Recombinant Chicken Protein LZIC (LZIC)

What are the functional domains and important structural features of chicken LZIC?

Chicken LZIC contains several important structural features:

• Leucine zipper domain: Critical for protein-protein interactions and potential dimerization

• CTNNBIP1 domain: Associated with interaction with beta-catenin pathway components

• Conserved regions: Several regions show high conservation across species, indicating functional importance

Understanding these domains is essential for designing experiments that investigate protein-protein interactions or functional studies. When planning mutational studies or truncation experiments, researchers should consider preserving these domains to maintain protein functionality. The protein belongs to the CTNNBIP1 family, which plays roles in cellular signaling pathways .

What expression systems are optimal for producing recombinant chicken LZIC?

Multiple expression systems have been successfully used for chicken LZIC production, each with distinct advantages:

For recombinant chicken LZIC specifically, successful expression has been documented in yeast systems with His-tag purification, achieving >90% purity suitable for applications such as ELISA .

What are the optimal purification strategies for recombinant chicken LZIC?

Purification of recombinant chicken LZIC typically involves the following methodology:

• Affinity chromatography: For His-tagged LZIC, Ni-NTA or similar metal affinity resins are most common

• Size exclusion chromatography: Often used as a secondary purification step

• Buffer optimization: Critical for maintaining protein stability

A validated protocol involves:

• Cell lysis in appropriate buffer (typically containing protease inhibitors)

• Clarification of lysate by centrifugation

• Affinity purification using the His-tag

• Buffer exchange to remove imidazole

• Quality control by SDS-PAGE analysis

SDS-PAGE analysis is essential to confirm purity, with recombinant chicken LZIC typically appearing as a single band corresponding to its expected molecular weight. Researchers have achieved >90% purity using these methods, suitable for most experimental applications .

How can recombinant chicken LZIC be effectively used in immunological studies?

Recombinant chicken LZIC serves as a valuable tool in immunological research through several applications:

• Antibody production: As an immunogen for raising antibodies against LZIC

• Blocking experiments: Pre-incubation of antibodies with recombinant LZIC can verify antibody specificity

• ELISA standard: As a calibration standard in quantitative assays

For blocking experiments, a recommended protocol involves:

• Pre-incubating the antibody with 100x molar excess of recombinant LZIC protein

• Incubating the mixture for 30 minutes at room temperature

• Using this pre-incubated mixture in your immunoassay

This approach is particularly valuable for validating antibody specificity in IHC/ICC and Western blot experiments . The recombinant protein can also serve as a positive control in assays detecting endogenous LZIC expression.

What methodologies are recommended for validating the structural integrity of recombinant chicken LZIC?

Validating the structural integrity of recombinant chicken LZIC requires multiple analytical approaches:

• SDS-PAGE analysis: For purity assessment and molecular weight confirmation

• Western blotting: For identity confirmation using anti-LZIC antibodies

• Mass spectrometry: For precise molecular weight determination and potential post-translational modification identification

• Circular dichroism: For secondary structure analysis

• Functional assays: To confirm biological activity

A comprehensive validation protocol should include:

• Running the purified protein on 15% SDS-PAGE with appropriate molecular weight markers

• Confirming a single band at the expected molecular weight (approximately 21 kDa plus any tag contribution)

• Performing mass spectrometry analysis to verify the intact mass matches the theoretical mass

• Testing functional activity relevant to known LZIC functions

These validation steps are essential to ensure experimental reproducibility and validity of subsequent research findings .

How can RT-PCR and qPCR be optimized for chicken LZIC expression analysis?

Optimizing RT-PCR and qPCR for chicken LZIC expression analysis requires careful consideration of several factors:

RT-PCR Protocol Optimization:

• RNA extraction from chicken tissues (e.g., ovarian follicular tissue, liver) using appropriate isolation kits

• Reverse transcription using oligo-dT primers to generate cDNA

• PCR amplification using LZIC-specific primers designed to span exon-exon junctions

• Thermal cycling conditions: Initial denaturation at 95°C for 120s, followed by 35-45 cycles (94°C for 15s, 57-60°C for 30s, 72°C for 15s)

qPCR Considerations:

• Reference gene selection: Multiple endogenous controls should be validated for stability in chicken tissues

• Primer efficiency testing: Standard curves should be generated for both target and reference genes

• Data analysis: Relative quantification using the 2^-ΔΔCt method for expression comparison

For accurate results, optimize primer annealing temperatures and validate specificity through melt curve analysis and product sequencing. All experiments should be performed in triplicate with appropriate negative controls to ensure reproducibility and accuracy .

What considerations are important when comparing chicken LZIC with mammalian orthologs in evolutionary studies?

When conducting evolutionary studies comparing chicken LZIC with mammalian orthologs, researchers should consider:

• Sequence alignment methodology: Multiple sequence alignment algorithms (e.g., MUSCLE, CLUSTAL) should be employed with appropriate gap penalties

• Phylogenetic analysis: Maximum likelihood or Bayesian methods provide robust evolutionary analyses

• Selection pressure analysis: dN/dS ratios can identify regions under positive or purifying selection

• Domain conservation: Analysis of functional domain conservation versus divergence informs functional evolution

Particular attention should be paid to:

• Conserved motifs that may indicate functional importance

• Differences in post-translational modification sites

• Variations in protein-protein interaction domains

• Species-specific insertions or deletions

Comparative analyses between chicken and human LZIC can reveal insights into the functional evolution of this protein family across vertebrate lineages. Notably, while human LZIC shares high identity with other mammalian orthologs (99% with mouse and rat), the chicken ortholog shows greater divergence while maintaining key structural elements .

What are the critical quality control parameters for recombinant chicken LZIC?

Critical quality control parameters for recombinant chicken LZIC include:

• Purity assessment:

  • SDS-PAGE analysis should demonstrate >90% purity

  • Absence of degradation products or contaminant bands

• Identity confirmation:

  • Western blot using anti-LZIC antibodies

  • Mass spectrometry validation of molecular weight and sequence coverage

• Functional validation:

  • Binding assays to known interaction partners

  • Activity assays relevant to known LZIC functions

• Endotoxin testing:

  • For applications sensitive to endotoxin contamination

  • Limulus Amebocyte Lysate (LAL) assay typically used

• Stability assessment:

  • Testing protein stability under various storage conditions

  • Freeze-thaw stability

For research applications, purity levels of >90% are generally acceptable, though higher purity (>95%) may be required for certain sensitive applications. Both yeast and E. coli expression systems have demonstrated the ability to produce high-purity recombinant chicken LZIC suitable for research applications .

What are common challenges in expressing recombinant chicken LZIC and how can they be addressed?

Researchers commonly encounter several challenges when expressing recombinant chicken LZIC:

When experiencing difficulties with E. coli expression, switching to a yeast expression system may provide benefits as it represents an economical and efficient eukaryotic system for both secretion and intracellular expression of chicken LZIC. For applications requiring more native-like protein, mammalian expression systems may be preferable despite higher costs and complexity .

Implementing these solutions requires systematic optimization, with each parameter changed individually while keeping others constant to determine optimal conditions for your specific research needs.