Recombinant Chicken Charged multivesicular body protein 4b (CHMP4B)

What is the molecular structure and function of Chicken CHMP4B?

CHMP4B is a homologue of yeast Snf7p (Vps32p) and belongs to the CHMP family implicated in multivesicular body (MVB) sorting. As a component of ESCRT-III (endosomal sorting complex required for transport III), it participates in the degradation of surface receptor proteins and formation of endocytic multivesicular bodies . The calculated molecular weight of CHMP4B is approximately 25 kDa, containing 224 amino acids, though post-translational modification can cause it to appear between 25-33 kDa in experimental analyses .

How does Chicken CHMP4B compare structurally to mammalian homologues?

Chicken CHMP4B shares significant sequence homology with mammalian CHMP4B variants, particularly in the core functional domains. Based on comparative analysis with human CHMP4B (UNIPROT ID: Q9H444), the chicken variant maintains conserved regions essential for interaction with Alix, a multifunctional adaptor protein . Unlike mammalian systems where CHMP4B interacts with specific partners including CHMP4A and CHMP4C, the chicken protein's interaction network may differ in certain aspects while maintaining core ESCRT-III functionality.

What are the key functional domains of Chicken CHMP4B?

Chicken CHMP4B contains several conserved functional domains typical of ESCRT-III family proteins:

• N-terminal core domain that mediates membrane association

• Central coiled-coil region involved in protein-protein interactions

• C-terminal autoinhibitory domain that regulates assembly

These domains collectively enable CHMP4B to participate in membrane deformation, vesicle formation, and recruitment of other ESCRT machinery components in cellular processes.

What are the optimal expression systems for recombinant Chicken CHMP4B production?

For optimal expression of recombinant Chicken CHMP4B, bacterial expression systems using E. coli (particularly BL21(DE3) strains) are commonly employed for basic research applications. For studies requiring post-translational modifications, insect cell expression systems (Sf9 or Hi5 cells with baculovirus vectors) or mammalian expression systems (HEK293 or CHO cells) are recommended. When designing expression constructs, incorporating a cleavable His-tag or Fc-chimera approach similar to other recombinant proteins can facilitate purification .

Expression yields typically vary by system:

How should recombinant Chicken CHMP4B be purified for maximum activity retention?

Purification of recombinant Chicken CHMP4B requires careful buffer selection to maintain protein stability and activity. A multi-step purification strategy is recommended:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

• Intermediate purification using ion exchange chromatography

• Polishing step using size exclusion chromatography

Buffer conditions should typically include:

• pH 7.4-8.0 phosphate or HEPES buffer

• 150-300 mM NaCl to maintain solubility

• 5-10% glycerol as a stabilizing agent

• 1-5 mM DTT or 2-ME to maintain reduced cysteine residues

For long-term storage, the purified protein should be lyophilized from a 0.2 μm filtered solution in PBS, similar to other recombinant proteins , and reconstituted at 100 μg/mL in sterile PBS for experimental use.

What quality control methods are essential for verifying recombinant Chicken CHMP4B integrity?

To ensure experimental reproducibility, multiple quality control analyses should be performed:

• SDS-PAGE and Western blot analysis using validated antibodies to confirm size and immunoreactivity

• Mass spectrometry for accurate molecular weight determination and sequence verification

• Dynamic light scattering to assess homogeneity and aggregation state

• Circular dichroism spectroscopy to verify secondary structure integrity

• Functional binding assays to confirm interaction with known partners

For western blot verification, antibodies validated against mammalian CHMP4B with cross-reactivity to chicken variants should be used at dilutions of 1:2000-1:10000 .

How can functional assays be designed to assess Chicken CHMP4B activity in vitro?

