting provides biochemical validation of microscopy findings. Sequential extraction with buffers of increasing detergent strength can differentiate between peripheral and integral membrane associations. Protease protection assays can determine the topology of membrane insertion by identifying which protein domains are accessible to proteolytic cleavage.

Advanced techniques like proximity labeling (BioID or APEX) are particularly valuable for uncharacterized proteins, as they identify neighboring proteins that may provide functional context. This approach has successfully identified novel components of mitochondrial nucleoids, as demonstrated for another uncharacterized protein, C17orf80 .

How can researchers validate expression and function of Recombinant Bovine Uncharacterized protein C17orf109 homolog?

Comprehensive validation of Recombinant Bovine Uncharacterized protein C17orf109 homolog requires multiple analytical approaches:

SDS-PAGE analysis confirms the expected molecular weight (approximately 9-10 kDa for the 78-amino acid protein, plus the tag size) and assesses purity, which should exceed 90% for functional studies . Western blotting using anti-tag antibodies (anti-His for His-tagged protein) provides additional confirmation of protein identity, while mass spectrometry analysis of tryptic peptides offers definitive sequence verification.

Structural integrity can be assessed using circular dichroism spectroscopy to confirm proper secondary structure formation, particularly important for proteins with predicted α-helical transmembrane domains. Dynamic light scattering evaluates sample homogeneity and detects potential aggregation.

Functional validation presents challenges for uncharacterized proteins. Approaches include:

• Protein-protein interaction studies using pull-down assays or co-immunoprecipitation to identify binding partners.

• Liposome binding assays to confirm membrane association predicted by sequence analysis.

• Cell-based assays examining effects of protein overexpression or depletion.

• Comparative analysis with structurally similar characterized proteins.

For truly uncharacterized proteins like C17orf109 homolog, an unbiased approach combining interactomics, localization studies, and phenotypic analysis of gene perturbation provides the most comprehensive functional insights.

What are the challenges in determining structure-function relationships for small membrane proteins like C17orf109 homolog?

Elucidating structure-function relationships for small membrane proteins like C17orf109 homolog presents several significant challenges requiring specialized methodological approaches. The hydrophobic nature of these proteins makes them difficult to express and purify in their native conformations, often necessitating detergent solubilization or lipid nanodisc incorporation that can potentially alter native structure.

Conventional structural biology techniques face limitations with such proteins. X-ray crystallography is challenging due to the difficulty in obtaining well-diffracting crystals of membrane proteins. Nuclear Magnetic Resonance (NMR) spectroscopy, while suitable for small proteins, requires isotopic labeling and optimization of membrane-mimetic environments. Cryo-electron microscopy, typically advantageous for larger complexes, may require fusion to larger scaffold proteins for small membrane proteins like C17orf109 homolog.

Computational prediction approaches such as AlphaFold2 have significantly advanced capabilities for structure prediction but may still face challenges with membrane proteins due to limited training data for small single-pass transmembrane proteins. Functional studies are complicated by the lack of obvious enzymatic activity, often requiring indirect approaches such as interaction partner identification or phenotypic analysis following gene perturbation.

To overcome these challenges, researchers should employ integrative structural biology approaches combining computational prediction, crosslinking mass spectrometry, HDX-MS (hydrogen-deuterium exchange mass spectrometry), and functional assays in membrane-mimetic environments.

How might post-translational modifications affect the structure and function of C17orf109 homolog?

Post-translational modifications (PTMs) potentially play crucial roles in regulating the structure, localization, and function of C17orf109 homolog. Sequence analysis reveals several candidate sites for modifications that merit investigation:

The cysteine-rich C-terminal region (CFCCCQYC) presents potential sites for palmitoylation, a lipid modification that could enhance membrane association or target the protein to specific membrane microdomains. Palmitoylation of multiple adjacent cysteines could create a lipid anchor influencing both protein localization and membrane curvature. Mass spectrometry-based palmitoyl-proteomics using hydroxylamine-sensitivity would be the method of choice for detecting these modifications.

Phosphorylation sites may exist within the N-terminal region, potentially regulating protein-protein interactions or subcellular trafficking. Phosphoproteomic analysis comparing protein expressed in different cellular contexts could identify regulated phosphorylation events. Site-directed mutagenesis of potential phosphorylation sites followed by functional assays would determine their physiological relevance.

