Recombinant Xenopus laevis Ankyrin repeat domain-containing protein 13C-B (ankrd13c-b), partial

What is Ankyrin repeat domain-containing protein 13C-B (ankrd13c-b) in Xenopus laevis?

Ankyrin repeat domain-containing protein 13C-B (ankrd13c-b) is a protein expressed in Xenopus laevis (African clawed frog) that belongs to the ankyrin repeat domain-containing family. Based on homology studies with mammalian counterparts, ankrd13c-b likely functions as a molecular chaperone associated with the endoplasmic reticulum (ER), where it appears to regulate the folding and trafficking of membrane proteins, particularly G protein-coupled receptors (GPCRs) . The protein contains characteristic ankyrin repeat motifs that mediate protein-protein interactions, making it important for cellular protein quality control mechanisms in the secretory pathway.

How is ankrd13c-b structurally characterized?

The ankrd13c-b protein from Xenopus laevis contains 513 amino acid residues based on the expression region information . Like other members of the ANKRD13 family, it likely contains multiple ankyrin repeat domains that form helix-turn-helix structures mediating protein-protein interactions. These structural motifs are essential for its chaperone function. Though the complete three-dimensional structure of ankrd13c-b has not been fully elucidated, homology modeling based on mammalian ANKRD13C suggests it contains a cytosolic domain that interacts with the C-terminus of GPCRs at the ER membrane interface .

What are the known functional roles of ankrd13c-b?

Based on studies of its mammalian homolog, ankrd13c-b likely functions as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and trafficking through the biosynthetic pathway . Specifically, it appears to:

• Interact with newly synthesized GPCRs at the ER membrane

• Promote receptor protein stability by inhibiting degradation of newly synthesized receptors

• Facilitate proper folding of GPCRs

• Regulate the exit of properly folded receptors from the ER

• Direct misfolded or unassembled receptors toward proteasome-mediated degradation pathways

What expression systems are most appropriate for recombinant ankrd13c-b production?

For recombinant ankrd13c-b expression, the baculovirus expression system has been successfully employed, as indicated by the product specifications . This system is particularly suitable for complex eukaryotic proteins that may require post-translational modifications. Alternative expression systems include:

Expression System Comparison Table for ankrd13c-b Production:

When selecting an expression system, researchers should consider their specific experimental requirements, including protein purity needs, functional integrity, and downstream applications.

What purification strategies are effective for recombinant ankrd13c-b?

Based on the methodologies described for related proteins, effective purification strategies for ankrd13c-b typically involve affinity chromatography techniques. For His-tagged versions, nickel-nitrilotriacetic acid-agarose resin is recommended, while GST-fusion proteins can be purified using glutathione-Sepharose . A typical purification workflow would include:

• Cell lysis under non-denaturing conditions in appropriate buffer systems

• Initial clarification by centrifugation

• Affinity chromatography based on the fusion tag

• Optional ion exchange chromatography for further purification

• Size exclusion chromatography for final polishing

• Quality assessment by SDS-PAGE and Western blotting

The purity should be verified through SDS-PAGE analysis, with expected purity levels exceeding 85% as indicated in the product specifications .

What are the optimal storage and handling conditions for recombinant ankrd13c-b?

According to the product specifications, recombinant ankrd13c-b requires careful handling to maintain stability and activity . Optimal storage and handling recommendations include:

• Avoid repeated freeze-thaw cycles as they significantly reduce protein stability

• For lyophilized protein: store at -20°C/-80°C with shelf life of approximately 12 months

• For protein in solution: store at -20°C/-80°C with expected shelf life of approximately 6 months

• Working aliquots may be kept at 4°C for up to one week

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

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

• Use appropriate buffer systems that maintain protein stability

How can recombinant ankrd13c-b be used in GPCR trafficking studies?

Recombinant ankrd13c-b can be employed to investigate GPCR trafficking mechanisms in several experimental approaches:

• Co-expression studies: Transfecting cells with ankrd13c-b and GPCRs to observe effects on receptor expression, localization, and degradation using confocal microscopy and subcellular fractionation

• Protein-protein interaction assays: Using purified recombinant ankrd13c-b in pull-down assays with GPCR C-terminal domains to map interaction sites and binding properties

• Loss-of-function experiments: Employing siRNA-mediated knockdown of endogenous ankrd13c-b to assess impact on GPCR biogenesis and trafficking

• Pulse-chase experiments: Determining the effects of ankrd13c-b on the stability and turnover rates of newly synthesized GPCRs

• Proteasomal degradation studies: Investigating how ankrd13c-b regulates ER-associated degradation of misfolded GPCRs through proteasome-mediated pathways

These approaches can provide insights into the fundamental mechanisms of GPCR quality control and trafficking that may be conserved across vertebrate species.

