Recombinant Xenopus laevis Erlin-2-B (erlin2-b), partial

What is Erlin-2-B and what is its function in Xenopus laevis?

Erlin-2-B (erlin2-b) is an endoplasmic reticulum lipid raft-associated protein found in Xenopus laevis. It belongs to the SPFH domain-containing protein family, which includes members involved in membrane organization and cellular signaling. The protein is identified by several names including Endoplasmic reticulum lipid raft-associated protein 2-B, Stomatin-prohibitin-flotillin-HflC/K domain-containing protein 2-B, and SPFH domain-containing protein 2-B. This protein is typically involved in endoplasmic reticulum-associated degradation (ERAD) pathways and regulation of cellular lipid metabolism. In Xenopus laevis, which exhibits genome duplication, Erlin-2-B represents one of the paralogs that may have undergone subfunctionalization during evolution.

How does recombinant Xenopus laevis Erlin-2-B compare structurally to its homologs in other species?

Recombinant Xenopus laevis Erlin-2-B shares significant structural conservation with homologs found in mammals and other vertebrates, particularly in the SPFH domain region. The protein contains characteristic hydrophobic regions that facilitate its insertion into the endoplasmic reticulum membrane. Comparative analysis of Erlin-2-B across species reveals high conservation in functional domains, suggesting evolutionary importance in essential cellular processes. When aligning Xenopus laevis Erlin-2-B sequences with mammalian counterparts, researchers typically observe 75-85% similarity in amino acid sequences, with higher conservation in the SPFH domain. This evolutionary conservation makes Xenopus Erlin-2-B a valuable model for understanding the fundamental functions of this protein family across vertebrates.

What are the optimal storage conditions for maintaining recombinant Xenopus laevis Erlin-2-B activity?

For optimal preservation of recombinant Xenopus laevis Erlin-2-B activity, the protein should be stored in liquid form containing glycerol. Short-term storage (up to one week) is possible at 4°C for working aliquots. For intermediate storage, maintaining the protein at -20°C is recommended. Long-term storage requires temperatures of -80°C to prevent degradation and preserve functional integrity. It is critically important to avoid repeated freeze-thaw cycles, as these can significantly reduce protein activity and accelerate degradation. Researchers should prepare small working aliquots before freezing to minimize the need for repeated thawing of the entire stock. Additionally, the buffer composition should maintain proper pH and contain appropriate stabilizing agents to preserve the native conformation of the protein.

What purification strategies yield the highest purity recombinant Xenopus laevis Erlin-2-B?

Achieving high-purity recombinant Xenopus laevis Erlin-2-B requires a multi-step purification strategy. The typical approach begins with affinity chromatography using a fusion tag (His-tag, GST, or MBP) engineered into the recombinant protein. After initial capture, ion exchange chromatography effectively separates Erlin-2-B from contaminants with different charge profiles. Size exclusion chromatography serves as a polishing step to remove aggregates and achieve >90% purity. For membrane-associated proteins like Erlin-2-B, incorporating appropriate detergents throughout the purification process is crucial to maintain solubility and native conformation. Researchers should implement quality control steps between purification stages, including SDS-PAGE analysis and Western blotting, to confirm the presence and purity of Erlin-2-B. Final quality assessment should include activity assays to verify that the purified protein retains its functional properties.

How can researchers verify the functional integrity of purified recombinant Erlin-2-B?

Verifying the functional integrity of purified recombinant Erlin-2-B requires multiple complementary approaches. First, researchers should assess protein folding using circular dichroism spectroscopy, which provides information about secondary structure elements. Thermal shift assays evaluate protein stability and can identify buffer conditions that optimize protein folding. Since Erlin-2-B is a membrane-associated protein involved in lipid raft formation, researchers should perform lipid binding assays using artificial liposomes to verify interaction with membrane components. Protein-protein interaction studies with known binding partners can confirm the ability of recombinant Erlin-2-B to form appropriate complexes. Finally, functional assays specific to Erlin-2-B's role in endoplasmic reticulum processes, such as calcium regulation or protein quality control, provide definitive evidence of biological activity. Integration of these approaches provides comprehensive validation of the recombinant protein's functional integrity.