The choice of expression system significantly impacts the PTM profile. E. coli expression systems typically lack eukaryotic PTM machinery, while mammalian expression systems provide the most authentic modification patterns . Comparative analysis of protein produced in different expression systems could reveal PTM-dependent functional properties. Specifically, expression in insect or mammalian cells would be essential to preserve modifications necessary for correct protein folding or retention of biological activity .

What comparative genomics approaches can reveal about the evolution and potential function of C17orf109 homolog?

Comparative genomics provides valuable insights into the evolutionary context and potential functions of uncharacterized proteins like C17orf109 homolog. Cross-species comparison of orthologs reveals that C17orf109 homolog is conserved across mammals, with identifiable orthologs in species ranging from bovine to mouse systems .

Sequence conservation analysis between bovine and mouse C17orf109 homologs shows preservation of key features: the transmembrane domain, the distinctive cysteine-rich C-terminal region, and specific motifs like LLLKLQ. Such evolutionary conservation suggests functional importance of these elements. Regions displaying higher sequence conservation typically correspond to functional domains or interaction interfaces essential for protein activity.

Synteny analysis examining the genomic context of C17orf109 across species can identify consistently neighboring genes that may share regulatory mechanisms or functional relationships. Co-expression network analysis using publicly available transcriptomic datasets can reveal genes consistently co-regulated with C17orf109, providing clues about biological pathways involving this protein.

Phylogenetic profiling, examining the presence/absence pattern of C17orf109 across diverse species, can identify organisms where the gene emerged or was lost, potentially correlating with specific biological capabilities. Evidence from another C17orf family member, C17orf80, indicates association with mitochondrial nucleoids , suggesting possible functional convergence or divergence within this gene family that merits investigation for C17orf109 homolog.

How can genetic manipulation techniques be applied to study the physiological role of C17orf109 homolog?

Genetic manipulation provides powerful approaches for investigating the physiological functions of uncharacterized proteins like C17orf109 homolog. CRISPR-Cas9 genome editing enables precise modification of the endogenous gene to create knockout cell lines, tag the native protein, or introduce specific mutations to disrupt predicted functional domains such as the transmembrane region or cysteine-rich motif.

For comprehensive functional analysis, researchers should implement the following strategy:

• Generate complete knockout cell lines using CRISPR-Cas9 targeting of early coding exons to create frameshift mutations. Validate knockout at DNA level (sequencing), RNA level (RT-PCR), and protein level (Western blot).

• Perform phenotypic characterization examining:

  • Cell viability and proliferation

  • Organelle morphology and function

  • Membrane integrity and organization

  • Response to cellular stressors

• Create knock-in cell lines with fluorescent or affinity tags at the endogenous locus for live-cell imaging and interactome analysis. This approach maintains physiological expression levels and regulatory control, avoiding artifacts associated with overexpression.

• Conduct rescue experiments with wild-type and mutant constructs to identify essential domains and residues.

Additionally, RNAi-based knockdown provides a complementary approach for studying dosage-dependent effects. Multi-omics analysis (transcriptomics, proteomics, lipidomics) of knockout cells can reveal dysregulated pathways, providing insights into the protein's function within cellular networks.

What strategies can address solubility challenges when expressing C17orf109 homolog?

Expressing small membrane proteins like C17orf109 homolog presents significant solubility challenges due to their hydrophobic nature. The protein contains a predicted transmembrane region (approximately residues 30-52) that may cause aggregation or inclusion body formation, particularly in bacterial expression systems. Several strategies can mitigate these issues:

Expression condition optimization represents the first approach. Reducing induction temperature (16-25°C instead of 37°C) slows protein synthesis, allowing more time for proper folding. Decreasing inducer concentration and extending expression time can also improve soluble yield. E. coli strains specifically designed for membrane proteins, such as C41/C43, often show superior performance for transmembrane proteins.

Fusion partners can dramatically enhance solubility. MBP (maltose-binding protein), SUMO, or Trx (thioredoxin) fusions placed at the N-terminus often improve folding and solubility. For C17orf109 homolog, placing the tag at the N-terminus would avoid interfering with the C-terminal cysteine-rich region.

Buffer optimization is critical for protein solubilization and stability. Mild detergents (0.1% DDM, CHAPS, or Triton X-100) can solubilize membrane-associated proteins while preserving native structure. Increased salt concentration (300-500 mM NaCl) and addition of glycerol (5-10%) reduce aggregation by masking hydrophobic interactions.

