Recombinant Bovine Coiled-coil domain-containing protein 149 (CCDC149)

What is Bovine CCDC149 and what structural features characterize this protein?

Bovine Coiled-coil domain-containing protein 149 (CCDC149) belongs to the CCDC protein family characterized by alpha-helical coiled-coil structural motifs. These motifs facilitate protein-protein interactions and can form dimeric or multimeric complexes essential for protein function. The coiled-coil domains typically contain heptad repeat patterns with hydrophobic residues in specific positions that create an amphipathic helix. While the specific sequence of bovine CCDC149 has similarities with human CCDC149, researchers should note that the full sequence conservation and structural elements may differ between species. For recombinant expression, understanding these structural characteristics is essential for proper folding and function .

What expression systems are most suitable for producing Recombinant Bovine CCDC149?

Based on established protocols for related CCDC proteins, E. coli remains the most widely used expression system for producing Recombinant Bovine CCDC149. For optimal expression, consider the following methodology:

• Vector selection: pET or pGEX vectors with T7 promoters offer high-level expression

• Tags: N-terminal His6 tags facilitate purification while minimally affecting protein structure

• Expression conditions: Induction at OD600 0.6-0.8 with 0.5-1.0 mM IPTG at 16-18°C overnight typically reduces inclusion body formation

• Buffer composition: PBS with 1M Urea at pH 7.4 is often suitable for stabilization

When expressing CCDC149, researchers should monitor protein solubility as coiled-coil domains can sometimes aggregate. Lower induction temperatures (16°C) and longer expression times often improve proper folding and solubility .

What purification methods yield the highest purity for Recombinant Bovine CCDC149?

Multi-step purification protocols yield the highest purity for Recombinant Bovine CCDC149:

For optimal storage after purification, maintain the protein in PBS with 1M urea at pH 7.4, aliquot to avoid freeze-thaw cycles, and store at -20°C. This methodology typically yields preparations with >80% purity suitable for most research applications .

What are the validated applications for Recombinant Bovine CCDC149 in research?

While specific bovine applications are still emerging, recombinant CCDC149 has been validated for several research applications that can be applied to bovine studies:

• Antibody validation and competition assays

• Protein-protein interaction studies

• Structural analysis through circular dichroism or X-ray crystallography

• Blocking/neutralizing experiments to assess functional roles

Researchers should consider using properly folded recombinant protein as a positive control in immunoassays and western blotting applications. Additionally, recombinant CCDC149 can serve as an antigen for generating specific antibodies for immunohistochemistry and localization studies in bovine tissues .

How can researchers investigate the functional role of CCDC149 in bovine cellular pathways?

To investigate CCDC149 function in bovine cellular systems, employ a multi-faceted approach:

• Gene expression manipulation:

  • CRISPR-Cas9 gene editing to introduce mutations or knockdown CCDC149

  • siRNA or shRNA-mediated knockdown in bovine cell lines

  • Overexpression using lentiviral vectors with inducible promoters

• Protein interaction studies:

  • Co-immunoprecipitation with potential binding partners

  • Proximity labeling techniques (BioID or APEX)

  • Yeast two-hybrid screening for novel interactions

• Functional assessment:

  • Subcellular localization through immunofluorescence

  • Response to cellular stressors (oxidative stress, heat shock)

  • Cell phenotype analysis after expression modulation

What transcriptomic approaches can elucidate CCDC149 expression patterns in bovine tissues?

Transcriptomic approaches to study CCDC149 expression in bovine systems should include:

• RNA-Seq analysis:

  • Compare expression across different bovine tissues and developmental stages

  • Analyze differential expression under disease conditions or treatments

  • Identify co-expressed genes that may function in the same pathways

• In situ hybridization techniques:

  • RNAscope for highly specific cellular localization

  • Fluorescent in situ hybridization for co-localization studies with other transcripts

  • Chromogenic in situ hybridization for tissue distribution analysis

• Single-cell RNA-Seq:

  • Identify cell type-specific expression patterns

  • Map CCDC149 expression in heterogeneous tissue populations

For reliable results, researchers should collect samples consistently, use appropriate normalization methods for RNA-Seq data, and validate findings through RT-qPCR or protein-level analysis .

How does bovine CCDC149 compare functionally with orthologs in other mammalian species?

For comparative functional analysis of CCDC149 across species:

When conducting cross-species functional comparisons, researchers should:

• Perform sequence alignments focusing on conserved domains

• Compare expression patterns in homologous tissues

• Test functional complementation in knockout/knockdown models

• Assess interaction partner conservation

This comparative approach can provide insights into evolutionarily conserved functions versus species-specific adaptations of CCDC149 .

What methodologies can assess potential roles of CCDC149 in bovine immune response pathways?

To investigate CCDC149's potential role in bovine immune responses:

• Immune cell expression profiling:

  • Isolate specific bovine immune cell populations (T cells, B cells, macrophages)

  • Quantify CCDC149 expression by RT-qPCR and western blotting

  • Compare expression levels before and after immune stimulation

• Functional immune assays:

  • Co-culture systems with macrophages and T cells with CCDC149 modulation

  • Cytokine production measurement after CCDC149 knockdown/overexpression

  • Phagocytosis and bacterial killing assays in CCDC149-modified cells

• Infection models:

  • Monitor CCDC149 expression changes during bacterial challenges (e.g., Mycobacterium bovis)

  • Assess correlation between CCDC149 expression and bacterial survival

  • Evaluate immune cell recruitment and activation in relation to CCDC149 levels

When designing immune function studies, researchers should consider the temporal dynamics of immune responses and use appropriate stimulation conditions that mimic physiological challenges .

How can structural biology approaches contribute to understanding bovine CCDC149 function?

Structural biology methodologies provide critical insights into CCDC149 function:

• Computational structure prediction:

  • Homology modeling based on known coiled-coil domain structures

  • Molecular dynamics simulations to predict conformational flexibility

  • Protein-protein docking to identify potential interaction interfaces

• Experimental structure determination:

  • X-ray crystallography of purified recombinant protein

  • Cryo-electron microscopy for larger complexes

  • NMR spectroscopy for dynamic structural analysis

• Structure-function analysis:

  • Site-directed mutagenesis of predicted functional residues

  • Domain deletion studies to map functional regions

  • Cross-linking mass spectrometry to identify interacting domains

For successful structural studies, researchers should focus on producing highly pure (>95%), homogeneous protein preparations and consider expressing individual domains separately if the full-length protein proves challenging to crystallize .

What considerations are important when designing antibodies against bovine CCDC149 for research applications?

When designing antibodies against bovine CCDC149:

• Epitope selection strategy:

  • Target unique, accessible regions not conserved in other CCDC family proteins

  • Avoid hydrophobic regions within coiled-coil domains that may be inaccessible

  • Consider both linear and conformational epitopes

  • Use this peptide sequence as a starting point for antibody generation: SVDELQDVKEERSSYQDKVERL

• Validation methodology:

  • Confirm specificity using recombinant CCDC149 as a positive control

  • Perform antibody competition assays with purified protein

  • Validate in multiple applications (western blot, immunoprecipitation, immunofluorescence)

  • Include knockout/knockdown controls to confirm specificity

• Polyclonal vs. monoclonal considerations:

  • Polyclonal antibodies offer broader epitope recognition but lower specificity

  • Monoclonal antibodies provide consistent results across experiments but may be sensitive to epitope masking

For optimal results, researchers should validate antibodies in the specific bovine tissue or cell type of interest and confirm cross-reactivity if using antibodies raised against human or mouse CCDC149 .