Recombinant Chicken NSFL1 cofactor p47 (NSFL1C)

What is NSFL1 cofactor p47 and what are its known functions in chicken?

NSFL1 cofactor p47 in chicken (UniProt ID: Q5ZK10) is a regulatory protein that performs several critical cellular functions. It reduces the ATPase activity of VCP (Valosin-containing protein) and plays essential roles in Golgi dynamics during cell division. Specifically, it facilitates the fragmentation of Golgi stacks during mitosis and participates in VCP-mediated reassembly of these structures after mitosis. The protein may also contribute to VCP-mediated formation of transitional endoplasmic reticulum (tER). Additionally, NSFL1C inhibits CTSL activity in vitro and, in conjunction with UBXN2B/p37, regulates centrosomal levels of AURKA/Aurora A kinase during mitotic progression by promoting AURKA removal from centrosomes in prophase. Furthermore, it helps regulate spindle orientation during mitosis .

What alternative names and identifiers are associated with chicken NSFL1 cofactor p47?

The chicken NSFL1 cofactor p47 protein is known by several synonyms in scientific literature:

This variety of identifiers reflects the protein's presence across multiple biological databases, facilitating cross-referencing in research literature and computational analyses .

How can recombinant chicken NSFL1 cofactor p47 be efficiently purified for research applications?

Purification of recombinant chicken NSFL1 cofactor p47 can be achieved through several techniques, with reversed-phase high-performance liquid chromatography (RP-HPLC) offering significant advantages over traditional affinity chromatography methods. The following one-step RP-HPLC protocol has demonstrated excellent potential for purification of recombinant proteins:

• Cell lysis: Extract cell contents with 0.1% aqueous trifluoroacetic acid (TFA)

• Direct application: Apply the extract to a narrow bore RP-HPLC C8 column (150 mm × 2.1 mm I.D.)

• Elution: Use a shallow gradient of acetonitrile (0.1%/min)

This approach has several advantages:

• Achieves >94% purity in a single step

• Maintains >90% recovery rates

• Eliminates the need for multiple purification steps, reducing sample loss

• Can purify even trace amounts (<0.1% of total contents) of target protein

• Easily scalable without requiring proportionally larger columns

For comparison, traditional affinity purification methods typically yield only 64% purity and require additional purification steps, resulting in considerable reduction in protein yield. The RP-HPLC method can be optimized by adjusting the gradient rate and sample load to balance purity and yield according to specific research requirements .

What techniques are most effective for detecting NSFL1 cofactor p47 in experimental systems?

Several detection methods can be employed for analyzing NSFL1 cofactor p47 in experimental systems, each with specific advantages depending on the research question:

For Western blotting, polyclonal antibodies generated from recombinant protein fragments containing sequences corresponding to amino acids 21-272 of human NSFL1C have shown good reactivity, though species-specific validation is recommended when working with chicken NSFL1C .

How does chicken NSFL1 cofactor p47 interact with VCP and what are the functional implications?

Chicken NSFL1 cofactor p47 functionally interacts with VCP (Valosin-containing protein), a highly conserved AAA+ ATPase involved in numerous cellular processes. This interaction is characterized by:

• Regulatory Mechanism: NSFL1 cofactor p47 reduces the ATPase activity of VCP, thereby modulating its function in various cellular contexts

• Golgi Dynamics: The interaction is necessary for VCP-mediated fragmentation of Golgi stacks during mitosis and subsequent reassembly after mitosis

• ER Formation: It may play a role in VCP-mediated formation of transitional endoplasmic reticulum (tER)

• Mitotic Regulation: Together with UBXN2B/p37, it regulates centrosomal levels of AURKA/Aurora A during mitotic progression

This interaction represents a critical regulatory node in membrane dynamics during cell division. To experimentally investigate this interaction, researchers typically employ co-immunoprecipitation, proximity ligation assays, or FRET-based approaches to detect physical association, alongside functional assays measuring ATPase activity to assess the regulatory impact of NSFL1C on VCP function .

What approaches can be used to study the role of NSFL1 cofactor p47 in mitotic progression?