If inclusion bodies form despite optimization efforts, protein can be extracted using strong denaturants (8M urea or 6M guanidine-HCl) followed by gradual dialysis to remove the denaturant and allow refolding. On-column refolding during purification often yields better results than dilution-based refolding.

How can researchers optimize yield and purity when purifying C17orf109 homolog?

Optimizing yield and purity for C17orf109 homolog requires systematic refinement of expression and purification parameters. For expression optimization, researchers should conduct a factorial experiment varying E. coli strain, media composition, temperature, induction timing, and inducer concentration to identify optimal conditions. Based on general principles and the hydrophobic nature of C17orf109 homolog, expression in C41/C43 strains at reduced temperature (18-25°C) in rich media like Terrific Broth often produces good results for membrane-associated proteins.

For purification optimization:

• Lysis buffer composition significantly impacts initial extraction efficiency. For membrane-associated proteins like C17orf109 homolog, inclusion of detergents is critical. A buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.1-0.5% detergent (DDM, CHAPS, or Triton X-100), and protease inhibitors typically provides good initial solubilization.

• Immobilized metal affinity chromatography (IMAC) conditions should be optimized by testing different imidazole concentrations in binding (10-20 mM), washing (20-50 mM), and elution buffers (250-500 mM). Gradient elution often provides better separation than step elution.

• Secondary purification through size exclusion chromatography separates monomeric protein from aggregates and removes imidazole from the preparation. For small proteins like C17orf109 homolog, columns with appropriate resolution in the 10-50 kDa range should be selected.

Final purity should exceed 90% as determined by SDS-PAGE . If higher purity is required, ion exchange chromatography can provide additional purification based on the protein's charge properties. The final product should be stored in Tris/PBS-based buffer with 50% glycerol at -20°C or -80°C .

What controls are essential when investigating protein-protein interactions of uncharacterized proteins?

When investigating protein-protein interactions of uncharacterized proteins like C17orf109 homolog, rigorous controls are essential to distinguish genuine interactions from experimental artifacts. The following control framework should be implemented:

Negative controls must include:

• Bait-only and prey-only controls to identify non-specific binding

• Irrelevant protein of similar size/structure to distinguish specific from non-specific interactions

• Tag-only controls to identify tag-mediated interactions

• Beads-only control to detect matrix binding proteins

Competition controls involve including excess untagged protein to compete with tagged bait protein, which should reduce specific interaction signals. Reciprocal co-immunoprecipitation, where each potential interacting partner is used as bait in separate experiments, provides strong validation of genuine interactions.

Interaction strength characterization through techniques like surface plasmon resonance or isothermal titration calorimetry determines binding affinity, allowing discrimination between high-affinity specific interactions and low-affinity non-specific associations. Structure-informed mutagenesis of predicted interaction interfaces provides functional validation by demonstrating that specific mutations disrupt the interaction.

For uncharacterized proteins, which may have unknown binding partners, unbiased approaches like proximity labeling (BioID, APEX) or co-immunoprecipitation followed by mass spectrometry are particularly valuable. These approaches have successfully identified interaction networks for other uncharacterized proteins, such as C17orf80's association with mitochondrial nucleoids .

How can researchers address reproducibility challenges when working with C17orf109 homolog?

Ensuring reproducibility when working with uncharacterized proteins like C17orf109 homolog requires systematic approach to experimental design, execution, and reporting. Several key strategies address specific challenges associated with this protein:

Standardization of recombinant protein preparation is essential. Detailed documentation of expression conditions, purification protocols, and quality control metrics enables consistent protein production across experiments. Quality control should include SDS-PAGE for purity assessment, mass spectrometry for identity confirmation, and dynamic light scattering for homogeneity analysis. Storage conditions should strictly follow recommendations to maintain -20°C/-80°C temperature with avoidance of freeze-thaw cycles .

Experimental design must include appropriate replicates: technical replicates within each experiment and biological replicates using independent protein preparations. Statistical power analysis should guide sample size determination, ensuring sufficient replicates to detect biologically meaningful effects.

Antibody validation is particularly critical for uncharacterized proteins. If using custom antibodies, validation should include western blots with recombinant protein positive controls, comparison with tagged protein detection, and ideally, absence of signal in knockout/knockdown samples. Given potential challenges with antibody generation for small proteins like C17orf109 homolog, epitope tagging strategies may offer more reliable detection.

