Recombinant Horse UPF0767 protein C1orf212 homolog

What is UPF0767 protein C1orf212 homolog and what is known about its function?

UPF0767 protein C1orf212 homolog belongs to the SMIM12 (Small integral membrane protein 12) family. While its exact function remains under investigation, it is part of the uncharacterized protein family (UPF) group, suggesting that its biological roles are still being elucidated. The protein has been identified in various species including mouse, xenopus laevis, and horse, indicating evolutionary conservation that may suggest important biological functions .

What are the structural characteristics of Horse UPF0767 protein C1orf212 homolog?

The Horse UPF0767 protein C1orf212 homolog is a partial recombinant protein available for research purposes. Though complete structural data for the equine variant is limited, we can gain insights from homologs in other species. For instance, the mouse homolog consists of 92 amino acids with the sequence: MWPVLWTVVRTYAPYVTFPVAFVVGAVGYHLEWFIRGKTPQPVEEEKSILERREDRKLDEMLGKDHTQVVSLKDKLEFAPKAVLNRNRPEKN . The protein likely has membrane-associated properties based on its classification as a small integral membrane protein .

How is Horse UPF0767 protein C1orf212 homolog identified in proteomic studies?

In proteomic studies, this protein can be identified using mass spectrometry-based methods. Its UniProt accession number is F6USH3 . When using resources like the Equine PeptideAtlas (which contains over 24,131 distinct peptides representing 2,636 canonical proteins), researchers can identify specific peptide sequences that uniquely match this protein. The Atlas serves as a reference for experimental planning and validation of new datasets in equine proteomics research .

What expression systems are optimal for producing recombinant Horse UPF0767 protein C1orf212 homolog?

Based on current research protocols, yeast expression systems have been successfully used to produce recombinant Horse UPF0767 protein C1orf212 homolog with purity levels exceeding 85% as determined by SDS-PAGE . For other species homologs, such as mouse and xenopus laevis variants, E. coli expression systems have been effectively employed . When designing your expression system, consider the protein's potential membrane-associated properties, which may affect solubility and folding during recombinant expression.

What are the recommended protocols for reconstitution and storage of the recombinant protein?

For optimal handling of recombinant Horse UPF0767 protein C1orf212 homolog:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is commonly used) for long-term storage

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep at -20°C/-80°C

The shelf life of the lyophilized form is typically 12 months at -20°C/-80°C, while the reconstituted liquid form generally maintains stability for 6 months under proper storage conditions .

What quality control measures should be implemented when working with this recombinant protein?

Quality control for recombinant Horse UPF0767 protein C1orf212 homolog should include:

• Purity assessment via SDS-PAGE (target >85%)

• Identity confirmation using mass spectrometry or western blot

• Functional validation appropriate to research context

• Batch-to-batch consistency testing

• Stability monitoring under storage conditions

When publishing research using this protein, document the UniProt accession number (F6USH3), expression system, tag information, and protein length (partial vs. full) to ensure reproducibility .

How can recombinant Horse UPF0767 protein C1orf212 homolog be utilized in comparative proteomics studies?

For comparative proteomics studies, researchers can use this recombinant protein as:

• A standard for quantification in targeted proteomic analyses

• A reference for validating antibody specificity in immunoassays

• A tool for studying protein-protein interactions specific to equine systems

• A basis for cross-species comparison with homologs from other organisms

When conducting comparative studies, consider integrating data from resources like the Equine PeptideAtlas, which provides extensive coverage of the equine proteome across 16 tissues and 7 body fluids. This approach allows researchers to contextualize findings about UPF0767 protein C1orf212 homolog within the broader equine proteome landscape .

What are the potential challenges in detecting endogenous UPF0767 protein C1orf212 homolog in equine samples?

Several challenges may arise when detecting endogenous UPF0767 protein C1orf212 homolog:

• Low abundance in native tissues, requiring sensitive detection methods

• Potential post-translational modifications not present in recombinant versions

• Cross-reactivity with homologous proteins when using antibody-based detection

• Sample preparation issues due to potential membrane association

• Limited availability of equine-specific detection reagents

To address these challenges, employ targeted mass spectrometry approaches using the Equine PeptideAtlas to identify unique peptide sequences. Consider developing custom antibodies using the recombinant protein as an immunogen, and validate specificity against potential cross-reactive proteins .

How might Horse UPF0767 protein C1orf212 homolog be incorporated into engineered antibody research?

The emerging field of engineered equine-human antibodies (termed "Centaur antibodies") offers intriguing possibilities for incorporating Horse UPF0767 protein C1orf212 homolog research. These chimeric antibodies carry equine V-genes and human Gamma1/Lambda constant genes . Researchers could:

• Develop antibodies against Horse UPF0767 protein C1orf212 homolog for diagnostic applications

• Explore potential epitopes within the protein that might serve as targets for therapeutic antibodies

• Use phage display libraries from immunized horses to generate high-affinity binders to this protein

• Study the immunogenicity profile of this protein in the context of equine disease models

The novel primer set EquPD v2020, designed for efficient amplification of rearranged horse antibody V-segments, could facilitate these approaches by enabling generation of diverse V-gene repertoires in phage display libraries .

What contaminant screening should be performed when using recombinant proteins in sensitive biological assays?