Investigating the role of NSFL1 cofactor p47 in mitotic progression requires a multifaceted approach:

• Live Cell Imaging:

  • Fluorescently tag NSFL1C to track its localization during different stages of mitosis

  • Co-visualization with markers for centrosomes, spindles, and Golgi to assess temporal relationships

  • Quantitative analysis of spindle orientation and positioning

• Functional Perturbation:

  • CRISPR-Cas9 gene editing to generate knockout or knockdown models

  • Expression of dominant-negative mutants to disrupt specific functional domains

  • Small molecule inhibitors of the NSFL1C-VCP interaction if available

• Interaction Studies:

  • Proximity-based proteomics (BioID, APEX) to identify mitosis-specific interaction partners

  • Co-immunoprecipitation studies during different cell cycle stages

  • Analysis of AURKA/Aurora A levels at centrosomes in the presence/absence of NSFL1C

• Biochemical Characterization:

  • In vitro reconstitution assays to measure effects on AURKA/Aurora A recruitment and removal

  • Phosphorylation profiling to identify regulatory post-translational modifications

  • Structural studies to determine binding interfaces with mitotic regulators

These approaches should be integrated within a comprehensive experimental design that includes appropriate controls and statistical validation to elucidate the precise mechanisms by which NSFL1C influences mitotic progression and spindle orientation .

What are the critical quality control parameters for recombinant chicken NSFL1 cofactor p47 preparation?

Quality control for recombinant chicken NSFL1 cofactor p47 preparations should address several key parameters:

Since chicken NSFL1 cofactor p47 functions by reducing the ATPase activity of VCP and participating in protein-protein interactions, functional assays should verify these capabilities. Stability testing under various storage conditions is also essential, with a recommendation for storage at -20°C for long-term preservation and 4°C for short-term use to maintain structural and functional integrity .

How can researchers resolve common issues in the expression and purification of recombinant NSFL1 cofactor p47?

Researchers frequently encounter challenges when expressing and purifying recombinant NSFL1 cofactor p47. The following troubleshooting guide addresses common issues:

• Poor Expression Yields:

  • Optimize codon usage for the expression host

  • Test multiple expression systems (bacterial, insect, mammalian)

  • Vary induction conditions (temperature, inducer concentration, duration)

  • Consider fusion tags that enhance solubility (MBP, SUMO, thioredoxin)

• Protein Insolubility:

  • Reduce expression temperature (e.g., 18-25°C instead of 37°C)

  • Include molecular chaperones in co-expression

  • Test different lysis buffers with various detergents and stabilizers

  • Express truncated constructs based on domain boundaries

• Purification Challenges:

  • For RP-HPLC purification, optimize the acetonitrile gradient (0.1%/min is recommended)

  • Adjust sample loading to prevent column saturation

  • Consider sample pre-treatment with 0.1% aqueous TFA for optimal binding

  • For multi-step approaches, minimize sample handling between steps

• Activity Loss:

  • Include protease inhibitors throughout purification

  • Test different buffer compositions for stabilization

  • Add glycerol (10%) to prevent aggregation

  • Determine optimal pH range for stability

Implementing the one-step RP-HPLC purification method described earlier can yield >94% purity with >90% recovery, significantly improving upon traditional affinity chromatography approaches that typically result in lower purity and yield due to multiple handling steps .

What experimental designs are recommended for investigating NSFL1 cofactor p47 interactions with binding partners?

When investigating interactions between NSFL1 cofactor p47 and its binding partners, researchers should consider the following experimental designs:

• Primary Interaction Screening:

  • Yeast two-hybrid screening to identify novel interaction partners

  • Pull-down assays using recombinant NSFL1C as bait

  • Protein microarrays to test interactions with known cellular proteins

• Validation and Characterization:

  • Co-immunoprecipitation with endogenous proteins

  • Proximity ligation assay for in situ visualization of interactions

  • FRET or BRET assays for real-time interaction dynamics

  • Surface plasmon resonance or isothermal titration calorimetry for binding kinetics

• Functional Impact Analysis:

  • VCP ATPase activity assays in the presence/absence of NSFL1C

  • Golgi fragmentation and reassembly assays during mitosis

  • AURKA/Aurora A centrosomal localization studies

  • Spindle orientation quantification in manipulated cells

• Structural Studies:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • X-ray crystallography or cryo-EM of complexes

  • NMR studies of domain-specific interactions

When designing these experiments, researchers should include appropriate controls to distinguish specific from non-specific interactions and consider the potential impact of tags or fusion proteins on interaction dynamics. Cellular context is also crucial, as interactions may be cell-cycle dependent or influenced by post-translational modifications .