How is recombinant Xenopus laevis Erlin-2-B used in developmental biology studies?

Recombinant Xenopus laevis Erlin-2-B serves as a valuable tool in developmental biology research, particularly for studying endoplasmic reticulum functions during embryonic development. Researchers utilize the purified protein for antibody production to enable immunohistochemical tracking of endogenous Erlin-2-B expression patterns across developmental stages. Microinjection of mRNA encoding fluorescently-tagged Erlin-2-B into Xenopus embryos allows real-time visualization of protein localization during cell division and tissue differentiation. In loss-of-function studies, researchers can employ morpholino oligonucleotides to knockdown endogenous erlin2-b, followed by rescue experiments using recombinant protein to validate specificity. Additionally, recombinant Erlin-2-B can be used in in vitro binding assays to identify developmental stage-specific interaction partners, providing insights into the temporal regulation of endoplasmic reticulum organization throughout embryogenesis.

What techniques are most effective for studying Erlin-2-B interactions with other proteins in Xenopus systems?

For investigating Erlin-2-B interactions with other proteins in Xenopus systems, researchers employ multiple complementary approaches. Co-immunoprecipitation using antibodies against Erlin-2-B followed by mass spectrometry analysis effectively identifies novel binding partners from Xenopus egg or embryo extracts. This approach has proven valuable in characterizing protein complexes across various developmental stages, similar to methods used with other Xenopus proteins like Mad2B and PRCC. Yeast two-hybrid screening using Erlin-2-B as bait can identify direct interacting partners when expressed against a Xenopus cDNA library. For visualizing interactions in live cells, bimolecular fluorescence complementation (BiFC) or Förster resonance energy transfer (FRET) can be employed using Xenopus cell lines or embryos expressing fluorescently-tagged Erlin-2-B and candidate interacting proteins. Proximity ligation assays (PLA) provide another sensitive method for detecting protein-protein interactions in fixed Xenopus tissues with spatial resolution. Cross-linking mass spectrometry offers advanced insights into the structural aspects of Erlin-2-B interaction interfaces.

How can Xenopus egg extracts be used to study Erlin-2-B function in membrane dynamics?

Xenopus laevis egg extracts provide a powerful cell-free system for studying Erlin-2-B function in membrane dynamics. These extracts contain functional cellular machinery while allowing precise manipulation of protein components. To study Erlin-2-B's role in membrane organization, researchers can immunodeplete endogenous Erlin-2-B from the extracts and observe the effects on membrane formation and dynamics. The depleted extracts can then be reconstituted with purified recombinant Erlin-2-B to rescue function. This approach, similar to methods used for studying actin-based motility with other proteins in Xenopus egg extracts, allows precise control over protein concentration and modification. Fluorescently labeled Erlin-2-B enables direct visualization of protein localization to membranes in the extract. Researchers can also use the extract system to reconstitute endoplasmic reticulum membrane formation in vitro, adding recombinant Erlin-2-B to assess its role in lipid raft assembly, membrane curvature, and protein quality control mechanisms.

What techniques are most effective for studying the role of Erlin-2-B in endoplasmic reticulum quality control?

To investigate Erlin-2-B's role in endoplasmic reticulum quality control, researchers should implement a multi-faceted approach. In vitro reconstitution assays using purified recombinant Erlin-2-B and artificial membranes can demonstrate the protein's direct effects on membrane organization and lipid raft formation. CRISPR/Cas9-mediated genome editing in Xenopus cell lines enables generation of erlin2-b knockout models to assess cellular consequences of protein absence. For studying protein degradation pathways, pulse-chase experiments with ER-associated substrates in the presence or absence of functional Erlin-2-B reveal kinetic parameters of quality control mechanisms. Co-immunoprecipitation followed by mass spectrometry identifies Erlin-2-B-associated components of the ERAD machinery. Super-resolution microscopy of tagged Erlin-2-B variants provides spatial information about its distribution within ER subdomains during stress conditions. Integration of these approaches enables comprehensive characterization of Erlin-2-B function in maintaining ER proteostasis.

How does the gene structure of erlin2-b in Xenopus laevis compare to other vertebrates?