Recent research on protein supplements has highlighted the importance of contaminant screening in recombinant protein preparations. When using recombinant Horse UPF0767 protein C1orf212 homolog in sensitive biological assays, consider screening for:

• Heavy metals (As, Cd, Pb, Hg, Tl, Cu) using inductively coupled plasma mass spectrometry

• Organic compounds including cycloheptatriene, spiroheptadiene, acetone, acetic acid, dichloromethane, and toluene via gas chromatography-mass spectrometry (GC-MS)

• Industrial solvents, alcohols, phenols, aldehydes, ketones, and esters that may be present from manufacturing processes

• Endotoxin contamination, particularly for E. coli-derived recombinant proteins

• Host cell proteins from the expression system

For maximum sensitivity, employ GC-MS with at least 70% match factor for component identification. This approach can reveal unexpected heterogeneity in composition that might affect experimental outcomes .

What strategies can address protein instability issues with UPF0767 protein C1orf212 homolog?

When working with potentially unstable proteins like UPF0767 protein C1orf212 homolog, consider these stabilization strategies:

• Buffer optimization: Test various buffer conditions including pH ranges (typically 7.0-8.0), salt concentrations, and additives

• Addition of stabilizing agents: 6% Trehalose has been effectively used in storage buffers for homologous proteins

• Glycerol supplementation: 5-50% glycerol significantly extends storage stability

• Lyophilization: The lyophilized form typically has a longer shelf life (12 months) compared to liquid formulations (6 months)

• Single-use aliquoting: Minimize freeze-thaw cycles which can significantly impact protein integrity

For long-term storage, maintain the protein at -20°C/-80°C in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, supplemented with 50% glycerol .

How does Horse UPF0767 protein C1orf212 homolog compare structurally to homologs in other species?

Comparative analysis of UPF0767 protein C1orf212 homolog across species reveals interesting evolutionary patterns:

While complete sequence data for the horse homolog is limited in the provided search results, comparative analysis of mouse and frog variants shows high conservation in key regions, particularly in the N-terminal domain. Both contain membrane-associated motifs consistent with their classification as small integral membrane proteins .

What insights can be gained from studying UPF0767 protein C1orf212 homolog across different species?

Cross-species analysis of UPF0767 protein C1orf212 homolog can provide valuable insights into:

• Evolutionary conservation of functional domains, suggesting biological importance

• Species-specific adaptations that might correlate with physiological differences

• Structural variations that could inform protein engineering approaches

• Potential binding partners and interaction networks that may be conserved across species

The high degree of sequence similarity between mouse and xenopus variants (despite evolutionary distance) suggests important conserved functions. Researchers can leverage this conservation to design experiments that translate findings across species models, potentially applying insights from better-characterized models to equine-specific research questions .

How can the Equine PeptideAtlas be utilized to improve detection and characterization of UPF0767 protein C1orf212 homolog?

The Equine PeptideAtlas represents a valuable resource for researchers working with Horse UPF0767 protein C1orf212 homolog:

• Mining peptide data: The atlas contains 24,131 distinct peptide sequences with a peptide-level FDR of 0.20%, mapping to 2,636 canonical proteins

• Tissue distribution analysis: With data from 16 tissues and 7 body fluids, researchers can identify tissues with highest expression

• Experimental design: The atlas provides empirically observed peptides for targeted assay development

• Dataset validation: New experimental data can be validated against this reference resource

When designing experiments, researchers should consider that the Atlas covers approximately 13% of the 20,449 predicted equine proteins. If UPF0767 protein C1orf212 homolog is represented in the Atlas, researchers can identify which specific peptides are most reliably detected in mass spectrometry experiments, improving assay development .

What are the most effective mass spectrometry approaches for identifying this protein in complex samples?

Based on methodologies used in developing the Equine PeptideAtlas, effective mass spectrometry approaches for UPF0767 protein C1orf212 homolog identification include:

• Sample preparation: Consider tissue-specific extraction protocols based on Atlas data showing protein distribution

• Instrumentation: High-resolution instruments such as TripleTOF or Q-Star Elite have been successfully used for equine proteome analysis

• Database searching: Use the Trans-Proteomic Pipeline (TPP) with a comprehensive equine database containing UniProt, Ensembl, and UniGene sequences

• Search parameters: Set peptide-level FDR to approximately 0.2% and protein-level FDR to 1.4% for reliable identification

• Data processing: Convert vendor files to mzML format before processing through the TPP workflow

For optimal results, use annotated sequence databases that incorporate the latest equine genome assemblies. This approach has successfully identified thousands of equine proteins with high confidence in the Equine PeptideAtlas project .

What are promising approaches for elucidating the biological function of UPF0767 protein C1orf212 homolog?

To determine the biological function of this poorly characterized protein, researchers might consider:

• Knockout/knockdown studies in model systems to observe phenotypic effects

• Protein interaction studies using techniques like BioID, AP-MS, or Y2H to identify binding partners

• Subcellular localization studies using fluorescent tags or immunofluorescence

• Comparative genomics and proteomics across species to identify conserved functional domains

• Expression profiling across different tissues and under various conditions to identify regulatory patterns

The classification as a small integral membrane protein (SMIM12) provides clues that it may function in membrane-associated processes, potentially in signaling or transport roles .

How might recombinant Horse UPF0767 protein C1orf212 homolog contribute to developing equine-specific research tools?

Recombinant Horse UPF0767 protein C1orf212 homolog could serve as a foundation for developing several equine-specific research tools:

• Generation of monoclonal or polyclonal antibodies for immunoassays and immunohistochemistry

• Development of ELISA kits for quantification in equine biological samples

• Creation of protein standards for targeted proteomics approaches

• Production of affinity columns for protein interaction studies

• Development of chimeric "Centaur antibodies" combining equine variable regions with human constant regions

Such tools would enhance equine proteomics research capabilities and potentially provide insights into equine-specific biological processes. The approaches used for developing chimeric equine-human recombinant antibodies, as described in recent literature, could be adapted for this purpose .