How can researchers utilize proteomics approaches to study NSFL1 cofactor p47 in cellular contexts?

Proteomics offers powerful approaches for studying NSFL1 cofactor p47 in cellular contexts:

• Interaction Proteomics:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Proximity-dependent biotin identification (BioID) or APEX labeling

  • Crosslinking mass spectrometry (XL-MS) to capture transient interactions

  • These methods can identify novel binding partners and characterize interaction networks

• Expression and Localization Profiling:

  • Cellular fractionation followed by Western blotting and LC-MS/MS

  • Targeted proteomics (MRM/PRM) for sensitive quantification

  • Spatial proteomics using organelle proteomics approaches

  • Data from such studies can reveal compartment-specific functions and dynamics

• Post-translational Modification Analysis:

  • Phosphoproteomics to identify regulatory modifications

  • Ubiquitinylation analysis to understand turnover mechanisms

  • Glycosylation profiling if relevant for secreted forms

  • These analyses help elucidate regulatory mechanisms

• Protein Turnover Studies:

  • Pulse-chase SILAC (Stable Isotope Labeling with Amino acids in Cell culture)

  • Dynamic SILAC to determine half-life

  • Thermal proteome profiling for stability analysis

  • Such approaches provide insights into protein dynamics and stability

Implementing these proteomics strategies requires careful experimental design, including appropriate controls, biological replicates, and statistical validation. For example, cellular fractionation studies of PCI-positive fractions have successfully identified NSFL1C in specific subcellular compartments, revealing insights about its localization and potential functional contexts .

What are the emerging techniques that could advance our understanding of NSFL1 cofactor p47 function?

Several cutting-edge techniques hold promise for deeper insights into NSFL1 cofactor p47 function:

• CRISPR-Based Approaches:

  • CRISPRi/CRISPRa for precise transcriptional control

  • Base editing for introducing specific mutations

  • Prime editing for targeted sequence replacements

  • These approaches enable more nuanced functional studies than traditional knockouts

• Advanced Imaging Techniques:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale localization

  • Lattice light-sheet microscopy for dynamic 3D imaging with minimal phototoxicity

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

  • These methods provide unprecedented spatial and temporal resolution

• Integrative Structural Biology:

  • AlphaFold2 and similar AI-based structural prediction

  • Integrative modeling combining multiple structural data types

  • In-cell NMR for structure determination in native environments

  • These approaches overcome limitations of traditional structural biology methods

• Systems Biology Integration:

  • Multi-omics integration to connect NSFL1C function to global cellular processes

  • Network analysis to identify critical nodes and pathways

  • Mathematical modeling of VCP-NSFL1C dynamics during cell cycle

  • These strategies place NSFL1C function in broader biological contexts

Application of these emerging technologies to NSFL1 cofactor p47 research could reveal new functional roles, regulatory mechanisms, and therapeutic possibilities that are currently hidden by methodological limitations .

What are the implications of studying chicken NSFL1 cofactor p47 for understanding conserved cellular processes?

Studying chicken NSFL1 cofactor p47 has significant implications for understanding evolutionarily conserved cellular processes:

• Evolutionary Conservation:

  • Chicken NSFL1C shares significant sequence and structural homology with mammalian orthologs

  • Functional domains are highly conserved across species, suggesting fundamental biological roles

  • Comparative studies can reveal essential vs. species-specific functions

• Model System Advantages:

  • Chicken systems offer unique advantages for developmental studies

  • The chicken embryo is readily accessible for manipulation and imaging

  • Cell lines derived from chicken tissues can provide insights applicable across species

• Fundamental Cell Biology:

  • Golgi dynamics during mitosis represent a conserved cellular challenge

  • VCP/p97 functions are critical across eukaryotes

  • Studying NSFL1C in chicken systems can illuminate universal mechanisms

• Translational Relevance:

  • Conservation of function suggests potential relevance to human disease

  • Insights from chicken NSFL1C may inform understanding of pathologies involving VCP dysfunction

  • Therapeutic approaches targeting this pathway might have broad applicability

By leveraging the advantages of chicken model systems while recognizing the conservation of NSFL1C function, researchers can gain insights that extend beyond species boundaries to illuminate fundamental aspects of cellular biology with potential implications for human health and disease .

