Recombinant Rat Emerin (Emd)

What are the key structural domains of Rat Emerin and their functional significance?

Rat Emerin contains several crucial domains:

The LEM domain is particularly significant as it provides direct interaction with BAF, while the AR region mediates emerin-emerin associations, which are critical for nuclear envelope structure .

How does Rat Emerin integrate into cellular membranes and what pathways are involved?

Rat Emerin, like human emerin, is a tail-anchored protein primarily found at the inner nuclear membrane (INM). Integration involves the TRC40/GET pathway for post-translational insertion into membranes. This process requires:

• ATP-dependent targeting

• Recognition by TRC40

• Membrane insertion via the TRC40-receptor proteins WRB and CAML

Proximity ligation assays demonstrate that emerin interacts with TRC40 in situ. Experiments show that emerin expressed in bacteria or cell-free lysates can be inserted into microsomal membranes in an ATP- and TRC40-dependent manner . Disruption of this pathway affects the proper localization of emerin to the INM, which may contribute to the pathology observed in EDMD .

What expression systems yield functional recombinant Rat Emerin with highest efficiency?

E. coli expression systems have been successfully used to produce recombinant Rat Emerin. The commercially available recombinant full-length Rat Emerin is produced in E. coli with an N-terminal His tag . When expressing Rat Emerin:

For structural studies or binding assays where post-translational modifications are less critical, E. coli expression offers the best balance of yield and functionality .

What purification strategy produces the highest quality Recombinant Rat Emerin for interaction studies?

A multi-step purification approach is recommended:

• Initial capture using affinity chromatography (His-tag based IMAC)

• Buffer exchange to remove imidazole

• Secondary purification via ion exchange chromatography

• Size exclusion chromatography to remove aggregates

Key buffer considerations include:

• Maintain pH 8.0 for optimal stability

• Include 6% trehalose to prevent aggregation during storage

• Consider Tris/PBS-based buffers for compatibility with downstream applications

After purification, protein quality should be verified by SDS-PAGE (>90% purity) and functional binding assays with known interaction partners such as BAF or lamins .

What are the optimal storage conditions for maintaining Recombinant Rat Emerin stability and activity?

Optimal storage conditions for Recombinant Rat Emerin include:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution in deionized sterile water (0.1-1.0 mg/mL), add glycerol to a final concentration of 50%

• Aliquot to avoid repeated freeze-thaw cycles

• For short-term storage (up to one week), working aliquots can be kept at 4°C

For reconstitution buffer, use:

• Tris/PBS-based buffer, pH 8.0

• 6% Trehalose to enhance stability

Repeated freeze-thaw cycles significantly impact protein integrity and should be avoided. Stability tests show that properly stored protein maintains activity for structural and interaction studies for at least 6 months.

How can researchers effectively study Emerin-Emerin interactions using recombinant protein?

Several complementary approaches are recommended:

• Peptide array analysis: Use GST-tagged emerin fragments (e.g., GST-emerin-170-220) to probe arrayed 20-mer emerin peptides spotted on cellulose membranes. This approach identified key interaction regions including the R-peptide (human residues 206-225) and SAYQ-region peptides .

• Co-immunoprecipitation assays: Express differentially tagged emerin constructs (e.g., GFP-emerin and Flag-emerin) in cell lines like HEK293T, followed by immunoprecipitation with anti-Flag antibodies and detection with anti-GFP antibodies. This approach confirmed intermolecular emerin-emerin association and identified regions critical for this interaction .

• In vitro binding assays: Purify recombinant emerin fragments and perform direct binding assays using techniques such as surface plasmon resonance or pull-down assays.

Data from such experiments revealed two modes of emerin-emerin association: one mediated by association between residues 170-220 in different molecules, and another involving residues 170-220 and 1-132 .

What methods can be used to study the interaction between Rat Emerin and nuclear lamins?

The following methodological approaches are effective:

• Direct binding assays: Recombinant emerin fragments (residues 1-132 and 159-220) have each been shown sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins .

• Co-immunoprecipitation: From cellular extracts of undifferentiated C2C12 myoblasts or purified hepatocyte nuclei, using emerin antibodies to pull down associated proteins. This technique demonstrated that both A- and B-type lamins interact with emerin .

• Proximity ligation assays: This technique can detect protein-protein interactions in situ with high sensitivity and specificity, as demonstrated with emerin and TRC40 .

• Immunofluorescence co-localization: In C. elegans, Ce-MAN1 (another LEM domain protein) was shown to interact directly with Ce-lamin and Ce-BAF in vitro and required Ce-lamin for its nuclear envelope localization, suggesting similar dependencies for emerin .

These techniques have revealed that emerin contains multiple regions capable of interacting with lamins, which is critical for its proper localization and function.

How can researchers effectively model EDMD mutations using Recombinant Rat Emerin?

To model EDMD mutations using Recombinant Rat Emerin:

• Site-directed mutagenesis: Introduce specific mutations corresponding to those found in EDMD patients, such as deletions in the transmembrane region (Del236-241) or mutations in the N-terminal domain .

• Expression and localization studies: Transfect GFP-emerin constructs reflecting these mutations into cell lines (e.g., undifferentiated C2C12 myoblasts) to assess localization. Studies show that while both wild-type and mutant emerins are targeted to the nuclear membrane, mutants show reduced localization efficiency .

• Functional assays: Assess the impact of mutations on:

  • Emerin-emerin interactions

  • Binding to lamins and BAF

  • Nuclear envelope stability

• Rapamycin-based dimerization assay: This technique can reveal correct transport of wild-type emerin to the INM, whereas TRC40-binding, membrane integration, and INM-targeting of emerin mutant proteins may be disturbed .

Research has shown that mutations affecting the transmembrane region have more severe effects on nuclear envelope targeting compared to mutations in the N-terminal domain .

How does Rat Emerin function in mitochondrial regulation and what experimental approaches can assess this?

Recent research has uncovered a previously unknown role for emerin in mitochondrial regulation:

• Mitochondrial oxidative phosphorylation: Knockdown of emerin in HL-1 or H9C2 cardiomyocytes leads to impaired mitochondrial oxidative phosphorylation capacity with:

  • Downregulation of electron transport chain complexes I and IV

  • Upregulation of complexes III and V

• Mitochondrial dynamics: Loss of emerin in HL-1 cells results in:

  • Collapsed mitochondrial membrane potential

  • Altered mitochondrial networks

  • Downregulation of factors involved in mitochondrial biogenesis, fission, and fusion (PGC1α, DRP1, MFF, MFN2)

Experimental approaches to assess this function include:

• RNA interference to knockdown emerin expression

• Measurement of mitochondrial membrane potential

• Analysis of mitochondrial network morphology

• Quantification of electron transport chain complex expression

• Assessment of factors regulating mitochondrial dynamics

This connection to mitochondrial function provides a novel perspective on the pathophysiology of EDMD and suggests targeting mitochondrial bioenergetics might be an effective strategy against cardiac disorders caused by EMD mutations .

What are the emerging techniques for studying the role of emerin in chromatin organization?

Advanced techniques for studying emerin's role in chromatin organization include:

• Chromosome conformation capture techniques (Hi-C, 4C-seq): These methods can map the three-dimensional organization of chromatin and assess how emerin depletion or mutation affects genome organization.

• ChIP-seq analysis: To identify genomic regions that interact with emerin, directly or indirectly through its binding partners.

• Live-cell imaging: Using fluorescently tagged emerin and chromatin markers to visualize dynamic interactions during cell cycle progression.

• BAF-emerin interaction studies: The LEM domain of emerin mediates direct binding to BAF, a chromatin-associated protein. Studies have shown that this interaction is critical for proper chromatin organization and nuclear assembly .

• Phenotypic analysis in model systems: In C. elegans, loss of both Ce-emerin and Ce-MAN1 (90% reduction) results in embryonic lethality with a phenotype involving repeated cycles of anaphase chromosome bridging and cytokinesis ("cell untimely torn" phenotype) .

These approaches can help elucidate how emerin contributes to nuclear architecture and genome organization, which is disrupted in EDMD.

What approaches can resolve contradictory findings about emerin localization outside the nuclear envelope?

Studies have reported seemingly contradictory findings about emerin localization outside the nuclear envelope. To resolve these contradictions:

• Multiple antibody validation: Use different well-characterized antibodies against distinct epitopes. Studies screening 15 monoclonal emerin antibodies by immunofluorescence showed varying results: two were clearly positive for intercalated disc (ICD) localization, five were faintly positive, while others showed no staining .

• Complementary detection methods: Combine techniques such as:

  • Indirect immunofluorescence

  • Immuno-gold EM labeling

  • Biochemical fractionation

  • Proximity ligation assays

• Tissue-specific analysis: Emerin has been detected at the plasma membrane in rat cardiomyocytes and in heart tissue from human, rat, and mouse, specifically at adhesive junctions of intercalated discs (ICDs) .

• Control for epitope masking: Lack of staining may be inconclusive since specific epitopes might be masked by location-specific partners or post-translational modifications .

• Expression of tagged emerin: Use fluorescently tagged emerin to track localization in live cells, complemented by fixation and immunostaining.

These methodological approaches can help reconcile findings suggesting emerin may have distinct localizations in different cell types or physiological states.

What are the common challenges in expressing soluble Recombinant Rat Emerin and how can they be addressed?

Common challenges and solutions include:

For membrane-spanning regions, specialized approaches such as expressing emerin without its transmembrane domain or using mild detergents like DDM or CHAPS may be necessary to maintain solubility while preserving function .