Why is Xenopus laevis an important model for studying ankrd13c-b?

Xenopus laevis serves as a valuable model organism for studying ankrd13c-b for several reasons:

• Evolutionary conservation: Studying ankrd13c-b in Xenopus provides insights into conserved mechanisms of protein quality control across vertebrates

• Experimental advantages: Xenopus offers large, abundant eggs and readily manipulated embryos, facilitating biochemical and developmental studies

• Cell-free extract systems: Xenopus egg extracts provide powerful biochemical systems for studying protein interactions and functions in vitro

• Complementary to mammalian studies: Findings in Xenopus can often be translated to mammalian systems due to conserved cellular, developmental, and genomic organization

• Enhanced by genetic tools: While Xenopus laevis has an allotetraploid genome, the related Xenopus tropicalis with its diploid genome offers complementary genetic approaches for functional studies

The combination of these advantages makes Xenopus an excellent system for exploring the fundamental roles of ankrd13c-b in cellular processes.

What technical challenges arise in studying protein-protein interactions involving ankrd13c-b?

Investigating protein-protein interactions involving ankrd13c-b presents several technical challenges that researchers should consider:

• Membrane protein complexes: Since ankrd13c-b likely interacts with membrane proteins at the ER interface, standard interaction assays may need modification to accommodate membrane environments

• Transient interactions: Chaperone-substrate interactions are often transient and may be difficult to capture without chemical crosslinking or specialized techniques

• Expression level considerations: Overexpression may lead to artificial interactions or ER retention, requiring careful titration of expression levels

• Native vs. recombinant proteins: Interactions observed with recombinant proteins may differ from those in the native cellular environment

• Buffer composition effects: The binding buffer composition (e.g., salt concentration, detergents, reducing agents) can significantly impact interaction detection

To address these challenges, researchers should employ multiple complementary approaches, including:

• In vitro binding assays with purified components

• Co-immunoprecipitation from cell lysates

• Proximity labeling techniques

• Fluorescence resonance energy transfer (FRET)

• Split-protein complementation assays

How can genetic approaches in Xenopus be applied to study ankrd13c-b function?

Several genetic approaches can be employed to study ankrd13c-b function in Xenopus:

• Morpholino oligonucleotides: These can be used to knock down ankrd13c-b expression post-fertilization, allowing for temporal control of gene expression

• CRISPR/Cas9 genome editing: While more challenging in Xenopus laevis due to its allotetraploid genome, CRISPR approaches can be used, particularly in Xenopus tropicalis, to generate targeted mutations

• Zinc-finger nucleases: These have been successfully used in Xenopus tropicalis for targeted gene disruption and could be applied to ankrd13c-b

• Transgenic approaches: Efficient transgenesis methods in Xenopus enable overexpression studies, dominant negative constructs, or reporter gene fusions

• TILLING (Targeting Induced Local Lesions in Genomes): Screening through mutagenized populations to identify mutations in ankrd13c-b

When designing genetic studies, researchers should consider the potential functional redundancy with other ankrd13 family members and adapt their approach accordingly.

What are the emerging techniques for studying ankrd13c-b's role in proteostasis?

Several cutting-edge approaches are advancing our understanding of chaperone proteins like ankrd13c-b:

• Proximity-dependent biotin identification (BioID): This technique can identify transient protein interactions in living cells by fusing ankrd13c-b to a biotin ligase that biotinylates nearby proteins

• Quantitative interactomics: Mass spectrometry-based approaches can identify the complete set of ankrd13c-b interacting partners and how these change under different conditions

• Live-cell imaging of protein trafficking: Using photoactivatable or photoconvertible fluorescent proteins fused to ankrd13c-b or its client proteins to track dynamic trafficking events

• Cryo-electron microscopy: This can potentially resolve the structure of ankrd13c-b in complex with client proteins at near-atomic resolution

• Fluorescence recovery after photobleaching (FRAP): This technique can measure the dynamics of ankrd13c-b associations with ER membranes and client proteins

These emerging techniques can provide unprecedented insights into the dynamics, specificity, and regulatory mechanisms of ankrd13c-b in cellular proteostasis.

What are common challenges in recombinant ankrd13c-b expression and how can they be overcome?

Researchers commonly encounter several challenges when expressing recombinant ankrd13c-b:

Troubleshooting Table for Recombinant ankrd13c-b Expression:

When optimizing expression, researchers should perform small-scale pilot experiments to identify optimal conditions before scaling up production.

How can specific antibodies against ankrd13c-b be generated and validated?

Generating specific antibodies against ankrd13c-b requires careful planning and validation:

• Antigen design strategies:

  • Synthetic peptides from unique regions (e.g., N-terminus residues 7-20, as used for mammalian ANKRD13C)

  • Recombinant protein fragments expressing distinct domains

  • Full-length protein for monoclonal antibody generation

• Antibody production approaches:

  • Polyclonal antibodies in rabbits using KLH-conjugated peptides

  • Monoclonal antibodies using purified recombinant protein

  • Recombinant antibodies through phage display technology

• Essential validation steps:

  • Western blot against recombinant protein and native tissue lysates

  • Peptide competition assays with increasing concentrations (0-1 mg/ml) of cognate peptide

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence with subcellular markers

  • Knockdown/knockout controls to confirm specificity

• Cross-reactivity considerations:

  • Test against related ANKRD family proteins

  • Validate across species if cross-reactivity is desired

  • Examine tissues known to express or not express the target

Proper antibody validation is critical for ensuring experimental reproducibility and reliable results in ankrd13c-b research.

What controls should be included in ankrd13c-b functional assays?

Robust experimental design for ankrd13c-b functional studies should include several critical controls:

• For protein-protein interaction studies:

  • GST-only or other tag-only controls to identify tag-mediated interactions

  • Unrelated proteins of similar size/structure to test specificity

  • Competition with excess unlabeled protein or interacting peptides

  • Mutated binding domain variants to map interaction sites

• For cellular localization experiments:

  • Co-localization with established ER markers

  • Fractionation controls to verify membrane association

  • Dominant negative constructs lacking functional domains

• For functional chaperone assays:

  • Positive controls with known chaperone proteins

  • Non-GPCR membrane proteins as specificity controls (e.g., VSVG)

  • Cytosolic proteins as negative controls (e.g., GFP, GRK2)

  • Time-course experiments to distinguish early vs. late effects

• For expression/degradation studies:

  • Proteasome inhibitors to confirm degradation pathway

  • Pulse-chase controls with established half-life proteins

  • Multiple GPCR substrates to establish range of activity

Inclusion of these controls ensures that experimental results can be accurately interpreted and that the specific functions of ankrd13c-b can be distinguished from non-specific effects.

What are promising areas for future ankrd13c-b research?

Several promising research directions could significantly advance our understanding of ankrd13c-b:

• Comparative studies across species: Investigating functional conservation and divergence between Xenopus ankrd13c-b and its mammalian counterparts could reveal evolutionarily conserved mechanisms of GPCR quality control

• Developmental regulation: Exploring how ankrd13c-b expression and function change throughout Xenopus development could uncover stage-specific roles in organogenesis and tissue differentiation

• Substrate specificity determinants: Identifying the molecular features that determine which GPCRs interact with ankrd13c-b could reveal fundamental principles of chaperone-client recognition

• Structural biology approaches: Resolving the three-dimensional structure of ankrd13c-b alone and in complex with client proteins would provide mechanistic insights into its chaperone function

• Integration with other quality control pathways: Investigating how ankrd13c-b coordinates with other ER quality control mechanisms would illuminate the broader proteostasis network

These research directions could not only expand our understanding of ankrd13c-b biology but also contribute to broader knowledge of protein quality control mechanisms across species.

How can findings from ankrd13c-b research be translated to human disease contexts?

Research on ankrd13c-b in Xenopus has potential translational implications for human disease:

• GPCR-related disorders: Since ankrd13c-b regulates GPCR biogenesis and trafficking, insights could inform therapeutic approaches for diseases involving GPCR dysfunction

• ER stress and proteostasis disorders: Understanding ankrd13c-b's role in ER quality control may provide insights into diseases characterized by protein misfolding and aggregation

• Developmental disorders: If ankrd13c-b plays critical roles in Xenopus development, human ANKRD13C might similarly impact developmental processes relevant to congenital disorders

• Drug development opportunities: The protein interaction interfaces between ankrd13c-b and its clients could represent novel therapeutic targets for modulating GPCR expression

• Biomarker potential: Expression patterns of ANKRD13C in human tissues could potentially serve as biomarkers for specific disease states

These translational aspects highlight the broader significance of basic research on ankrd13c-b beyond its immediate biological context.