How can recombinant chicken NSFL1 cofactor p47 be used as a tool in cell biology research?

Recombinant chicken NSFL1 cofactor p47 can serve as a valuable research tool in several applications:

• As a Molecular Probe:

  • Fluorescently labeled NSFL1C for tracking Golgi dynamics in live cells

  • Biotinylated NSFL1C for pull-down assays to identify interaction partners

  • Peptide fragments as competitive inhibitors of specific protein-protein interactions

  • These applications provide insights into subcellular dynamics and molecular networks

• For Functional Assays:

  • In vitro reconstitution of VCP-mediated membrane fusion/fission

  • Cell-free assays of Golgi fragmentation and reassembly

  • Measurements of ATPase activity modulation

  • These approaches enable mechanistic studies under controlled conditions

• As a Standard:

  • Positive control for antibody validation

  • Reference for absolute quantification in proteomics

  • Calibration standard for binding kinetics measurements

  • These applications enhance experimental reliability and comparability

• For Method Development:

  • Model protein for optimizing purification protocols

  • Test substrate for developing novel interaction detection methods

  • Structural template for computational drug design

  • These uses advance methodological innovation

Recombinant chicken NSFL1C, particularly when produced through optimized RP-HPLC purification methods, provides a consistent and well-characterized reagent for these diverse research applications, enhancing reproducibility and facilitating new discoveries in cell biology .

How should researchers interpret conflicting data regarding NSFL1 cofactor p47 function across different experimental systems?

When confronted with conflicting data regarding NSFL1 cofactor p47 function, researchers should implement a systematic approach to interpretation:

• Methodological Analysis:

  • Evaluate differences in experimental techniques (in vitro vs. cellular, different detection methods)

  • Consider the impact of tags, fusion proteins, or antibodies on protein function

  • Assess cellular contexts (overexpression vs. endogenous levels, cell type differences)

  • These factors can significantly influence experimental outcomes

• Biological Complexity Recognition:

  • Consider cell cycle, differentiation state, or developmental context

  • Evaluate potential redundancy with other UBX domain-containing proteins

  • Assess the influence of post-translational modifications

  • These biological variables may explain apparent contradictions

• Integrative Data Assessment:

  • Perform meta-analysis of available data, weighing quality and reproducibility

  • Generate testable models that could reconcile conflicting observations

  • Design critical experiments that directly address contradictions

  • This approach transforms conflicts into opportunities for deeper understanding

• Reporting Recommendations:

  • Clearly communicate experimental conditions and limitations

  • Present alternative interpretations of data

  • Acknowledge conflicts with existing literature

  • These practices enhance scientific transparency and progress

By approaching conflicting data as an opportunity rather than an obstacle, researchers can develop more nuanced models of NSFL1 cofactor p47 function that accommodate context-dependent activities and regulatory mechanisms .

What standards should be applied when validating antibodies for chicken NSFL1 cofactor p47 research?

Rigorous antibody validation is essential for reliable NSFL1 cofactor p47 research. The following standards should be applied:

• Specificity Verification:

  • Western blot against recombinant protein and tissue/cell lysates

  • Immunoprecipitation followed by mass spectrometry

  • Testing in knockout/knockdown models or with blocking peptides

  • These approaches confirm binding to the intended target

• Application-Specific Validation:

  • For Western blotting: verify single band of correct molecular weight

  • For immunohistochemistry: demonstrate specific staining pattern

  • For immunoprecipitation: confirm enrichment of target protein

  • Each application requires dedicated validation protocols

• Cross-Species Reactivity Assessment:

  • Test antibodies raised against human NSFL1C on chicken samples

  • Verify epitope conservation through sequence alignment

  • Consider generating species-specific antibodies if needed

  • This is crucial when using antibodies across evolutionary boundaries

• Proper Controls:

  • Positive controls: tissues/cells known to express NSFL1C

  • Negative controls: knockout samples or tissues without expression

  • Technical controls: secondary antibody-only, isotype controls

  • These minimize false positives and negatives