Recombinant Chicken Kelch-like protein 13 (KLHL13), partial

What is Kelch-like protein 13 (KLHL13) and what is its fundamental role in cellular processes?

KLHL13 functions as a substrate adaptor protein that, in complex with KLHL9 and Cullin 3 (CUL3), forms an E3 ubiquitin ligase complex. This complex plays a crucial role in the ubiquitin-proteasome system, targeting specific proteins for degradation. Research has confirmed that KLHL13, together with KLHL9 and CUL3, interacts with proteins such as Insulin Receptor Substrate-1 (IRS1), promoting its proteasomal degradation . The KLHL13-containing E3 ligase complex therefore participates in post-translational regulation of protein abundance, which can significantly impact downstream signaling pathways including insulin signaling. Understanding this function is essential for researchers investigating protein quality control mechanisms and signaling pathway regulation in chicken tissues.

How does the structural organization of KLHL13 contribute to its function?

Like other Kelch-like family proteins, KLHL13 contains multiple functional domains that enable its role in the ubiquitin-proteasome system. While the chicken-specific structure requires experimental verification, comparative analysis with other species suggests KLHL13 likely contains: (1) an N-terminal BTB (Broad Complex, Tramtrack, and Bric-a-brac) domain that mediates binding to CUL3; (2) a central BACK domain that contributes to complex formation; and (3) a C-terminal Kelch domain consisting of multiple Kelch repeats that form a β-propeller structure involved in substrate recognition and binding. This multi-domain structure allows KLHL13 to simultaneously interact with both the E3 ligase machinery (via the BTB domain) and specific substrate proteins (via the Kelch domain), thereby functioning as a critical mediator in targeted protein degradation pathways.

What experimental approaches can determine the expression pattern of KLHL13 in chicken tissues?

Researchers investigating chicken KLHL13 expression patterns should consider a multi-faceted approach combining transcriptomic and proteomic methodologies. RNA-seq analysis of different chicken tissues can establish baseline mRNA expression levels across tissue types. This should be complemented by quantitative PCR (qPCR) with primers specific to chicken KLHL13 for more targeted expression analysis. At the protein level, Western blotting and immunohistochemistry using validated antibodies against chicken KLHL13 can visualize protein distribution in tissues. More advanced techniques like mass spectrometry-based proteomics can provide quantitative expression data, similar to the Orbitrap tandem mass spectrometry approach described for confirming protein identity in recombinant protein studies . These complementary techniques enable researchers to develop comprehensive tissue expression maps that inform downstream functional studies.

How does KLHL13 contribute to substrate specificity in E3 ubiquitin ligase complexes?

The substrate specificity of KLHL13-containing E3 ligase complexes emerges from the coordinated activity of multiple proteins working in concert. BioID analysis and co-immunoprecipitation experiments have confirmed that KLHL13 and KLHL9 function as substrate-specific adaptor proteins that associate with CUL3 and mediate interactions with target proteins such as IRS1 . The precise mechanism of substrate recognition likely involves: (1) the formation of specific binding interfaces created by the β-propeller structure of the Kelch domain; (2) recognition of specific structural motifs or post-translational modifications on target proteins; and (3) potentially cooperative binding with KLHL9, which may expand the range of recognizable substrates. Researchers studying recombinant chicken KLHL13 should consider designing experiments that investigate how sequence variations in the Kelch domain might affect substrate recognition profiles compared to mammalian orthologs.

What are the functional consequences of manipulating KLHL13 expression levels in cellular systems?

Experimental manipulation of KLHL13 expression has significant downstream effects on cellular signaling pathways. siRNA-mediated knockdown of KLHL13 resulted in approximately 1.5-fold increase in IRS1 protein content in wild-type cells and a more pronounced 2-fold increase in ATG16L1 knockout cells . This elevation in IRS1 levels was accompanied by enhanced insulin signaling, demonstrating KLHL13's role in regulating metabolic processes. When designing experiments with recombinant chicken KLHL13, researchers should anticipate that overexpression may accelerate degradation of target substrates, potentially suppressing pathways dependent on those substrates. Conversely, dominant-negative approaches using partial KLHL13 constructs might disrupt endogenous KLHL13 function, stabilizing target proteins and amplifying dependent signaling cascades. These experimental manipulations provide valuable tools for dissecting KLHL13's role in various cellular contexts.