Functional characterization of recombinant Chicken CHMP4B can be performed through several complementary approaches:

• Liposome Binding Assays: Using giant unilamellar vesicles (GUVs) containing specific phospholipids to assess membrane binding capacity

• ESCRT-III Assembly Assays: Monitoring polymer formation through light scattering or electron microscopy

• Protein-Protein Interaction Studies: Using pull-down assays, surface plasmon resonance, or isothermal titration calorimetry to quantify binding to partners like Alix

• Membrane Deformation Assays: Assessing the protein's ability to induce curvature in model membranes

For binding assays, the protocol should be adapted from established functional ELISA methods, where immobilized recombinant CHMP4B (at 5 μg/mL, 100 μL per well) can be used to evaluate binding to interaction partners across a concentration range of 0.8-100 ng/mL .

What are the established protocols for studying Chicken CHMP4B intracellular localization?

For intracellular localization studies of Chicken CHMP4B:

• Immunofluorescence Microscopy:

  • Fix cells using 4% paraformaldehyde (10 minutes, room temperature)

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

  • Block with 3% BSA in PBS (1 hour)

  • Incubate with primary antibodies at 1:200-1:500 dilution (overnight, 4°C)

  • Wash and add fluorophore-conjugated secondary antibodies

  • Counterstain with DAPI and mount for imaging

• Live Cell Imaging:

  • Generate fusion constructs with fluorescent tags (GFP, mCherry)

  • Transfect chicken cell lines (e.g., DF-1 fibroblasts) using lipofection

  • Perform time-lapse microscopy to track protein dynamics

  • Co-express with markers for endosomes, multivesicular bodies, or other ESCRT components

For flow cytometry applications, similar to mammalian CHMP4B studies, use 0.25 μg of antibody per 10^6 cells in a 100 μl suspension .

How should recombinant Chicken CHMP4B be used in ESCRT-III reconstitution experiments?

For in vitro reconstitution of ESCRT-III function using recombinant Chicken CHMP4B:

• Preparation of Model Membranes:

  • Generate liposomes containing 60% DOPC, 30% DOPS, and 10% PI(3)P

  • Extrude through 200 nm filters to create uniform size distribution

  • Label with fluorescent lipids for visualization if needed

• Sequential Addition Protocol:

  • Add early ESCRT components (ESCRT-0, I, II) at 0.5-1 μM

  • Introduce recombinant Chicken CHMP4B at 1-5 μM concentration

  • Add remaining ESCRT-III subunits sequentially

  • Include ATP and appropriate cofactors

• Analysis Methods:

  • Negative-stain electron microscopy to visualize assembled structures

  • Fluorescence microscopy for labeled components

  • Light scattering to monitor assembly kinetics

  • Atomic force microscopy to assess membrane deformation

How does Chicken CHMP4B function differ in embryonic versus adult tissues?

Chicken CHMP4B shows developmental stage-specific expression and functional patterns. In embryonic tissues, CHMP4B is typically expressed at higher levels, reflecting the increased membrane remodeling activity during development. The protein's subcellular distribution may vary between embryonic and adult tissues, with embryonic cells showing more dynamic localization patterns associated with rapid cell division and tissue morphogenesis.

Comparative analysis of embryonic versus adult expression profiles:

What is the comparative role of CHMP4B in avian versus mammalian developmental contexts?

In avian systems, CHMP4B appears to have both conserved and divergent functions compared to mammalian systems. While core ESCRT-III functions are preserved, chicken CHMP4B may have unique roles in avian-specific developmental processes:

• Left-Right Asymmetry: Similar to the role of chicken Caronte in left-right patterning through BMP antagonism , CHMP4B might participate in specialized vesicular transport events that establish embryonic asymmetry.

• Neural Crest Development: Avian models have distinctive neural crest migration patterns, and CHMP4B may regulate receptor trafficking that guides these migrations differently than in mammals.

• Feather Morphogenesis: CHMP4B potentially contributes to the specialized membrane dynamics required for feather bud formation and growth.

The binding profiles of chicken versus mammalian CHMP4B variants show interesting differences in affinity and specificity, possibly reflecting evolutionary adaptations to species-specific developmental requirements.

How does CHMP4B interact with other ESCRT components in chicken development?