Comprehensive reporting following established guidelines (e.g., ARRIVE for animal studies, MIAPE for proteomics) ensures that all methodological details necessary for reproduction are documented. For uncharacterized proteins, special attention should be given to reporting expression construct details, tag position and sequence, and buffer compositions including detergents if used.

How does bovine C17orf109 homolog compare structurally and functionally to the mouse version?

Sequence alignment reveals several highly conserved features:

• A central hydrophobic region (approximately residues 30-52) with high conservation, likely representing a transmembrane domain

• The motif LLLKLQ (positions 16-21) shows perfect or near-perfect conservation

• The region LPQAEPVE (positions 22-29) preceding the transmembrane domain is highly conserved

• Both proteins contain cysteine-rich C-terminal regions, though with some species-specific variations

The mouse sequence (MAATDFVGEIRSVGERLLLKLQQLPQAEPVELVAFSIIVLFTATVLVLGLIACSCCCAHCCCSESRQRKIPVRPTKPR) and bovine sequence (MAASKLMQEIHSIGDRLLLKLQRLPQAEPVEILAFSVLVVFTATVVLLLLIACGFCCCQYCWPRRRGRRTQVGPMTPP) show greatest divergence at the N- and C-termini, while the central regions display higher conservation . This pattern typically indicates that the conserved regions are functionally important, while terminal regions may have species-specific adaptations.

The high conservation of the predicted transmembrane domain suggests membrane integration is a fundamental aspect of this protein's function across species. The conserved cysteine-rich region likely plays an important role, possibly in protein-protein interactions, metal binding, or post-translational modifications, though the specific pattern of cysteines shows some species variation.

What insights can be gained by comparing C17orf109 homolog with other C17orf family proteins?

Comparative analysis of C17orf109 homolog with other proteins from the C17orf family reveals distinct structural features and potential functional relationships. The C17orf family contains several uncharacterized proteins of varying sizes and predicted functions, providing context for understanding C17orf109's potential roles.

C17orf109 homolog (78 amino acids) is significantly smaller than other family members like C17orf78 (286 amino acids) and C17orf80 (609 amino acids) , suggesting distinct functional roles. Despite size differences, some shared features exist: C17orf80 contains a predicted transmembrane helix similar to C17orf109, indicating that membrane association may be a common characteristic among some family members .

Functional studies of C17orf80 demonstrate its association with mitochondrial nucleoids and interaction with the inner mitochondrial membrane . C17orf80 was detected in proximity to nucleoid components by mass spectrometry, similar to how interaction partners are identified for other uncharacterized proteins . This suggests similar interactomics approaches would be valuable for elucidating C17orf109's functional network.

While C17orf80 contains a zinc finger motif indicating potential nucleic acid binding capability , no such motif is evident in the smaller C17orf109 homolog, pointing to distinct molecular functions. Additionally, C17orf80 contains intrinsically disordered regions , a feature less likely in the much smaller C17orf109 homolog.

This comparative analysis suggests that while these proteins share the "uncharacterized" designation and potential membrane association, they likely perform distinct cellular functions, with C17orf109's small size and membrane-spanning potential indicating possible roles in signaling or membrane organization rather than nucleoid association.

What structural features distinguish C17orf109 homolog from other small membrane proteins?

C17orf109 homolog exhibits distinctive structural features that differentiate it from other small membrane proteins, providing clues about its potential specialized functions. The most notable feature is its compact size (78 amino acids) combined with both a predicted transmembrane domain and a cysteine-rich C-terminal region.

The predicted single transmembrane domain (approximately residues 30-52) is flanked by distinctive regions: an N-terminal domain containing the conserved LLLKLQ motif and a C-terminal domain enriched in cysteine residues. This organization differs from many small membrane proteins that often contain either multiple transmembrane spans or a single transmembrane anchor with a globular functional domain.

The cysteine-rich C-terminal region (CFCCCQYC in bovine, CSCCCAHC in mouse) represents a particularly distinctive feature . This unusual clustering of cysteines suggests potential metal coordination (similar to zinc finger proteins), disulfide bond formation, or sites for lipid modifications like palmitoylation. This feature is uncommon among typical single-pass membrane proteins and may indicate specialized functions in redox sensing, metal binding, or membrane microdomain targeting.

• Small membrane regulators of ion channels or transporters

• Membrane-tethered signaling adaptors

• Redox sensors with membrane anchors

• SNARE regulators involved in membrane fusion events

This distinctive combination of features suggests C17orf109 homolog may perform specialized functions at membrane interfaces rather than serving as a structural membrane protein.