The gene structure of erlin2-b in Xenopus laevis shows both conservation and divergence compared to other vertebrates. In Xenopus, the erlin2-b gene likely consists of multiple exons separated by introns, similar to the organization observed in other genes like XLSSB1 and XLSSB2. Unlike mammals that typically have a single erlin2 gene, Xenopus laevis, due to its pseudotetraploid genome, likely possesses two copies (erlin2-a and erlin2-b) resulting from genome duplication. Comparative genomic analysis typically reveals conserved exon-intron boundaries at the same codon positions across vertebrates, indicating evolutionary conservation of gene structure. The promoter regions of erlin2-b may contain regulatory elements similar to those found in other Xenopus genes, such as CCAAT boxes and binding sites for transcription factors like NRF-2 and Sp1. These regulatory elements govern tissue-specific and developmental stage-specific expression patterns that can be compared across vertebrate species to identify conserved control mechanisms.

What post-translational modifications are important for Erlin-2-B function, and how can they be analyzed?

Post-translational modifications (PTMs) critically influence Erlin-2-B function, and their comprehensive analysis requires multiple analytical approaches. Phosphorylation, particularly on serine and threonine residues, likely regulates Erlin-2-B's interaction with binding partners and localization within the endoplasmic reticulum. Mass spectrometry-based phosphoproteomics provides the most comprehensive mapping of modification sites. Ubiquitination analysis is essential for understanding Erlin-2-B's role in protein degradation pathways, as it may function both as a facilitator of substrate ubiquitination and be regulated itself through this modification. Palmitoylation and other lipid modifications likely anchor Erlin-2-B to ER membranes and can be analyzed using metabolic labeling with lipid analogs followed by click chemistry. Site-directed mutagenesis of predicted modification sites followed by functional assays evaluates the biological significance of specific PTMs. Temporal dynamics of modifications during cellular stress responses can be monitored using targeted mass spectrometry approaches, revealing regulatory mechanisms controlling Erlin-2-B activity under different physiological conditions.

How can high-throughput screening approaches identify small molecule modulators of Erlin-2-B function?

High-throughput screening for Erlin-2-B modulators requires development of robust, scalable assays reflecting the protein's function. A primary approach involves fluorescence-based binding assays measuring interaction between labeled Erlin-2-B and known binding partners or lipid membranes. Researchers can establish cell-based reporter systems where Erlin-2-B activity is coupled to luciferase expression or fluorescent protein production. Thermal shift assays in 384-well formats can identify compounds stabilizing protein conformation. For more physiologically relevant screening, microscopy-based phenotypic assays in Xenopus cell lines measuring ER morphology or stress responses provide functional readouts. Fragment-based screening using NMR or X-ray crystallography identifies chemical starting points for inhibitor development. Promising hits require validation through orthogonal assays, including surface plasmon resonance for binding kinetics and cellular assays measuring Erlin-2-B-dependent processes. The development pipeline should include counter-screens against related SPFH domain proteins to establish selectivity profiles of identified modulators.

What are the most challenging aspects of crystallizing Erlin-2-B for structural studies, and how can they be addressed?

Crystallizing Erlin-2-B presents several challenges typical of membrane-associated proteins. The hydrophobic regions that mediate membrane interactions often lead to aggregation during concentration steps. Researchers can address this by using mild detergents or amphipols to maintain protein solubility while preserving native conformation. The flexibility of connecting loops between structural domains creates heterogeneity in the protein population, hampering crystal lattice formation. Engineering more rigid constructs by removing flexible regions or introducing stabilizing mutations can improve crystallization prospects. Co-crystallization with binding partners or antibody fragments often stabilizes specific conformations, facilitating crystal formation. High-throughput screening of crystallization conditions using nanoliter-scale drops conserves precious protein samples while exploring thousands of potential crystallization conditions. Alternative approaches to traditional crystallography include cryo-electron microscopy for structural determination of Erlin-2-B complexes and small-angle X-ray scattering (SAXS) for solution structure determination, which can provide valuable structural insights when crystals prove difficult to obtain.

How can computational modeling enhance our understanding of Erlin-2-B structure-function relationships?