During chicken development, CHMP4B forms dynamic interaction networks with other ESCRT machinery components:

• Complex Assembly Sequence:

  • ESCRT-0: Initial recognition of ubiquitinated cargo

  • ESCRT-I/II: Membrane deformation initiation

  • ESCRT-III (including CHMP4B): Membrane scission

  • Vps4: Disassembly and recycling

• Developmental Regulation:

  • Stage-specific phosphorylation of CHMP4B modulates its interaction with other ESCRT-III subunits

  • Alternative splicing generates developmental variants with altered binding properties

  • Expression timing of CHMP4B relative to other ESCRT components affects complex assembly efficiency

• Tissue-Specific Interactions:

  • Neural tissue: Enhanced interaction with neuronal-specific ESCRT adaptors

  • Immune tissues: Modified interactions supporting specialized endosomal sorting in developing B and T cells

  • Epithelial tissues: Interactions promoting polarized protein trafficking

How can gene editing techniques be applied to study Chicken CHMP4B function?

Modern gene editing approaches provide powerful tools for investigating Chicken CHMP4B function:

• CRISPR-Cas9 Strategies:

  • Generate knockout chicken cell lines by targeting conserved exons

  • Create point mutations to disrupt specific domains (N-terminal core, C-terminal MIM domain)

  • Introduce tags for endogenous protein visualization

  • Develop conditional knockouts using floxed alleles

• Recommended gRNA Design:

  • Target sequences with high conservation between chicken and mammalian CHMP4B

  • Avoid regions with potential off-target effects in the chicken genome

  • Consider PAM accessibility in the genomic context

• Validation Methods:

  • Western blot verification using dilutions of 1:2000-1:10000 as established for mammalian CHMP4B

  • Genomic sequencing to confirm editing efficiency

  • Functional assays to assess phenotypic consequences

What proteomics approaches are most effective for identifying Chicken CHMP4B interaction partners?

To comprehensively map the Chicken CHMP4B interactome:

• Proximity-Based Labeling:

  • BioID fusion constructs expressing CHMP4B-BirA to biotinylate proximal proteins

  • APEX2 fusion for electron microscopy-compatible proximity labeling

  • TurboID for rapid in vivo labeling of transient interactions

• Affinity Purification-Mass Spectrometry:

  • Tandem affinity purification using dual tags on CHMP4B

  • Quantitative analysis using SILAC or TMT labeling

  • Crosslinking mass spectrometry to capture transient complexes

• Interaction Network Analysis:

  • Computational prediction of conserved interaction motifs

  • Comparative analysis with mammalian CHMP4B interactomes

  • Validation of key interactions using recombinant proteins

  • Functional classification of interaction partners

How can high-resolution imaging techniques advance our understanding of Chicken CHMP4B dynamics?

Advanced imaging approaches offer unprecedented insights into CHMP4B dynamics:

• Super-Resolution Microscopy:

  • STORM/PALM: Achieve 20-30 nm resolution to visualize individual ESCRT-III filaments

  • SIM: Capture dynamic assembly/disassembly with improved temporal resolution

  • Expansion microscopy: Physically expand samples for enhanced resolution of CHMP4B structures

• Live-Cell Advanced Imaging:

  • Lattice light-sheet microscopy for rapid 3D imaging with reduced phototoxicity

  • FRAP/FLIP analyses to measure CHMP4B turnover rates on membranes

  • Single-particle tracking to follow individual CHMP4B molecules

• Correlative Light and Electron Microscopy (CLEM):

  • Combine fluorescence localization with ultrastructural context

  • Visualize CHMP4B in relation to membrane deformation events

  • Map protein distribution within MVB formation sites

What are common challenges in producing active recombinant Chicken CHMP4B?

