Recombinant Sclerotinia sclerotiorum Eukaryotic translation initiation factor 3 subunit C (nip1), partial

What is eukaryotic translation initiation factor 3 subunit C (eIF3c/nip1) and what is its significance in Sclerotinia sclerotiorum?

Eukaryotic translation initiation factor 3 subunit C (eIF3c) is a critical component of the translation machinery that facilitates protein synthesis by promoting the formation of initiation complexes. In pathogenic organisms like S. sclerotiorum, this protein may play additional roles beyond translation initiation. Research suggests that eIF3c can influence cellular processes including autophagy regulation, as demonstrated in studies of other eukaryotic systems . In S. sclerotiorum, a necrotrophic fungal pathogen that causes diseases in over 400 plant species, nip1 may contribute to virulence mechanisms, though this requires further investigation.

What expression systems are recommended for recombinant nip1 production?

Based on experimental approaches used for similar proteins, E. coli expression systems are commonly employed for initial recombinant protein production attempts. For optimal expression of functional recombinant nip1, consider the following validated parameters based on similar recombinant protein expression studies:

• Growth conditions: Culture until OD600 of 0.8 before induction

• Inducer concentration: 0.1 mM IPTG

• Induction duration: 4 hours

• Temperature during induction: 25°C

• Medium composition: 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose, with appropriate antibiotic selection

These conditions have been shown to yield high levels (up to 250 mg/L) of soluble, functional recombinant protein with similar characteristics .

How can I verify the identity and purity of recombinant nip1?

Multiple complementary techniques should be employed:

• SDS-PAGE and Western blotting using anti-His tag or specific anti-nip1 antibodies

• Mass spectrometry for protein identification and verification

• Size-exclusion chromatography to assess protein homogeneity

• Circular dichroism spectroscopy to confirm proper folding

• Functional assays specific to translation initiation factors

For preliminary confirmation, Western blot analysis using antibodies against the fusion tag (if applicable) or the protein itself can provide initial verification of successful expression, as demonstrated in similar recombinant protein studies .

What are critical factors in experimental design for functional studies of recombinant nip1?

Effective experimental design for functional characterization of recombinant nip1 should implement factorial design approaches to optimize multiple variables simultaneously. Key considerations include:

• Expression parameters optimization: Apply a multifactorial experimental design approach (like the 2^8-4 factorial design used in similar studies) to simultaneously evaluate the effects of:

  • Medium composition

  • Induction timing

  • IPTG concentration

  • Post-induction temperature

  • Induction duration

• Activity assessment: Develop specific functional assays relevant to translation initiation factors, potentially including:

  • In vitro translation efficiency assays

  • RNA binding capacity

  • Protein-protein interaction studies with other initiation factors

• Proper controls: Include both positive controls (known functional proteins) and negative controls (inactive mutants) for validation .

How does nip1/eIF3c interact with cellular pathways based on current research?

Current research indicates that eIF3c plays roles beyond translation initiation. Key findings include:

• Autophagy regulation: Studies have shown that eIF3c upregulation is associated with enhanced autophagy, as evidenced by increased LC3B-II levels. This mechanism has been linked to drug resistance in cancer cells .

• Cellular differentiation pathways: In mammalian systems, related proteins like Numb-interacting Protein 1 (Nip1) regulate cell differentiation through:

  • Modulation of reactive oxygen species (ROS) production via interaction with dual oxidase systems

  • Influence on cytoskeletal organization, particularly through intermediate filaments

  • Interaction with nuclear lamina components like lamin A/C

These findings suggest that fungal nip1 proteins may similarly interact with multiple cellular pathways beyond their canonical roles in translation.

What experimental approaches are most effective for investigating potential protein interactions of recombinant nip1?

To investigate interactions between recombinant nip1 and other proteins, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Using tagged recombinant nip1 to pull down interacting proteins, followed by mass spectrometry identification.

• Yeast two-hybrid screening: Useful for identifying direct protein-protein interactions in a cellular context.

• Biolayer interferometry or surface plasmon resonance: For quantitative measurement of binding kinetics.

• Proximity-based labeling methods: Such as BioID or APEX2 to identify proteins in close proximity to nip1 in live cells.