What are the optimal expression systems for producing recombinant chicken KLHL13?

For efficient production of recombinant chicken KLHL13, E. coli-based expression systems offer several advantages. Bacterial systems such as E. coli Rosetta (DE3) are particularly suitable due to their ease of handling, safety profile, and genetic traits that make them receptive to foreign DNA . These systems can produce large quantities of protein cost-effectively compared to alternative expression hosts like yeast or mammalian cells . When expressing chicken KLHL13, researchers should consider codon optimization to address potential codon bias issues, as has been necessary for other A/T-rich genes. Expression vectors containing solubility-enhancing tags (such as SUMO or MBP) can improve yield of correctly folded protein. For partial KLHL13 constructs, careful design of domain boundaries is critical to ensure proper folding of the expressed protein fragment. Temperature optimization during induction is also essential, as lower temperatures (16-18°C) often improve solubility for challenging proteins.

What purification strategies are most effective for recombinant chicken KLHL13?

A multi-step purification approach is recommended for obtaining high-purity recombinant chicken KLHL13. Initial purification via Immobilized Metal Affinity Chromatography (IMAC) provides efficient capture of His-tagged protein constructs, as demonstrated in similar proteins . After IMAC, size exclusion chromatography (SEC) helps remove aggregates and contaminating proteins of different molecular weights. For higher purity requirements, ion exchange chromatography can provide additional resolution based on the protein's charge properties. Throughout the purification process, samples should be analyzed by SDS-PAGE to monitor purity and integrity. Final verification through Orbitrap tandem mass spectrometry, as utilized for PfK13 proteins, confirms protein identity with high confidence . The successful purification of recombinant KLHL13 enables downstream functional studies, interaction analyses, and structural investigations.

How can researchers verify the quality and integrity of purified recombinant chicken KLHL13?

Quality assessment of purified recombinant chicken KLHL13 requires multiple complementary approaches. SDS-PAGE analysis provides initial verification of protein size and purity, with chicken KLHL13 expected to migrate at approximately 65-70 kDa (for full-length) or at the appropriate size for partial constructs. Western blotting with anti-KLHL13 antibodies confirms protein identity. Mass spectrometry analysis, particularly Orbitrap tandem mass spectrometry as described for PfK13 proteins , offers definitive identification through peptide matching. Researchers should examine parameters such as experimental q-value, number of peptides identified, and unique peptide sequences, similar to the analysis shown for PfK13 variants where successful identification resulted in zero q-values . Circular dichroism spectroscopy can verify proper secondary structure folding, while dynamic light scattering assesses sample homogeneity and aggregation state. These quality control steps are essential before proceeding to functional and interaction studies.

What methodologies are most appropriate for studying KLHL13 interactions with potential substrate proteins?

Multiple complementary techniques enable comprehensive characterization of KLHL13 protein interactions. Proximity-dependent biotin identification (BioID) has proven valuable for identifying novel KLHL13 interaction partners, including its association with IRS1 . This approach allows for detection of both stable and transient interactions in the cellular context. Co-immunoprecipitation experiments provide validation of direct protein-protein interactions, as successfully demonstrated for the KLHL13-IRS1 interaction . For quantitative binding parameters, researchers should consider biophysical methods including Isothermal Titration Calorimetry (ITC) and fluorescence spectroscopy, which have been effectively applied to analyze binding interactions for related proteins . Surface Plasmon Resonance (SPR) offers additional advantages by measuring association and dissociation kinetics in real-time. When designing interaction studies, researchers should include appropriate controls, such as testing binding to known interaction partners (KLHL9, CUL3) alongside novel candidate substrates.

How can researchers assess the functional activity of recombinant chicken KLHL13 in ubiquitination assays?

