Recombinant Osmerus mordax Ubiquitin-fold modifier-conjugating enzyme 1 (ufc1)

What is Osmerus mordax UFC1 and what is its biological function?

Ubiquitin-Fold Modifier Conjugating Enzyme 1 (UFC1) from Osmerus mordax (Rainbow smelt) is a protein involved in the UFM1 conjugation pathway, which is a ubiquitin-like post-translational modification system. This enzyme functions as an E2-like conjugating enzyme that accepts the ubiquitin-fold modifier (UFM1) from UBA5 (E1-activating enzyme) and transfers it to target proteins, typically facilitated by an E3 ligase. The UFC1 protein is part of the broader ubiquitin-like protein modification system that regulates various cellular processes including protein quality control, endoplasmic reticulum homeostasis, and cellular stress responses. The Osmerus mordax variant provides researchers with a model to study evolutionary conservation of the UFM1 pathway across species .

How is recombinant Osmerus mordax UFC1 produced and purified?

Recombinant Osmerus mordax UFC1 is commonly expressed using a yeast expression system, which provides several advantages for this protein. The production process typically includes:

• Cloning: The UFC1 gene from Osmerus mordax is cloned into a yeast expression vector containing a His-tag sequence.

• Transformation: The expression construct is transformed into yeast cells.

• Expression: Transformed yeast cultures are grown under controlled conditions to express the recombinant protein.

• Extraction: Cells are lysed to release the expressed protein.

• Purification: The protein undergoes affinity chromatography using the His-tag, followed by additional purification steps.

• Quality Control: The purified protein is assessed for purity (>90% by SDS-PAGE) and functionality.

The yeast expression system is particularly valuable for UFC1 as it allows for eukaryotic post-translational modifications while being more economical than mammalian expression systems. This results in a protein that closely resembles the native structure and function while maintaining reasonable production costs .

What are the available storage and handling recommendations for recombinant UFC1?

For optimal stability and functionality of recombinant Osmerus mordax UFC1, the following storage and handling protocols are recommended:

• Storage Format: The protein is typically supplied in lyophilized form or in a Tris-based buffer with 50% glycerol.

• Storage Temperature: Long-term storage should be at -20°C or -80°C for extended periods.

• Working Aliquots: Prepare small working aliquots that can be stored at 4°C for up to one week.

• Stability Considerations: Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and activity.

• Reconstitution: Lyophilized protein should be reconstituted in appropriate buffers (typically Tris-based) before use.

Following these guidelines ensures maintenance of protein structure and biological activity during storage and experimental use .

How can recombinant Osmerus mordax UFC1 be utilized in ELISA-based experimental designs?

Recombinant Osmerus mordax UFC1 can be employed in several ELISA configurations to study protein-protein interactions and enzymatic activities:

• Direct ELISA: Immobilize recombinant UFC1 on a plate surface to detect interaction with UFM1, UBA5, or substrate proteins. This approach can be used to:

  • Screen for novel UFC1-interacting proteins

  • Quantify binding affinities between UFC1 and known partners

  • Evaluate specificity of anti-UFC1 antibodies

• Sandwich ELISA: Use anti-UFC1 antibodies as capture antibodies and detect with antibodies against interaction partners to study complex formation in solution.

• Activity-Based ELISA: Design assays where UFC1 conjugating activity is measured by detecting UFM1 transfer to substrate proteins.

Methodological considerations should include:

• Optimization of coating concentrations (typically 1-10 μg/ml)

• Selection of appropriate blocking agents to minimize background

• Validation of antibody specificity using appropriate controls

• Inclusion of positive and negative controls to ensure assay reliability

These approaches can yield quantitative data on UFC1 interactions and enzymatic activities in controlled experimental conditions .

What are the comparative advantages of different expression systems for recombinant UFC1?

Different expression systems offer distinct advantages for recombinant UFC1 production, which researchers should consider based on their specific experimental requirements:

The choice of expression system should be based on the specific research goals, budget constraints, and the importance of post-translational modifications for the particular experimental design .

What methodological approaches can be used to evaluate the enzymatic activity of recombinant UFC1?

