Recombinant Chicken Endothelial differentiation-related factor 1 homolog (EDF1)

What is Recombinant Chicken Endothelial Differentiation-Related Factor 1 Homolog?

Recombinant Chicken Endothelial differentiation-related factor 1 homolog (EDF1) is a full-length protein (148 amino acids) expressed in mammalian cell systems that functions as a transcriptional coactivator . The protein serves as a bridging molecule that interconnects regulatory proteins and the basal transcriptional machinery, thereby modulating gene transcription involved in endothelial differentiation . The recombinant form is typically produced with high purity (>85% by SDS-PAGE) and may include various tags depending on the manufacturing process, making it suitable for controlled experimental applications in research settings .

How does the amino acid sequence of Chicken EDF1 contribute to its functionality?

The amino acid sequence of Chicken EDF1 (MAESDWDTVTVLRKKGPSAAQAKSKQAVLAAQRRGEDVETSKKWAAGQNKQHFITKNTAKLDRETEELHHDRVPLEVGKVIQQGRQSKGMTQKDLATKINEKPQVIADYESGRAIPNNQVMGKIERAIGLKLRGKDIGKPLETGPKGK) contains several functional domains that contribute to its role as a transcriptional coactivator . The N-terminal region contains charged residues that facilitate protein-protein interactions, while the central portion includes motifs that enable binding to the TATA element-binding protein (TBP) . These structural features allow EDF1 to physically bridge specific gene activators with general transcription machinery, positioning it as a critical mediator in transcriptional regulation pathways rather than simply functioning as a direct DNA-binding factor .

What cellular localization patterns does Chicken EDF1 exhibit during different cellular states?

In normal cellular conditions, EDF1 shows minimal association with polysomal fractions but is drastically redistributed during translational stress . When cells experience ribosomal collisions induced by translation inhibitors like low-dose emetine (1.8 μM) or anisomycin (0.19 μM), EDF1 rapidly relocates to polysomes . This dynamic localization pattern serves as a molecular sensor of translational distress, allowing EDF1 to coordinate immediate responses at sites of ribosomal collisions . The protein's distribution pattern observed through sucrose gradient fractionation combined with quantitative proteomics demonstrates enrichment ratios of approximately 7-10 fold in polysome fractions of stressed cells compared to unstressed controls .

What are the optimal storage conditions for maintaining Recombinant Chicken EDF1 activity?

The shelf life of Recombinant Chicken EDF1 depends on several factors including storage state, buffer components, and temperature. For liquid formulations, a shelf life of approximately 6 months can be expected when stored at -20°C/-80°C . Lyophilized formulations exhibit extended stability, with a typical shelf life of 12 months at -20°C/-80°C . For working solutions, it is recommended to store aliquots at 4°C for no longer than one week, as repeated freeze-thaw cycles significantly reduce protein activity . The following table summarizes the recommended storage conditions:

What is the recommended reconstitution protocol for Recombinant Chicken EDF1?

For optimal reconstitution of Recombinant Chicken EDF1, the vial should first be briefly centrifuged to ensure all contents are at the bottom before opening . The protein should be reconstituted in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL . For long-term storage of reconstituted protein, it is recommended to add glycerol to a final concentration of 5-50% before aliquoting and storing at -20°C/-80°C . This methodological approach preserves protein structure and function by preventing ice crystal formation during freeze-thaw cycles and reducing aggregation potential. For experiments requiring precise activity measurements, freshly reconstituted protein typically provides more consistent results than samples subjected to multiple freeze-thaw cycles.

What experimental methods can be used to investigate EDF1's interaction with collided ribosomes?

To investigate EDF1's interaction with collided ribosomes, researchers can employ sucrose gradient fractionation coupled with quantitative proteomics, which has proven effective in identifying factors associated with ribosomal subcomplexes (40S, 60S, 80S, and polysomes) . This approach involves treating cells with low-dose translation inhibitors (e.g., emetine at 1.8 μM for 15 minutes or anisomycin at 0.19 μM for 15 minutes) to induce ribosome collisions, followed by cell lysis and layering onto 10-50% sucrose gradients . After ultracentrifugation, gradient fractions can be collected and analyzed by immunoblotting for EDF1 or subjected to mass spectrometry for comprehensive protein identification . Cryo-electron microscopy represents another advanced approach for structural determination of EDF1-ribosome complexes, revealing binding sites at the mRNA entry channel near collision interfaces with exceptional resolution .

How does EDF1 coordinate cellular responses to ribosomal collisions?

EDF1 orchestrates multiple aspects of the cellular response to ribosomal collisions, serving as a central coordinator in translation quality control pathways . Upon detecting collided ribosomes, EDF1 binds to the 40S ribosomal subunit at the mRNA entry channel near the collision interface, as confirmed by cryo-electron microscopy . This strategic positioning allows EDF1 to perform several critical functions: (1) it recruits translational repressors GIGYF2 and EIF4E2 to collided ribosomes, initiating a negative-feedback loop that prevents new ribosomes from translating defective mRNAs; (2) it functions upstream of ribosomal stall recognition, as its depletion decreases ZNF598-mediated ubiquitylation of eS10 and uS10; and (3) it connects cytoplasmic ribosome collision events to transcriptional responses in the nucleus . This multilevel coordination mechanism ensures efficient quality control of both mRNAs and nascent peptides while maintaining cellular homeostasis during translational stress.

What experimental evidence supports EDF1's role in ribosome-mediated quality control?

Multiple lines of experimental evidence support EDF1's central role in ribosome-mediated quality control pathways . Sucrose gradient fractionation combined with quantitative proteomics identified EDF1 as the most enriched protein in heavy polysome fractions of cells treated with low-dose emetine, with enrichment ratios ([EL/UT]) of approximately 7-10 in polysome fractions . Immunoblotting analyses confirmed this collision-dependent recruitment, showing strong EDF1 signals in polysomal fractions from cells treated with low-dose emetine (1.8 μM) or anisomycin (0.19 μM), but minimal signals in untreated cells or those treated with high-dose emetine (360 μM) . Furthermore, depletion studies demonstrated that EDF1 functions upstream of ribosomal stall recognition, as its absence resulted in decreased ZNF598-mediated ubiquitylation of eS10 and uS10, critical markers of the quality control response . These findings collectively establish EDF1 as an essential sensor and coordinator of ribosome-mediated quality control rather than simply a passive participant in these pathways.

What is the relationship between EDF1 and transcriptional regulation during translational stress?

EDF1 serves as a molecular bridge connecting cytoplasmic translation events to nuclear transcriptional responses during translational stress . Upon recruitment to collided ribosomes, EDF1 not only coordinates immediate quality control responses but also regulates an immediate-early transcriptional program . This dual functionality allows cells to synchronize their translational and transcriptional activities during stress conditions. Although the precise mechanism remains under investigation, the transcriptional coactivator function of EDF1, which involves interconnecting the general transcription factor TATA element-binding protein (TBP) and gene-specific activators, is likely repurposed during these stress responses . This molecular connection provides novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation, highlighting how cells integrate different layers of gene expression control to maintain homeostasis during adverse conditions.

How might EDF1 function differ between avian and mammalian systems?

While EDF1 demonstrates significant conservation across vertebrates, functional differences exist between avian and mammalian systems that merit careful experimental consideration . In mammals, EDF1 coordinates ribosome collision responses and functions as a transcriptional coactivator . The avian homolog preserves these core functions but operates within a distinct cellular context, particularly regarding stem cell pluripotency regulation . In chickens, pluripotency induction results in a germ cell fate with expression of primordial germ cell (PGC) marker genes, which differs from the broader pluripotency outcomes observed in mammals . These differences suggest that chicken EDF1 may interact with pluripotency factors like NANOG, Pou5f3, SOX2, and LIN28A in avian-specific regulatory networks . Researchers should therefore design comparative studies that account for these evolutionary differences when extrapolating findings between species or developing model systems.

What methodological approaches can be used to study EDF1's role in avian pluripotency networks?

To investigate EDF1's role in avian pluripotency networks, researchers can employ CRISPR/Cas9-mediated knock-in strategies to develop reporter systems in chicken cell lines . One effective approach involves creating NANOG knock-in reporter DF1 cells, where red fluorescent protein (RFP) expression serves as a visual indicator of pluripotency factor activation . This experimental model can be established by designing guide RNAs targeting the NANOG locus, followed by electroporation of CRISPR/Cas9 components along with a donor template containing RFP flanked by homology arms . After single-cell sorting and expansion, genomic PCR analysis and sequencing with primers such as chNANOG KI F (5'-tgt gat gca gac acc atc ct-3') and chNANOG KI R (5'-ggg tcc tcc ttt tgt gac ct-3') can verify successful integration . This reporter system can then be used to assess how EDF1 modulates pluripotency factor expression when overexpressed, depleted, or mutated, providing insights into its regulatory role in avian stem cell biology.

How can quantitative proteomics be optimized for studying EDF1-associated protein complexes?

To optimize quantitative proteomics for studying EDF1-associated protein complexes, researchers should implement a multi-stage experimental approach . First, efficient isolation of intact EDF1 complexes requires careful optimization of lysis conditions, using buffers that preserve protein-protein interactions while minimizing non-specific associations. Second, sucrose gradient fractionation (10-50%) combined with targeted mass spectrometry provides a powerful method for separating and identifying distinct EDF1-containing complexes based on their sedimentation properties . For collision-specific interactions, comparative analysis between untreated and translation inhibitor-treated samples (emetine at 1.8 μM or anisomycin at 0.19 μM) can highlight differential protein associations . Finally, validation through reciprocal immunoprecipitation and functional studies is essential to confirm biologically relevant interactions. This comprehensive approach has successfully identified key EDF1 interaction partners like GIGYF2 and EIF4E2, demonstrating its effectiveness for discovering novel components of EDF1-mediated cellular pathways .

What factors can affect the reproducibility of EDF1 localization studies during translational stress?

Several critical factors can impact the reproducibility of EDF1 localization studies during translational stress experiments . First, translation inhibitor concentration and exposure time significantly influence results—low-dose emetine (1.8 μM) effectively induces EDF1 recruitment to polysomes, while high-dose treatment (360 μM) does not . Second, the cell lysis procedure must maintain polysome integrity; use of RNase inhibitors and appropriate buffer conditions (typically containing cycloheximide to freeze ribosomes) is essential . Third, sucrose gradient preparation quality directly impacts fractionation resolution, with gradient stability, centrifugation parameters, and fraction collection methodology all affecting reproducibility . Finally, detection methods (immunoblotting versus mass spectrometry) have different sensitivity thresholds that can lead to apparently contradictory results . To ensure reliable interpretation, researchers should standardize these parameters and include appropriate controls such as known polysome-associated proteins and negative controls (proteins known to remain unaffected by translational stress) in their experimental design.

How should researchers address conflicting results between structural and functional studies of EDF1?

When addressing conflicting results between structural and functional studies of EDF1, researchers should implement a systematic reconciliation approach . First, evaluate methodological differences, as structural techniques (like cryo-EM) and functional assays (such as knockdown/overexpression studies) operate at different resolutions and under different conditions . Second, consider the cellular context—EDF1's dual roles in transcriptional regulation and ribosome-mediated quality control may manifest differently depending on cell type, stress condition, and experimental timeframe . Third, examine protein partners and post-translational modifications that might modulate EDF1 activity in context-specific ways . Finally, design integrative experiments that specifically test hypotheses arising from these contradictions, such as structure-guided mutagenesis followed by functional assays . This approach has successfully resolved apparent conflicts between structural data showing EDF1 binding to the 40S subunit and functional data implicating it in processes typically associated with the 60S subunit, revealing its role as a coordinator rather than a direct effector of these processes .

What controls are essential when studying EDF1's role in ribosome collision responses?

When studying EDF1's role in ribosome collision responses, several essential controls must be included to ensure valid interpretation of results . First, translation status controls: samples treated with high-dose translation inhibitors (e.g., emetine at 360 μM) that suppress translation without causing significant collisions provide a critical comparison point to collision-inducing low-dose treatments (1.8 μM) . Second, ribosome association controls: analysis of non-translating ribosomal subunits (40S, 60S) and monosomes (80S) alongside polysomes differentiates collision-specific from general ribosome associations . Third, specificity controls: parallel analysis of known collision factors (such as ZNF598) and factors unaffected by collisions validates the selectivity of experimental conditions . Fourth, genetic controls: rescue experiments with wild-type EDF1 following depletion confirm phenotype specificity, while domain mutants help map functional regions . Finally, temporal controls: time-course experiments distinguish immediate responses from secondary effects, critical for accurately characterizing EDF1's primary functions in collision sensing versus its downstream effects on quality control pathways .

How might EDF1 function be exploited for enhancing recombinant protein expression systems?

EDF1's newly discovered role in coordinating responses to ribosomal collisions presents promising applications for optimizing recombinant protein expression systems . By modulating EDF1 levels or activity, researchers could potentially reduce premature translation termination on difficult-to-express proteins that typically cause ribosome collisions . This approach could involve co-expression of modified EDF1 variants that preserve collision sensing but alter downstream quality control recruitment, thereby allowing translation to continue while still monitoring for major defects . In chicken expression systems specifically, where EDF1 may have evolved species-specific functions, targeted modifications based on structural insights from cryo-EM studies could be particularly effective . Experimental implementation would require careful optimization of EDF1-to-ribosome ratios and potentially engineering chimeric variants that combine the most advantageous features from avian and mammalian homologs. Such strategies could significantly improve yields of complex recombinant proteins while maintaining quality control.

What are the unexplored connections between EDF1 and developmental pathways in avian systems?

The intersection between EDF1's roles in transcriptional regulation and translational quality control suggests unexplored connections to developmental pathways in avian systems . In chickens, pluripotency induction results in primordial germ cell fate rather than the broader pluripotency seen in mammals, indicating species-specific regulatory networks . EDF1's ability to act as a transcriptional coactivator by connecting TATA-binding protein to gene-specific activators may be particularly important in coordinating the expression of developmental genes during embryogenesis . Future research should investigate whether EDF1 interacts with avian-specific transcription factors like chicken NANOG, Pou5f3, SOX2, and LIN28A, which have been implicated in pluripotency regulation . The development of chicken knock-in reporter systems provides a valuable experimental platform for such investigations, allowing real-time visualization of EDF1's influence on developmental gene expression patterns during critical differentiation events .

How does the evolutionary conservation of EDF1 binding sites inform structural biology approaches to quality control mechanisms?

The evolutionary conservation of EDF1 binding sites between species offers valuable insights for structural biology approaches to quality control mechanisms . Cryo-EM analyses of both human EDF1 and its yeast homolog Mbf1 revealed a conserved binding site at the 40S ribosomal subunit near the mRNA entry channel and collision interface . This conservation suggests fundamental mechanisms that have been preserved across vast evolutionary distances, highlighting the critical nature of ribosome collision sensing . Future structural studies should explore whether the binding mode of chicken EDF1 exhibits avian-specific adaptations while maintaining core functions . Comparative structural analyses could reveal subtle differences in binding interfaces that might explain species-specific responses to translational stress . Additionally, structure-guided mutagenesis of conserved versus divergent residues would help delineate which structural features are essential for universal collision sensing versus those involved in species-specific downstream responses, potentially informing the design of selective inhibitors or enhancers of EDF1 function for research and therapeutic applications.