In vitro ubiquitination assays provide direct evidence of KLHL13's functional role within the E3 ubiquitin ligase complex. A complete reconstituted system requires purified E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, recombinant KLHL13, CUL3, RBX1, and potential substrate proteins like IRS1. The reaction is initiated by adding ubiquitin and ATP, with ubiquitination detected by Western blotting using antibodies against the substrate or ubiquitin. Time-course experiments can reveal the kinetics of ubiquitination, while comparing wild-type KLHL13 with mutant versions can identify critical residues for substrate recognition or CUL3 binding. Cell-based ubiquitination assays complement in vitro approaches by expressing recombinant chicken KLHL13 in appropriate cell lines and monitoring substrate ubiquitination and degradation. Proteasome inhibitors (e.g., MG132) can be used to accumulate ubiquitinated substrates, facilitating their detection and confirming KLHL13's role in targeting specific proteins for degradation.

What cell-based systems are most suitable for studying recombinant chicken KLHL13 function?

The selection of appropriate cellular systems is critical for studying recombinant chicken KLHL13 function. Chicken cell lines (such as DF-1 fibroblasts or HD11 macrophage-like cells) provide the most relevant cellular context with compatible signaling networks. Alternatively, mammalian cell lines with KLHL13 knockout can serve as a "clean background" for introducing chicken KLHL13 variants. The research on KLHL13's role in insulin signaling utilized ATG16L1 knockout mouse embryonic fibroblasts (MEFs) to reveal its function in IRS1 regulation , suggesting that MEFs can be suitable hosts for functional studies. When designing cell-based assays, researchers should consider: (1) transfection efficiency of the cell line; (2) expression levels of endogenous KLHL13 and potential redundant proteins; (3) presence of relevant pathway components (e.g., insulin signaling machinery); and (4) availability of appropriate assay readouts. Inducible expression systems allow for controlled timing and level of recombinant KLHL13 expression, minimizing potential artifacts from constitutive overexpression.

How should researchers analyze and interpret fluorescence spectroscopy data for KLHL13-substrate interactions?

Fluorescence spectroscopy provides valuable insights into protein-protein interactions through changes in intrinsic fluorescence upon binding. When analyzing fluorescence data for KLHL13-substrate interactions, researchers should: (1) establish baseline measurements for recombinant KLHL13 alone; (2) perform titration experiments with potential binding partners; and (3) analyze changes in fluorescence intensity and emission maxima. Increased fluorescence intensity, as observed with PfK13 mutants (N537I: 83 a.u., V494I: 143 a.u.) compared to wild-type (33 a.u.), can indicate conformational changes affecting tryptophan exposure . Shifts in emission maximum wavelength provide additional information about changes in the microenvironment of fluorescent residues. Data should be fitted to appropriate binding models to determine dissociation constants (Kd) and other thermodynamic parameters. Statistical analysis should include multiple independent experiments with error calculations and significance testing to ensure reproducibility and reliability of the observed interaction parameters.

What considerations are important when interpreting the impact of KLHL13 on cellular signaling pathways?

Interpreting KLHL13's impact on cellular signaling requires careful experimental design and comprehensive pathway analysis. When manipulating KLHL13 levels (through overexpression or knockdown), researchers should: (1) confirm changes in KLHL13 expression at both mRNA and protein levels; (2) assess effects on multiple pathway components, not just direct substrates; and (3) include time-course analyses to distinguish primary from secondary effects. For insulin signaling, research has shown that KLHL13 knockdown increased IRS1 protein levels and enhanced downstream insulin signaling . Similar approaches can be applied to investigate chicken KLHL13's role in various pathways. Integration of transcriptomic and proteomic data provides a systems-level view of pathway alterations. When comparing results across experimental systems or species, researchers should consider potential differences in KLHL13 substrate specificity, expression levels, or pathway architecture that might influence functional outcomes.

How can researchers differentiate between direct and indirect effects of KLHL13 on cellular pathways?

Distinguishing direct from indirect effects of KLHL13 requires strategic experimental approaches and careful data interpretation. To identify direct effects, researchers should consider: (1) in vitro binding and ubiquitination assays with purified components; (2) proximity labeling approaches like BioID to identify proteins physically close to KLHL13 in cells ; and (3) rapid induction systems that allow monitoring of immediate responses before secondary effects occur. Substrate trap approaches, using KLHL13 mutants that bind but cannot promote degradation of targets, can stabilize normally transient interactions. Quantitative proteomics comparing KLHL13 knockdown/knockout with controls can identify proteins whose abundance changes most rapidly, suggesting direct regulation. The table below outlines experimental strategies for differentiating direct and indirect KLHL13 effects:

These combined approaches enable researchers to build a comprehensive picture of KLHL13's direct substrates and distinguish them from downstream pathway effects .

