Recombinant Neurospora crassa WD repeat-containing protein jip-5 (jip-5)

What is the jip-5 protein and why is it significant for research?

The jip-5 protein is a WD repeat-containing protein found in Neurospora crassa, a filamentous fungus widely used as a model organism in genetics research. WD repeat proteins are characterized by their distinctive structural motifs that form β-propeller structures and commonly function in protein-protein interactions, signal transduction, and cellular regulation mechanisms . The significance of jip-5 lies in its potential role in fundamental cellular processes, making it valuable for studying protein-protein interactions in eukaryotic systems.

Neurospora crassa has been established as a key model organism since the 1930s, with groundbreaking studies by scientists like Beadle and Tatum establishing the "one gene-one protein" principle . The organism offers advantages including rapid growth, non-toxicity, and ease of genetic manipulation, with a fully sequenced genome containing approximately 10,000 predicted protein-coding genes .

What expression systems are typically used for producing recombinant jip-5?

Recombinant jip-5 protein can be produced using several expression systems, with Escherichia coli being the most commonly employed for WD repeat proteins. Based on established protocols for similar proteins, the methodology typically involves:

• E. coli expression system: Using BL21(DE3) or similar strains with expression vectors containing T7 promoters (pET series) for high-level expression .

• Neurospora crassa homologous expression: Can be used when post-translational modifications are critical, using the fungus's own machinery to express the native protein with proper folding and modifications .

• Other eukaryotic systems: Including yeast, baculovirus-infected insect cells, and mammalian cells for more complex proteins requiring specific modifications .

For the expression of jip-5 in E. coli, the typical protocol involves:

• