Recombinant Drosophila virilis DDRGK domain-containing protein 1 (GJ23857)

What expression systems are recommended for producing recombinant DDRGK domain-containing protein 1?

Several expression systems can be used for producing recombinant DDRGK domain-containing protein 1, each with specific advantages:

E. coli and yeast systems offer the best yields and shorter turnaround times and are suitable for applications where post-translational modifications are not critical . For studies requiring functional activity dependent on proper protein folding or post-translational modifications, insect cell or mammalian expression systems are recommended despite their lower yields and longer processing times .

What are the optimal storage conditions for recombinant DDRGK domain-containing protein 1?

For optimal stability of recombinant DDRGK domain-containing protein 1, the following storage conditions are recommended:

• Short-term storage (up to one week): 4°C

• Standard storage: -20°C

• Extended storage: -20°C or -80°C

• Avoid repeated freeze-thaw cycles as they can lead to protein degradation and loss of activity

For long-term storage, the protein should be reconstituted and aliquoted with glycerol (recommended final concentration 50%) to prevent freeze-thaw damage . The shelf life is approximately 6 months for liquid form at -20°C/-80°C and 12 months for lyophilized form at -20°C/-80°C. Shelf life depends on multiple factors including buffer ingredients, storage temperature, and the intrinsic stability of the protein itself .

How should recombinant DDRGK domain-containing protein 1 be reconstituted for experimental use?

For optimal reconstitution of recombinant DDRGK domain-containing protein 1:

• Initial preparation: Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitution buffer: Use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Glycerol addition: Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquoting: Divide into small working aliquots to minimize freeze-thaw cycles

• Storage: Store reconstituted aliquots at -20°C/-80°C for long-term storage

For functional assays, consider using buffers that maintain physiological pH (typically 7.2-7.4) and adding protease inhibitors to prevent degradation. The specific buffer composition may need optimization depending on the intended experimental application, as buffer components can affect protein activity and stability.

What experimental approaches can be used to study DDRGK domain-containing protein 1 interactions with other proteins?

Several experimental approaches can be employed to study protein-protein interactions involving DDRGK domain-containing protein 1:

Research has shown that human DDRGK1 interacts with components of the UFM1 pathway, particularly UFL1 . In studies examining similar interactions, GST-tagged proteins were used in pull-down assays to verify interactions between UFL1, C53, and other proteins . When designing experiments, researchers should consider that DDRGK domain-containing protein 1 likely participates in complexes involving UFM1 pathway components, and these interactions may be conserved across species.

How can researchers verify the functional activity of recombinant DDRGK domain-containing protein 1?

Verifying the functional activity of recombinant DDRGK domain-containing protein 1 requires multiple approaches:

• Binding assays: Assess the ability of the recombinant protein to bind known interaction partners such as UFL1 using biochemical techniques (pull-down assays, surface plasmon resonance)

• Ufmylation assays: Test the protein's ability to participate in the ufmylation pathway by reconstituting the reaction in vitro with purified components (UFM1, UBA5, UFC1, UFL1)

• Cellular complementation: Express the recombinant protein in DDRGK1-depleted cells to determine if it restores phenotypes associated with DDRGK1 deficiency

• Structural integrity assessment: Use circular dichroism or thermal shift assays to verify proper folding

• Subcellular localization: Confirm proper localization using fluorescently-tagged versions of the protein to ensure it targets the expected cellular compartments (endoplasmic reticulum)

Research in human cells has shown that DDRGK1 functions in the UFM1 pathway and is involved in endoplasmic reticulum homeostasis . While functional assays would ideally be species-specific, the conservation of the DDRGK domain suggests that assays developed for human DDRGK1 may be adapted for the Drosophila virilis ortholog.

What considerations should guide the choice between prokaryotic and eukaryotic expression systems?

When choosing between prokaryotic and eukaryotic expression systems for DDRGK domain-containing protein 1, researchers should consider:

The decision should be based on the research question being addressed. If structural studies or antibody production are planned, E. coli may be sufficient. For functional studies examining protein-protein interactions or cellular activities, eukaryotic systems that provide appropriate post-translational modifications would be preferable . Evidence suggests that proper folding and modifications may be crucial for DDRGK domain-containing protein 1 function, particularly in its role in the UFM1 pathway.

How does DDRGK domain-containing protein 1 contribute to endoplasmic reticulum homeostasis and stress responses?