What can researchers learn from comparing partial versus full-length recombinant chicken KLHL13?

Comparing partial and full-length recombinant chicken KLHL13 constructs provides critical insights into domain-specific functions and protein engineering strategies. Partial constructs focusing on the Kelch domain enable specific study of substrate recognition without BTB domain-mediated dimerization or CUL3 binding. Conversely, BTB-only constructs can investigate CUL3 interactions independent of substrate binding. When interpreting results from partial constructs, researchers should consider: (1) potential disruption of interdomain interactions that might affect protein folding or stability; (2) altered subcellular localization due to missing targeting sequences; and (3) dominant-negative effects when partial constructs sequester interaction partners without fulfilling complete functional roles. Domain-swapping experiments between chicken and mammalian KLHL13 can identify species-specific functional determinants. The successful expression and characterization of partial recombinant proteins, as demonstrated with the PfK13 propeller domain , provides a template for similar approaches with chicken KLHL13.

What strategies can address common challenges in recombinant chicken KLHL13 expression and purification?

Researchers frequently encounter challenges when expressing and purifying recombinant Kelch-like proteins. Based on experiences with related proteins, several strategies can improve outcomes for chicken KLHL13: (1) Optimization of expression conditions, including temperature (16-18°C often improves solubility), induction timing, and inducer concentration; (2) Testing multiple solubility-enhancing tags (e.g., SUMO, MBP, GST) to identify optimal constructs; (3) Addition of stabilizing agents (glycerol, reducing agents) to purification buffers; and (4) Considering insect cell or mammalian expression systems for proteins recalcitrant to bacterial expression. The expression challenges noted for P. falciparum proteins - which included A/T-rich genes encoding low-complexity amino acid sequences - highlight how genetic features can impact heterologous expression. While chicken genes typically have less extreme codon bias than Plasmodium, codon optimization may still improve expression. Systematic troubleshooting of each expression and purification step, with SDS-PAGE analysis at each stage, allows identification of specific bottlenecks requiring optimization.

How can researchers address non-specific binding issues in KLHL13 interaction studies?

Non-specific binding presents a significant challenge in protein interaction studies involving KLHL13. To minimize false positives and ensure reliable interaction data, researchers should implement: (1) Stringent washing conditions in pull-down or co-immunoprecipitation experiments, optimized to maintain specific interactions while reducing background; (2) Competition assays with unlabeled proteins to demonstrate binding specificity; (3) Multiple negative controls, including unrelated proteins with similar physicochemical properties; and (4) Reciprocal interaction studies (e.g., pulling down with KLHL13 and then with the putative partner). When using proximity labeling methods like BioID, as applied for KLHL13 interaction mapping , careful selection of appropriate controls is essential for distinguishing true interactors from background. Quantitative approaches comparing wild-type KLHL13 with binding-deficient mutants can help differentiate specific from non-specific interactions. Cross-validation of interactions using multiple independent techniques (e.g., co-immunoprecipitation, direct binding assays, functional studies) provides the strongest evidence for physiologically relevant interactions.

What quality control measures ensure reproducibility in KLHL13 functional studies?

Ensuring reproducibility in KLHL13 functional studies requires rigorous quality control measures throughout the experimental workflow. Key considerations include: (1) Thorough validation of recombinant protein identity and integrity using mass spectrometry, as demonstrated for related proteins ; (2) Verification of protein folding through circular dichroism or limited proteolysis; (3) Batch-to-batch consistency checks for recombinant proteins; and (4) Inclusion of positive controls with known activity in functional assays. For cell-based studies, researchers should verify KLHL13 expression levels and subcellular localization across experiments. When studying KLHL13's role in ubiquitination, controls should include omission of critical components (E1, E2, ATP) to confirm specificity. Documentation of detailed protocols, including buffer compositions, incubation times, and temperatures, enables others to reproduce the work. Statistical analysis should include appropriate tests for the experimental design, with clear reporting of sample sizes, replicates, and variability measures to facilitate interpretation and reproduction of results.