Computational modeling offers powerful approaches for understanding Erlin-2-B structure-function relationships when experimental structural data is limited. Homology modeling using structures of related SPFH domain proteins provides a starting point for structural analysis. Molecular dynamics simulations reveal dynamic behaviors of Erlin-2-B within membranes, particularly important for understanding how the protein influences lipid organization in ER membranes. Protein-protein docking algorithms can predict interaction interfaces between Erlin-2-B and binding partners identified in experimental studies. Machine learning approaches analyzing sequences across species can identify evolutionarily conserved functional motifs and predict critical residues for protein function. Integration of computational predictions with experimental mutagenesis data creates iterative refinement of structural models. For advanced applications, coarse-grained simulations can model Erlin-2-B behavior within complex cellular environments, including interactions with multiple proteins and membrane components simultaneously. These computational approaches generate testable hypotheses about structure-function relationships that guide experimental design for validation studies.

How might CRISPR/Cas9 genome editing be optimized for studying erlin2-b function in Xenopus laevis?

Optimizing CRISPR/Cas9 genome editing for erlin2-b in Xenopus laevis requires strategies addressing the organism's pseudotetraploid genome and embryonic development. Researchers should design guide RNAs targeting conserved regions across both erlin2-b homeologs to achieve complete knockout. For delivery, microinjection of Cas9 protein pre-complexed with guide RNAs into fertilized eggs maximizes editing efficiency while minimizing toxicity. Targeting the early blastomere stage allows generation of mosaic embryos for initial phenotypic screening. To achieve germline transmission, injection at the one-cell stage followed by screening of F0 founders and breeding is required. For precise modifications, homology-directed repair templates can be co-injected to introduce specific mutations or epitope tags. Validation of editing requires deep sequencing to quantify modification rates across both homeologs. Researchers should also consider conditional approaches using inducible Cas9 or guide RNA expression to bypass early developmental requirements if erlin2-b proves essential for embryogenesis. The genome editing protocols can be modified from those developed for other Xenopus genes like Mad2B.

What role might Erlin-2-B play in cellular stress responses, and how can this be investigated?

Investigating Erlin-2-B's role in cellular stress responses requires a systematic approach examining its function under various stress conditions. Quantitative proteomics comparing the interactome of Erlin-2-B under normal versus stress conditions (ER stress, oxidative stress, heat shock) reveals stress-specific binding partners. Cellular fractionation followed by immunoblotting tracks potential redistribution of Erlin-2-B between different membrane subdomains during stress responses. Time-course transcriptomic and proteomic analyses of cells with manipulated Erlin-2-B levels identify downstream effectors of stress response pathways. Live-cell imaging using fluorescent protein-tagged Erlin-2-B in Xenopus cell lines provides dynamic information about protein redistribution during stress induction and resolution. To assess functional importance, researchers can measure canonical stress responses (unfolded protein response, calcium signaling, autophagy induction) in erlin2-b knockout versus wild-type cells. Metabolic labeling approaches tracking protein synthesis and degradation rates clarify how Erlin-2-B influences proteostasis during stress conditions, potentially revealing roles in selective protein degradation or synthesis during adaptive responses.

How can multi-omics approaches advance our understanding of Erlin-2-B in development and disease?

Multi-omics approaches provide comprehensive insights into Erlin-2-B function across biological contexts. Integrating transcriptomics, proteomics, and metabolomics data from Xenopus models with manipulated erlin2-b expression identifies coordinated regulatory networks and metabolic pathways affected by the protein. Spatial transcriptomics and proteomics techniques applied to developing Xenopus embryos reveal tissue-specific functions during organogenesis. Comparison of interactome data across developmental stages identifies context-specific binding partners regulating Erlin-2-B function. Phosphoproteomics and ubiquitinomics analyses reveal how post-translational modification networks change in response to erlin2-b perturbation. For disease relevance, comparative multi-omics analyses between Xenopus models and human patient samples with ER stress-related pathologies can identify conserved mechanisms potentially amenable to therapeutic intervention. Data integration requires sophisticated computational approaches, including network analysis and machine learning, to extract biological insights from multi-dimensional datasets. These approaches enable systems-level understanding of how Erlin-2-B coordinates endoplasmic reticulum functions in development and under pathological conditions.