To assess the enzymatic activity of recombinant Osmerus mordax UFC1, researchers can employ several complementary methodological approaches:

• In vitro Conjugation Assays:

  • Combine purified recombinant UFC1 with UFM1, UBA5 (E1), ATP, and substrate proteins

  • Monitor formation of UFC1~UFM1 thioester intermediates and final substrate conjugation

  • Detection methods include Western blotting, fluorescence-based assays, or mass spectrometry

• Thioester Bond Formation Analysis:

  • Assess the ability of UFC1 to form thioester bonds with UFM1

  • Use non-reducing SDS-PAGE followed by Western blotting to detect UFC1~UFM1 intermediates

  • Compare samples with and without reducing agents (e.g., DTT or β-mercaptoethanol)

• ATP Consumption Assays:

  • Measure ATP hydrolysis as an indirect indicator of UFC1 activity

  • Use luminescence-based ATP detection kits to quantify remaining ATP

  • Include appropriate controls (no enzyme, heat-inactivated enzyme)

• Fluorescence Resonance Energy Transfer (FRET):

  • Label UFM1 and potential substrates with fluorophore pairs

  • Monitor real-time conjugation through changes in FRET signal

  • Calculate reaction kinetics from time-course data

• Mass Spectrometry:

  • Identify UFM1 attachment sites on substrates following in vitro reactions

  • Quantify the stoichiometry of modification

  • Compare wild-type UFC1 with mutant variants to assess specificity

These approaches provide complementary data on different aspects of UFC1 enzymatic function and can be selected based on available equipment, expertise, and specific research questions .

How can researchers design cross-species comparative studies using Osmerus mordax UFC1?

Designing robust cross-species comparative studies with Osmerus mordax UFC1 requires systematic methodological approaches:

• Sequence Analysis and Alignment:

  • Align UFC1 sequences from multiple species (e.g., human, mouse, zebrafish, Osmerus mordax)

  • Identify conserved domains, active sites, and species-specific variations

  • Use phylogenetic analysis to establish evolutionary relationships

• Structural Comparison:

  • Generate homology models of Osmerus mordax UFC1 based on available UFC1 structures

  • Compare predicted structures to identify conserved structural features

  • Analyze potential differences in substrate binding regions

• Functional Conservation Assays:

  • Assess the ability of Osmerus mordax UFC1 to complement UFC1 deficiency in other species

  • Use cell lines with UFC1 knockdown/knockout to test functional rescue

  • Compare enzymatic parameters (Km, Vmax, substrate specificity) across species

• Interactome Analysis:

  • Perform pull-down experiments with tagged Osmerus mordax UFC1 in different species' cell extracts

  • Identify conserved and species-specific interaction partners by mass spectrometry

  • Validate key interactions through co-immunoprecipitation or yeast two-hybrid assays

• Environmental Adaptation Studies:

  • Investigate how UFC1 function may have adapted to the cold-water environment of rainbow smelt

  • Compare thermal stability and activity profiles across temperature ranges

  • Assess potential cold-adaptation features in the protein structure

These approaches allow researchers to gain insights into the evolutionary conservation and divergence of the UFM1 pathway across species, potentially revealing fundamental aspects of this ubiquitin-like modification system .

What are the considerations for developing antibodies against Osmerus mordax UFC1?

Developing specific and effective antibodies against Osmerus mordax UFC1 requires careful planning and validation:

• Epitope Selection and Antigen Preparation:

  • Use bioinformatic tools to identify unique, surface-exposed epitopes

  • Consider producing both full-length protein and specific peptide antigens

  • Ensure high purity (>90%) of recombinant UFC1 used for immunization

  • Verify proper folding of the antigen to generate antibodies recognizing native conformations

• Immunization Strategies:

  • Select appropriate animal models (rabbits for polyclonal; mice or rats for monoclonal)

  • Design immunization schedules with proper adjuvants

  • Monitor antibody titers throughout the immunization process

  • Consider the advantages of recombinant antibody technology as an alternative

• Cross-Reactivity Analysis:

  • Test antibodies against UFC1 from multiple species to determine specificity

  • Create a cross-reactivity profile using Western blot, ELISA, and immunoprecipitation

  • Determine whether antibodies recognize specific domains or conformations

• Validation Methods:

  • Western blot with recombinant and native UFC1 proteins

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence in cells expressing UFC1

  • Negative controls using UFC1-depleted samples

• Application-Specific Validation:

  • For each intended application (WB, IP, IF, IHC, ELISA), perform specific validation tests

  • Determine optimal working conditions (concentrations, buffers, incubation times)

  • Establish detection limits and quantification ranges

A systematic approach to antibody development and validation ensures reliable tools for UFC1 research across multiple experimental platforms .