Based on research with human DDRGK1, the protein appears to play critical roles in ER homeostasis and stress responses:

• Ribosome recycling: DDRGK1 participates in ribosome recycling by mediating mono-ufmylation of the RPL26/uL24 subunit of the 60S ribosome following ribosome dissociation. This ufmylation weakens the junction between post-termination 60S subunits and SEC61 translocons, promoting release and recycling of the large ribosomal subunit from the ER membrane

• ER-associated degradation: The protein is involved in ufmylation-dependent reticulophagy that promotes lysosomal degradation of ufmylated proteins

• Unfolded protein response regulation: DDRGK1 participates in inhibiting the unfolded protein response (UPR) by regulating ERN1/IRE1-alpha stability

• Selective autophagy: Required for stabilization and ufmylation of ATG9A, suggesting a role in autophagy regulation

These functions position DDRGK domain-containing protein 1 as a critical factor in maintaining ER homeostasis, particularly under stress conditions. The specific conservation of these functions in Drosophila virilis remains to be fully characterized, presenting an opportunity for comparative studies of ER stress responses across species.

What evolutionary insights can be gained from studying DDRGK domain-containing protein 1 in Drosophila virilis compared to other Drosophila species?

Studying DDRGK domain-containing protein 1 in Drosophila virilis offers valuable evolutionary insights:

• Comparative genomics: D. virilis is a prominent reference species for comparison with D. melanogaster in patterns and mechanisms of molecular and genomic evolution . The genome of D. virilis shows extensive rearrangements relative to D. melanogaster, making it valuable for studying gene evolution and conservation

• Functional conservation: Comparing DDRGK domain-containing protein 1 function between these species can reveal the core conserved functions versus species-specific adaptations

• Regulatory evolution: The regulation of DDRGK domain-containing protein 1 may differ between species, providing insights into the evolution of gene regulatory networks

• Protein interaction network evolution: Differences in protein-protein interactions involving DDRGK domain-containing protein 1 between species can illuminate how interaction networks evolve

• Adaptation to cellular stress: Species-specific roles in stress responses may reflect evolutionary adaptations to different ecological niches

Drosophila virilis diverged from D. melanogaster approximately 40-60 million years ago, making it well-suited for evolutionary studies. The virilis and montana clades are estimated to have diverged about 9.0 ± 0.7 million years ago , providing an appropriate evolutionary distance for comparative functional studies.

How might the function of DDRGK domain-containing protein 1 relate to the unique histone organization in Drosophila virilis?

Drosophila virilis possesses a unique histone gene organization compared to other Drosophila species that may intersect with DDRGK domain-containing protein 1 function:

• Atypical histone arrangements: Unlike D. melanogaster, D. virilis contains two distinct histone loci with different organization: one with quartet repeats lacking the H1 gene and another with multi-length variant quintet repeats containing the H1 gene

• MSL2 targeting: Interestingly, MSL2 (a component of the dosage compensation complex) targets one of the two autosomal histone loci in D. virilis but not in other Drosophila species

• Potential regulatory connections: While direct links between DDRGK domain-containing protein 1 and histone regulation in D. virilis have not been established, human DDRGK1 has roles in chromatin regulation through protein ufmylation

• ER-nuclear communication: Human DDRGK1 participates in ER-nuclear communication pathways, and similar functions in D. virilis could potentially influence chromatin organization

• Stress response integration: Both histone modifications and DDRGK1 functions are implicated in stress responses, suggesting possible integration of these pathways

The unique organization of histone genes in D. virilis and the targeting of one histone locus by MSL2 present an intriguing system for studying potential connections between DDRGK domain-containing protein 1, chromatin regulation, and species-specific genomic adaptations. This represents an area where further research could reveal novel functional connections.

How can researchers address protein degradation issues when working with DDRGK domain-containing protein 1?

Protein degradation is a common challenge when working with recombinant proteins like DDRGK domain-containing protein 1. To address this issue:

• Optimize storage conditions:

  • Store at -80°C for long-term storage

  • Use appropriate cryoprotectants (50% glycerol recommended)

  • Prepare single-use aliquots to avoid freeze-thaw cycles

• Buffer optimization:

  • Include protease inhibitors (PMSF, leupeptin, aprotinin, or commercial cocktails)

  • Maintain pH stability (typically pH 7.2-7.8)

  • Consider adding stabilizing agents (glycerol, sucrose, or specific salt concentrations)

• Handling practices:

  • Keep samples on ice when working

  • Use low-protein-binding tubes

  • Minimize pipetting and vortexing to reduce mechanical stress

• Purification strategies:

  • Consider including additional purification steps to remove contaminating proteases

  • Evaluate different purification tags (His, GST, MBP) that may enhance stability

  • Optimize elution conditions to minimize exposure to harsh chemicals

• Expression optimization:

  • Test different expression temperatures (lower temperatures often reduce aggregation)

  • Co-express with chaperones in recombinant systems

  • Consider fusion partners that enhance solubility (MBP, SUMO, TRX)

Monitoring degradation through SDS-PAGE analysis at different time points can help identify when degradation occurs and inform preventive strategies.

What strategies can optimize protein yield during recombinant expression of DDRGK domain-containing protein 1?

