Recombinant Bovine UPF0767 protein C1orf212 homolog

How conserved is UPF0767 protein C1orf212 homolog across mammalian species?

Sequence alignment reveals high conservation across mammalian species with some notable differences:

These minor variations suggest evolutionary conservation of critical functional domains across mammals, indicating potential biological importance of this protein.

What is currently known about the biological function of UPF0767 protein C1orf212 homolog?

This protein belongs to the UPF (Uncharacterized Protein Family) designation, indicating limited functional characterization to date. As a SMIM12 homolog, it likely has membrane-associated properties, potentially involved in cellular signaling or membrane organization. Current research is focused on expression and purification methodologies as a foundation for functional studies . Research gaps exist in determining tissue-specific expression patterns, interacting partners, and physiological roles in bovine systems.

Which expression systems are most suitable for producing recombinant bovine UPF0767 protein C1orf212 homolog?

Multiple expression systems can be employed, each with distinct advantages:

For most basic research applications, E. coli expression with an N-terminal His-tag has been successfully employed, as demonstrated in multiple commercial preparations .

What are effective purification strategies for recombinant bovine UPF0767 protein C1orf212 homolog?

A standard purification protocol involves:

• Expression in E. coli with N-terminal His-tag

• Cell lysis using appropriate buffer systems

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Optional secondary purification step using size exclusion chromatography

• Buffer exchange to storage conditions (typically Tris/PBS-based buffer with 6% trehalose at pH 8.0)

For highest purity requirements (>95%), consider additional ion exchange chromatography steps. Quality control via SDS-PAGE should confirm >90% purity of the final preparation.

What are optimal storage conditions for maintaining stability of purified recombinant protein?

To maximize stability and shelf-life:

• Store the protein at -20°C/-80°C for long-term storage

• Add glycerol to a final concentration of 50% for cryoprotection

• Aliquot to avoid repeated freeze-thaw cycles

• For working stocks, store aliquots at 4°C for up to one week

• When reconstituting lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

Experimental data indicates that repeated freeze-thaw cycles significantly reduce protein stability and activity, making proper aliquoting essential for research reproducibility.

What analytical methods are recommended for confirming the identity and purity of recombinant bovine UPF0767 protein C1orf212 homolog?

A comprehensive analytical characterization should include:

• SDS-PAGE: Confirms the expected molecular weight and provides initial purity assessment (>90% purity is typically acceptable for most applications)

• Western blotting: Verifies protein identity using either anti-His antibodies (to detect the tag) or specific antibodies against the protein

• Mass spectrometry:

  • Intact mass analysis to confirm molecular weight

  • Peptide mapping following tryptic digestion to verify sequence coverage

• Circular dichroism (CD): Provides information about secondary structure elements

• Dynamic light scattering (DLS): Assesses homogeneity and detects potential aggregation

For functional validation, binding assays with known or predicted interaction partners would provide additional characterization.

How can researchers assess the folding and structural integrity of the recombinant protein?

Multiple complementary approaches are recommended:

• Circular dichroism spectroscopy: Provides information about secondary structure content (α-helices, β-sheets)

• Fluorescence spectroscopy: Intrinsic tryptophan fluorescence can indicate tertiary structure integrity

• Size exclusion chromatography: Evaluates oligomeric state and aggregation propensity

• Thermal shift assays: Measures protein stability and can identify buffer conditions that enhance thermal stability

• Limited proteolysis: Well-folded proteins typically show resistance to proteolytic digestion compared to misfolded variants

These methods collectively provide a comprehensive assessment of protein structural integrity, essential for downstream functional studies.

How can recombinant bovine UPF0767 protein C1orf212 homolog be utilized for antibody development?

Developing specific antibodies requires careful planning:

• Immunization strategy:

  • Use purified recombinant protein (>90% purity) with appropriate adjuvant

  • Consider both the full-length protein and synthetic peptides corresponding to predicted antigenic regions

  • Implement a 28-42 day immunization protocol with 3-4 booster injections

• Screening methodology:

  • Develop ELISA assays using the recombinant protein as a coating antigen

  • Validate antibody specificity using Western blotting against recombinant protein and native tissue extracts

  • Test cross-reactivity with homologous proteins from other species

• Validation approaches:

  • Immunoprecipitation to confirm native protein recognition

  • Immunohistochemistry to determine tissue localization patterns

  • Flow cytometry for cell surface expression analysis (if membrane association is confirmed)

Similar approaches have been successfully employed for other bovine recombinant proteins, such as the Gag protein in BFV studies .