What are common challenges in maintaining recombinant UFC1 stability and how can they be addressed?

Researchers working with recombinant Osmerus mordax UFC1 may encounter several stability challenges that can be systematically addressed:

• Protein Aggregation:

  • Problem: Formation of insoluble aggregates during storage or experimental handling

  • Solution: Add 5-10% glycerol to storage buffers; maintain protein at concentrations below 2 mg/mL; include reducing agents like DTT (0.5-1 mM); optimize buffer pH based on protein's isoelectric point

• Activity Loss During Storage:

  • Problem: Decreased enzymatic function after storage periods

  • Solution: Store as aliquots at -80°C; avoid repeated freeze-thaw cycles; add stabilizing agents like trehalose (10%); monitor activity using functional assays before experiments

• Thiol Oxidation:

  • Problem: Oxidation of catalytically important cysteine residues

  • Solution: Include reducing agents in buffers; prepare fresh solutions before experiments; consider argon/nitrogen-purged buffers for sensitive applications

• Proteolytic Degradation:

  • Problem: Appearance of degradation products during storage or experiments

  • Solution: Add protease inhibitor cocktails to working solutions; maintain samples at 4°C during experiments; minimize exposure to room temperature

• Tag Interference with Function:

  • Problem: His-tag affecting protein activity or interactions

  • Solution: Compare tagged and tag-cleaved versions; position tags away from functional domains; verify that tag does not interfere with activity through parallel assays

Monitoring protein quality through analytical techniques (SEC, DLS, activity assays) before critical experiments ensures reliable and reproducible results when working with recombinant UFC1 .

How can researchers optimize buffer conditions for recombinant UFC1 enzymatic assays?

Optimizing buffer conditions is critical for accurate assessment of recombinant Osmerus mordax UFC1 enzymatic activity. A systematic approach includes:

• Buffer Type Screening:

  • Test multiple buffer systems (Tris, HEPES, phosphate, MES) at 50-100 mM

  • Evaluate activity across pH range 6.5-8.5 in 0.5 pH increments

  • Determine temperature stability in each buffer system (4°C, 25°C, 37°C)

• Salt Concentration Optimization:

  • Test NaCl concentrations from 0-500 mM to identify optimal ionic strength

  • Evaluate effects of different cations (K+, Na+, Mg2+) on enzyme performance

  • Consider the impact of salt on protein-protein interactions in multi-component assays

• Reducing Conditions:

  • Assess activity with various reducing agents (DTT, β-mercaptoethanol, TCEP)

  • Determine optimal reducing agent concentration (typically 0.5-5 mM)

  • Consider the impact of reducing conditions on thioester bond stability

• Stabilizing Additives:

  • Test effects of glycerol (5-20%), BSA (0.1-1 mg/mL), and other stabilizers

  • Evaluate whether additives affect enzyme kinetics or just stability

  • Include appropriate controls to account for additive effects

• Systematic Optimization Example:

Methodical optimization of these parameters ensures maximum enzyme performance and reproducible results across experimental replicates .

What controls should be included when studying recombinant UFC1-mediated protein interactions?

Rigorous experimental design for studying recombinant Osmerus mordax UFC1 interactions requires comprehensive controls:

• Negative Controls:

  • Catalytically inactive UFC1 mutant (active site cysteine to alanine/serine)

  • Heat-denatured UFC1 protein (95°C for 10 minutes)

  • Unrelated protein with similar size and tag (e.g., His-tagged GFP)

  • Reaction mixtures lacking essential components (ATP, E1 enzyme, UFM1)

• Positive Controls:

  • Known UFC1 interaction partners (e.g., UFM1, UBA5)

  • Pre-validated substrate proteins with confirmed UFMylation sites

  • Commercially available UFC1 from related species with established activity

• Specificity Controls:

  • Competitive inhibition with excess unlabeled protein

  • Dose-response relationships with varying UFC1 concentrations

  • Pre-blocking with specific antibodies to prevent interactions

  • Tag-only controls to rule out tag-mediated interactions

• Technical Controls:

  • Input samples to verify protein presence before pull-down/IP

  • Loading controls for consistent protein amounts across samples

  • Replicates across multiple protein preparations

  • Time-course analyses to establish reaction kinetics

• Buffer and Condition Controls:

  • Parallel reactions at different salt/pH conditions

  • Reactions with and without reducing agents

  • Controls for potential effects of detergents or stabilizing agents

A systematic control framework ensures that observed interactions are specific, reproducible, and physiologically relevant rather than artifacts of the experimental system .

What are emerging applications of recombinant UFC1 in studying cellular stress response mechanisms?

Recombinant Osmerus mordax UFC1 presents valuable opportunities for investigating stress response mechanisms across species:

• Comparative Cold Stress Adaptation:

  • Investigate how UFC1 from the cold-water rainbow smelt may participate in adaptation to low temperatures

  • Compare UFMylation patterns between cold-adapted and temperate species under temperature stress

  • Examine how UFMylation machinery may be modified for function in extreme environments

• ER Stress Response Pathways:

  • Use recombinant UFC1 to reconstitute UFMylation of key ER-resident proteins in vitro

  • Compare how UFMylation patterns change under various ER stress conditions

  • Develop reporter systems to monitor UFC1 activity during unfolded protein response activation

• Oxidative Stress Signaling:

  • Investigate whether UFC1 activity or substrate specificity changes under oxidative conditions

  • Examine potential redox regulation of the UFC1 active site cysteine

  • Map the relationship between UFMylation and other stress-responsive post-translational modifications

• Tissue-Specific Stress Responses:

  • Develop tools to monitor UFC1 activity in different tissues under stress conditions

  • Compare UFMylation patterns in metabolically active versus quiescent tissues

  • Investigate potential roles in tissue-specific stress adaptation mechanisms

• Cross-Talk with Other Ubiquitin-Like Modifiers:

  • Examine how the UFM1 pathway cooperates with or antagonizes other ubiquitin-like modification systems

  • Map the interactome of UFC1 under various stress conditions

  • Develop multiplexed assays to monitor multiple modification pathways simultaneously

These research directions could reveal fundamental aspects of how post-translational modification systems contribute to cellular resilience under adverse conditions, with potential implications for understanding stress adaptation across species .

How might recombinant UFC1 contribute to understanding evolutionary conservation of ubiquitin-like modification systems?

Recombinant Osmerus mordax UFC1 provides a valuable tool for evolutionary studies of ubiquitin-like modification systems:

• Phylogenetic Analysis and Functional Conservation:

  • Reconstruct the evolutionary history of the UFM1 pathway across aquatic and terrestrial vertebrates

  • Compare substrate specificities between UFC1 orthologs from diverse species

  • Investigate whether environmental adaptations have shaped UFC1 function in different lineages

• Structure-Function Relationships Across Species:

  • Perform comparative structural analysis of UFC1 proteins from diverse species

  • Identify conserved surfaces for protein-protein interactions

  • Map species-specific variations to functional differences through mutagenesis studies

• Methodological Approach to Cross-Species Complementation:

  • Test whether Osmerus mordax UFC1 can complement UFC1 deficiency in mammalian cells

  • Analyze which domains or residues are essential for cross-species functionality

  • Create chimeric proteins to map species-specific functional elements

• Comparative Interactome Analysis:

  • Identify conserved and species-specific UFC1 interaction partners

  • Investigate how interaction networks have evolved across species

  • Correlate interactome changes with species-specific physiological adaptations

• Environmental Adaptation of the UFM1 Pathway:

  • Compare UFC1 from species living in different environments (marine, freshwater, terrestrial)

  • Investigate temperature, pH, or salinity adaptations in UFC1 function

  • Develop experimental systems to test environmental influences on UFMylation

These approaches can reveal fundamental principles of how ubiquitin-like modification systems have evolved while maintaining essential functions, potentially identifying both conserved mechanisms and species-specific adaptations .