Optimizing protein yield requires a systematic approach addressing multiple factors:

For E. coli expression specifically:

• Test multiple strains (BL21(DE3), Rosetta, Arctic Express)

• Optimize induction parameters (OD600 0.6-0.8, IPTG concentration 0.1-1.0 mM)

• Consider auto-induction media for high-density cultures

• Test expression at reduced temperatures (16-30°C) to improve folding

• Co-express with chaperones (GroEL/ES, DnaK) to enhance solubility

For insect/mammalian cell expression:

• Optimize viral titer or DNA:transfection reagent ratio

• Determine optimal harvest time post-infection/transfection

• Consider serum-free formulations for simplified purification

• Evaluate different cell lines for highest expression

What controls should be included in experiments involving DDRGK domain-containing protein 1?

Robust experimental design for DDRGK domain-containing protein 1 studies should include appropriate controls:

For protein-protein interaction studies:

• Negative binding controls: GST or other tag-only controls to assess non-specific binding

• Irrelevant protein controls: Unrelated proteins (e.g., calcineurin has been used as a negative control)

• Positive binding controls: Known interaction partners (e.g., UFL1 if studying DDRGK1)

• Input controls: Analysis of starting material before precipitation/pulldown

• Competition controls: Excess untagged protein to demonstrate binding specificity

For functional assays:

• Catalytically inactive mutants: Mutations in key residues of the DDRGK domain

• Domain deletion variants: Constructs lacking specific functional domains

• Pathway inhibition controls: Chemical inhibitors of relevant pathways

• Knockdown/knockout validation: Cells lacking endogenous protein to confirm specificity

For cellular localization studies:

• Compartment markers: Organelle-specific markers (e.g., ER markers like calnexin)

• Mislocalization controls: Constructs with mutated localization signals

• Fractionation controls: Markers for different cellular compartments in biochemical fractionation

General controls:

• Loading controls: Housekeeping proteins (actin, GAPDH) for western blots

• Expression verification: Antibodies against tags or the protein itself

• Buffer controls: Vehicle-only treatments matching experimental conditions

Including these controls ensures that experimental results can be interpreted with confidence and specificity.

How can researchers assess post-translational modifications of DDRGK domain-containing protein 1?

Assessing post-translational modifications (PTMs) of DDRGK domain-containing protein 1 requires a multi-technique approach:

• Mass spectrometry analysis:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for comprehensive PTM mapping

  • Targeted multiple reaction monitoring (MRM) for specific modifications

  • Top-down proteomics for intact protein analysis

• Modification-specific detection methods:

  • Phosphorylation: Phos-tag gels, phospho-specific antibodies

  • Ubiquitin-like modifications: Antibodies against UFM1

  • Glycosylation: Lectin blotting, PNGase F treatment

  • General PTM shifts: 2D gel electrophoresis

• Functional validation approaches:

  • Site-directed mutagenesis of modified residues

  • In vitro modification assays with purified enzymes

  • Inhibitor studies targeting specific modification pathways

• Comparative analyses:

  • Expression in different systems with varying PTM capabilities

  • Analysis under different cellular conditions (normal vs. stress)

  • Comparison between wild-type and mutant forms

Human DDRGK1 undergoes ufmylation, which affects its stability and function . The Drosophila ortholog may have similar modifications, but species-specific differences may exist. Differences in PTMs between prokaryotic and eukaryotic expression systems should be considered when interpreting functional data, as E. coli-expressed protein will lack many eukaryotic modifications.

What approaches can address solubility challenges with recombinant DDRGK domain-containing protein 1?

Solubility challenges are common with recombinant proteins and can be addressed through multiple strategies:

• Expression conditions optimization:

  • Reduce expression temperature (16-25°C)

  • Lower inducer concentration

  • Use different E. coli strains (Arctic Express, Origami, SHuffle)

  • Co-express with molecular chaperones

• Construct design strategies:

  • Express protein domains separately

  • Use solubility-enhancing fusion tags (MBP, SUMO, TRX, GST)

  • Remove hydrophobic regions through truncation

  • Optimize construct boundaries based on structural predictions

• Buffer optimization:

  • Screen different pH conditions (typically pH 6.0-8.5)

  • Test various salt concentrations (50-500 mM)

  • Add solubilizing agents (0.5-1% Triton X-100, low concentrations of urea)

  • Include stabilizing additives (10-20% glycerol, arginine, proline)

• Refolding approaches:

  • Solubilize inclusion bodies with 6-8M urea or guanidine hydrochloride

  • Use step-wise dialysis for gradual refolding

  • Employ rapid dilution refolding methods

  • Add redox pairs (GSH/GSSG) to facilitate disulfide bond formation

• Alternative expression systems:

  • Switch to eukaryotic expression systems for challenging proteins

  • Try cell-free expression systems

  • Consider specialized bacterial strains for membrane or toxic proteins

A systematic approach testing multiple conditions is often necessary to identify optimal solubilization conditions for DDRGK domain-containing protein 1.