• Functional validation experiments: Knockdown/overexpression studies to confirm the biological relevance of identified interactions.

Research on related proteins has successfully employed these approaches to identify functional interactions, such as the interaction between Nip1 and lamin A/C associated with neuronal differentiation .

How can the functional impact of post-translational modifications on recombinant nip1 be assessed?

Post-translational modifications (PTMs) may significantly impact nip1 function. To investigate this:

• Identification of PTMs: Use mass spectrometry to identify phosphorylation, acetylation, ubiquitination, or other modifications.

• Site-directed mutagenesis: Generate mutants at potential modification sites to assess functional consequences.

• In vitro modification systems: Apply specific kinases, acetylases, or other modifying enzymes to recombinant nip1 and assess functional changes.

• Comparative analysis: Compare modifications between recombinant protein and native protein from S. sclerotiorum.

• Functional correlation: Correlate specific modifications with functional outcomes using activity assays.

Research on related proteins has shown that post-translational modifications can significantly alter protein function, such as the relationship between phosphorylation status and protein-protein interactions observed in Nip1-related pathways .

What are the recommended approaches for investigating nip1's role in pathogenicity mechanisms?

To investigate potential roles of nip1 in S. sclerotiorum pathogenicity:

• Gene knockout/knockdown studies: Using CRISPR-Cas9 or RNAi to assess the impact of nip1 deficiency on virulence.

• Domain function analysis: Generate recombinant proteins with specific domain deletions or mutations to identify regions critical for virulence-related functions.

• Host-pathogen interaction assays: Examine how recombinant nip1 interacts with host plant proteins using pull-down assays and functional studies.

• Transcriptomics and proteomics: Apply multi-omics approaches to identify genes and proteins affected by nip1 expression levels.

• Structural analysis: Determine the three-dimensional structure of nip1 to identify potential functional sites and compare with mammalian counterparts.

The methodological approaches should be similar to those used in studies of other fungal virulence factors, focusing on both molecular mechanisms and phenotypic outcomes.

What purification strategy yields the highest activity for recombinant nip1?

For optimal purification of active recombinant nip1, a multi-step approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged proteins or glutathione affinity chromatography for GST-tagged proteins.

• Intermediate purification: Ion exchange chromatography based on the theoretical isoelectric point of nip1.

• Polishing step: Size exclusion chromatography to separate monomeric protein from aggregates and remove remaining impurities.

• Buffer optimization: Test multiple buffer compositions, focusing on:

  • pH range: 6.5-8.0

  • Salt concentration: 100-500 mM NaCl

  • Additives: 5-10% glycerol, reducing agents (1-5 mM DTT or β-mercaptoethanol)

• Activity preservation: Include stabilizing agents such as glycerol (10-20%) for long-term storage and consider flash-freezing in liquid nitrogen for cryopreservation.

Similar recombinant protein purification approaches have yielded functional proteins with approximately 75% homogeneity .

How can I overcome common challenges in expressing recombinant eIF3c/nip1?

Common challenges in expressing eukaryotic proteins like nip1 in bacterial systems include insolubility, improper folding, and toxicity to the host. Recommended solutions include:

• For inclusion body formation:

  • Lower induction temperature (16-25°C)

  • Reduce inducer concentration (0.01-0.1 mM IPTG)

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J, trigger factor)

  • Use solubility-enhancing fusion partners (SUMO, MBP, TrxA)

• For toxicity issues:

  • Use tightly regulated expression systems

  • Express in specialized E. coli strains (e.g., C41/C43(DE3) for toxic proteins)

  • Consider cell-free expression systems

• For codon bias:

  • Use codon-optimized synthetic genes

  • Express in E. coli strains with extra copies of rare tRNAs (e.g., Rosetta, CodonPlus)

Factorial design approaches have been successful in optimizing conditions for challenging recombinant proteins, achieving soluble expression levels of 250 mg/L .

What analytical techniques are recommended for assessing eIF3c/nip1 function in translation and beyond?