What considerations are important when designing interaction studies with bovine UPF0767 protein C1orf212 homolog?

For protein interaction studies:

• Pull-down assays:

  • Immobilize His-tagged protein on Ni-NTA resin

  • Incubate with potential binding partners (cellular lysates or purified proteins)

  • Analyze bound proteins by SDS-PAGE and mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant protein on sensor chip

  • Flow potential binding partners over the surface

  • Determine binding kinetics and affinities

• Co-immunoprecipitation:

  • Express tagged protein in appropriate cell lines

  • Immunoprecipitate using tag-specific antibodies

  • Identify interacting partners by mass spectrometry

• Yeast two-hybrid screening:

  • Use protein as bait to screen bovine cDNA libraries

  • Validate interactions using alternative methods

When designing these experiments, consider the potential impact of the His-tag on protein interactions and include appropriate controls to account for non-specific binding.

How can researchers effectively compare UPF0767 protein C1orf212 homolog function across different species?

Inter-species functional comparison requires:

• Sequence-based analysis:

  • Multiple sequence alignment to identify conserved regions

  • Identification of species-specific variations that might impact function

  • Phylogenetic analysis to understand evolutionary relationships

• Expression pattern comparison:

  • RT-PCR or RNA-seq analysis of tissue distribution across species

  • Comparison of promoter regions to identify conserved regulatory elements

• Functional assays:

  • Parallel expression and purification of homologs from multiple species

  • Side-by-side comparison in relevant functional assays

  • Chimeric protein construction to map species-specific functional domains

• Structural comparison:

  • 3D structure prediction and comparison

  • Identification of conserved surface patches likely involved in interactions

The amino acid sequences from bovine, mouse, and dog show high conservation with few variations (positions 45, 51, 69, and 70), suggesting potentially similar functions across mammalian species .

What methodological approaches are recommended for studying post-translational modifications across species variants?

For comprehensive PTM analysis:

• Mass spectrometry-based approaches:

  • Enrichment strategies for specific modifications (phosphorylation, glycosylation)

  • Multiple fragmentation techniques (CID, ETD, HCD) for comprehensive coverage

  • Quantitative comparison of modification sites and stoichiometry

• Expression system considerations:

  • Express each species variant in homologous and heterologous systems

  • Compare PTM patterns when expressed in E. coli, yeast, insect, and mammalian cells

  • Assess impact of different cellular machinery on modification patterns

• Functional impact assessment:

  • Generate site-directed mutants at predicted modification sites

  • Compare activity, localization, and interaction profiles of wild-type and mutant proteins

  • Develop modification-specific antibodies for immunological detection

The selection of expression system significantly impacts PTM profiles, with mammalian systems providing the most authentic modifications for functional studies .

What strategies can address poor solubility of recombinant bovine UPF0767 protein C1orf212 homolog?

Solubility challenges can be addressed through:

• Expression optimization:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Optimize induction conditions (temperature, IPTG concentration, duration)

  • Co-express with chaperones to assist folding

• Fusion tag approaches:

  • Test solubility-enhancing tags (SUMO, MBP, TRX) alongside His-tag

  • Compare N-terminal versus C-terminal tag placement

  • Include TEV or other protease sites for tag removal

• Buffer optimization:

  • Screen different pH conditions (typically 7.0-8.5)

  • Test various salt concentrations (150-500 mM NaCl)

  • Include stabilizing additives (glycerol, trehalose, arginine)

• Refolding strategies:

  • Purify under denaturing conditions if inclusion bodies form

  • Test gradual dialysis or rapid dilution refolding methods

  • Validate refolded protein structure and function

Commercial preparations typically employ a Tris/PBS-based buffer with 6% trehalose at pH 8.0, suggesting these conditions favor protein stability .

How can researchers validate that E. coli-expressed recombinant bovine UPF0767 protein C1orf212 homolog retains native-like properties?

Validation approaches include:

• Structural comparison:

  • Compare CD spectra between E. coli-expressed protein and protein expressed in mammalian systems

  • Assess thermal stability profiles across expression systems

• Functional assessments:

  • Develop activity assays based on predicted function

  • Compare activity metrics between proteins from different expression systems

• Binding partner validation:

  • Test interaction with known or predicted binding partners

  • Compare binding affinities across expression systems

• Cell-based functional rescue:

  • Use the recombinant protein in cellular knockdown/knockout systems

  • Assess ability to rescue phenotypes compared to native protein

When complete functional characterization is required, parallel expression in multiple systems (E. coli, insect, and mammalian cells) is recommended to compare potential functional differences .