How can researchers validate the functionality of purified Recombinant Rat Emerin?

Functionality validation requires multiple complementary approaches:

• Binding assays with known partners:

  • BAF binding assay using purified recombinant BAF

  • Lamin binding assay with lamin A/C and B1 tails

  • Self-association assays to confirm emerin-emerin interactions

• Structural integrity assessment:

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to confirm proper folding

  • Thermal shift assays to determine stability

• Functional reconstitution:

  • Membrane integration assays using microsomes

  • ATP- and TRC40-dependent membrane insertion assays

• Activity in cellular context:

  • Transfection into emerin-null cells to rescue phenotypes

  • Assessment of nuclear envelope reformation after mitosis

  • Restoration of proper chromatin organization

These validation steps ensure that the recombinant protein retains both structural integrity and functional activity comparable to native emerin.

How might emerging structural biology techniques advance our understanding of Rat Emerin function?

Emerging structural biology techniques offer promising avenues for emerin research:

• Cryo-electron microscopy (Cryo-EM): Could resolve the structure of emerin within the context of nuclear envelope complexes, particularly challenging given emerin's predicted intrinsic disorder .

• Integrative structural biology: Combining X-ray crystallography of ordered domains with NMR spectroscopy of flexible regions to build comprehensive structural models.

• Single-molecule FRET: To investigate the dynamics of emerin conformational changes during interactions with binding partners like BAF and lamins.

• In-cell NMR: Could provide insights into emerin structure and dynamics in the native cellular environment.

• AlphaFold and other AI-based prediction methods: To model the tertiary structure of emerin and predict interaction interfaces with binding partners.

These approaches would help overcome current limitations in understanding emerin's molecular mechanism, particularly given evidence of multiple "backbone" and LEM-domain configurations in the proposed intermolecular emerin network at the nuclear envelope .

What is the significance of LEM domain protein functional overlap and how can it be studied experimentally?

Studies in C. elegans have revealed crucial functional overlap between LEM domain proteins:

• Significance:

  • While loss of Ce-emerin alone has no detectable phenotype in C. elegans, partial reduction (90%) of Ce-MAN1 in emerin-null cells is lethal to all embryos by the 100-cell stage

  • This "enhanced lethality" demonstrates that LEM domain proteins are essential for cell division

  • Emerin has at least one significant function that overlaps with MAN1, preventing death of MAN1-reduced cells

• Experimental approaches:

  • Combined RNAi/genetic knockouts: Study phenotypes when multiple LEM proteins are simultaneously reduced/eliminated

  • Rescue experiments: Test whether overexpression of one LEM protein can rescue defects caused by loss of another

  • Domain swapping: Create chimeric proteins to identify which domains confer redundant functions

  • Immunostaining analysis: In C. elegans, anaphase-bridged chromatin retained mitosis-specific phosphohistone H3 epitopes and failed to recruit detectable Ce-lamin or Ce-BAF

  • Proteomic analysis: Identify common binding partners between different LEM proteins

• Relevance to EDMD:

  • Understanding functional overlap might explain tissue-specific pathology in EDMD despite emerin's ubiquitous expression

  • Could reveal compensatory mechanisms that might be therapeutically enhanced

  • May provide insight into why mutations in different proteins (emerin and lamins) can cause similar disease phenotypes

This research direction is particularly relevant for understanding the complex pathophysiology of EDMD and developing potential therapeutic approaches.

How do the biochemical properties of Recombinant Rat Emerin compare to human emerin for modeling EDMD mutations?

Comparative analysis between rat and human emerin:

What insights have been gained from comparative studies of emerin across species for evolutionary conservation of function?

Comparative studies have revealed:

• Conserved functional domains across species:

  • Manual alignment of human, rat, nine-banded armadillo, and zebrafish emerin sequences shows conservation of key functional regions

  • The R-peptide region and portions of the SAYQ-region show qualitatively stronger conservation, suggesting functional importance

• Evolutionary significance of LEM domain proteins:

  • Only three LEM proteins are conserved in C. elegans: Ce-MAN1, Ce-emerin, and lem-3

  • This reduced complexity in C. elegans has facilitated study of LEM protein functions and interactions in vivo

• Functional conservation despite sequence divergence:

  • Despite sequence variations, emerin's interactions with BAF and lamins are conserved across species

  • The functional redundancy between emerin and MAN1 is also conserved, suggesting evolutionary pressure to maintain nuclear envelope integrity

• Species-specific variations in emerin localization:

  • In rat cardiomyocytes, emerin was detected at intercalated discs and the nuclear envelope

  • These differential localizations may reflect species-specific adaptations in certain tissues

These comparative studies provide context for understanding the essential and conserved functions of emerin versus those that may have evolved for species-specific requirements.