To comprehensively analyze nip1 function, employ multiple complementary approaches:

• Translation activity assays:

  • In vitro translation systems using rabbit reticulocyte lysate or wheat germ extract

  • Polysome profiling to assess impact on translation initiation

  • Ribosome binding assays

• Molecular interaction studies:

  • RNA binding assays (EMSA, filter binding)

  • Interaction studies with other translation factors

  • Pull-down assays to identify novel binding partners

• Cellular function assessment:

  • ROS measurement assays, as eIF3c-related proteins have been linked to ROS production

  • Autophagy assessment (LC3B-II detection by Western blot)

  • Cytoskeletal organization analysis through immunofluorescence microscopy

• Structural characterization:

  • Circular dichroism for secondary structure analysis

  • Thermal shift assays for stability assessment

  • X-ray crystallography or cryo-EM for three-dimensional structure determination

Research on related proteins has shown that they can influence multiple cellular pathways, including ROS generation through interaction with dual oxidase systems and modulation of intermediate filaments .

How should I interpret contradictory results between in vitro and in vivo studies of recombinant nip1?

When facing contradictory results between in vitro and in vivo studies:

• Evaluate protein integrity: Confirm that the recombinant protein maintains its native structure and modifications in both contexts.

• Consider contextual factors: Examine whether cellular components present in vivo but absent in vitro might influence nip1 function.

• Assess experimental conditions: Determine if differences in concentration, buffer composition, or experimental setup could explain discrepancies.

• Examine time-dependent effects: Consider whether the observed effects occur on different timescales in the two contexts.

• Design bridging experiments: Use cell extracts or reconstituted systems that incorporate key components from the in vivo environment to bridge the gap between fully purified and cellular systems.

Research on Nip1-family proteins has demonstrated that their function can be highly context-dependent, with different outcomes observed in different cellular environments .

What statistical approaches are most appropriate for analyzing factorial design experiments with recombinant nip1?

For factorial design experiments studying recombinant nip1 expression and function:

• Analysis of variance (ANOVA): To identify significant factors and interactions affecting protein expression and activity.

• Response surface methodology (RSM): For optimization of continuous variables like temperature, inducer concentration, and incubation time.

• Principal component analysis (PCA): To identify patterns in multivariate data and reduce dimensionality.

• Multiple linear regression: To develop predictive models of protein yield and activity based on experimental factors.

• Design of experiments software: Consider specialized software like JMP, Design-Expert, or R packages for comprehensive analysis.

Similar statistical approaches were successfully used in optimizing recombinant protein expression, where a 2^8-4 factorial design identified key variables affecting soluble protein yield .

What are promising research directions for investigating differential functions of nip1 across species?

Several promising research directions include:

• Comparative genomics and proteomics: Systematic comparison of nip1 sequences, structures, and interactomes across fungal, plant, and animal species.

• Domain swap experiments: Create chimeric proteins combining domains from different species to identify species-specific functional elements.

• Host-pathogen interaction studies: Investigate how nip1 from pathogenic fungi like S. sclerotiorum interacts with host plant proteins compared to endogenous plant eIF3c.

• Evolutionary analysis: Trace the evolutionary history of nip1/eIF3c to identify conserved and divergent functions.

• Systems biology approaches: Integrate transcriptomics, proteomics, and metabolomics data to build comprehensive models of nip1 function in different organisms.

Research on related proteins has revealed diverse functions beyond translation initiation, suggesting that fungal nip1 may similarly have evolved specialized roles .

How might findings about nip1/eIF3c mechanisms inform broader understanding of translation regulation in eukaryotic pathogens?

Research on nip1/eIF3c mechanisms can advance understanding of translation regulation in several ways:

• Pathogen-specific translation control: Identify unique features of translation initiation in fungal pathogens that could be exploited for targeted interventions.

• Stress response mechanisms: Elucidate how translation is regulated during pathogen stress responses, including host defense encounters.

• Regulatory networks: Map the integration of translation control with other cellular processes in pathogens, such as autophagy and ROS production .

• Non-canonical functions: Discover pathogen-specific non-canonical functions of translation factors that may contribute to virulence.

• Evolutionary adaptations: Understand how translation machinery has adapted in different pathogens to optimize survival and host colonization.

Current research suggests that translation factors like eIF3c play roles beyond protein synthesis, including involvement in autophagy regulation and cellular differentiation pathways , which may have particular significance in pathogen biology.