Researchers frequently encounter several challenges when working with recombinant Chicken CHMP4B:

• Protein Aggregation Issues:

  • Problem: CHMP4B has intrinsic tendency to self-associate

  • Solution: Include 5-10% glycerol in buffers; maintain protein at low concentrations (<1 mg/ml); use mild detergents (0.01% DDM) for stabilization

• Incorrect Folding:

  • Problem: Bacterial expression systems may produce misfolded protein

  • Solution: Co-express with chaperones (GroEL/ES); use slower induction protocols (reduce IPTG to 0.1 mM, lower temperature to 18°C)

• Low Solubility:

  • Problem: The hydrophobic regions of CHMP4B reduce solubility

  • Solution: Express as fusion with solubility enhancers (MBP, SUMO); optimize salt concentration in buffers (test range from 150-500 mM NaCl)

• Proteolytic Degradation:

  • Problem: CHMP4B can be susceptible to proteolysis

  • Solution: Include protease inhibitor cocktails during purification; minimize handling time; store in aliquots to avoid freeze-thaw cycles

How can researchers resolve Western blot detection issues with Chicken CHMP4B antibodies?

When encountering detection problems using antibodies against Chicken CHMP4B:

• Low Signal Intensity:

  • Optimize primary antibody concentration (start with 1:2000 and adjust as needed)

  • Increase incubation time (overnight at 4°C)

  • Use signal enhancement systems (HRP amplification reagents)

  • Consider loading more protein (up to 50 μg per lane)

• Non-specific Binding:

  • Increase blocking stringency (5% milk or BSA in TBST)

  • Add 0.1-0.5% Tween-20 in wash buffers

  • Pre-absorb antibody with non-relevant tissues

  • Include competing proteins (1% BSA) during antibody incubation

• Unexpected Band Patterns:

  • Remember that post-translational modifications can cause CHMP4B to appear between 25-33 kDa

  • Use positive controls (human or mouse samples) for comparison

  • Consider testing multiple antibodies targeting different epitopes

  • Verify with knockout controls or siRNA-treated samples

What strategies address variability in CHMP4B functional assays?

To improve reproducibility in functional characterization of Chicken CHMP4B:

• Membrane Binding Assays:

  • Standardize liposome composition and preparation methods

  • Include internal controls (known ESCRT proteins) in each experiment

  • Use multiple complementary techniques (flotation, pelleting, FRET-based assays)

  • Analyze binding kinetics rather than endpoint measurements only

• Cellular Localization Studies:

  • Establish clear criteria for scoring localization patterns

  • Use automated image analysis algorithms to reduce subjective interpretation

  • Include multiple time points to capture dynamic processes

  • Validate with multiple cell types to ensure generalizability

• Protein-Protein Interaction Measurements:

  • Perform assays at multiple protein concentrations to generate binding curves

  • Control for buffer conditions that may affect interactions

  • Use multiple methods (co-IP, ELISA, SPR) for cross-validation

  • Include both positive and negative interaction controls

What are emerging trends in Chicken CHMP4B research?

The field of Chicken CHMP4B research is evolving rapidly with several promising directions:

• Avian-Specific Developmental Roles: Investigations into unique functions during chicken embryogenesis that may differ from mammalian systems.

• Comparative Evolutionary Studies: Analysis of CHMP4B structural and functional conservation across vertebrate lineages to understand evolutionary adaptations.

• Agricultural Applications: Potential roles in egg formation, embryo development, and disease resistance relevant to poultry science.

• Model System Advantages: Leveraging the accessibility of the chicken embryo for in vivo manipulation and visualization of ESCRT-dependent processes.

What methodological advances will accelerate Chicken CHMP4B research?

Future progress in this field will be driven by several methodological innovations:

• Advanced Genetic Tools:

  • Improved CRISPR delivery methods for chicken embryos and cell lines

  • Inducible expression systems for temporal control of CHMP4B variants

  • Single-cell technologies to analyze CHMP4B function in heterogeneous tissues

• Structural Biology Approaches:

  • Cryo-EM analysis of chicken ESCRT-III assemblies

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Integrative structural modeling combining multiple experimental datasets

• Cellular Engineering Platforms:

  • Organoid systems recapitulating chicken tissue architecture

  • Microfluidic devices for controlled manipulation of ESCRT-dependent processes

  • Synthetic biology approaches to reconstitute minimal ESCRT systems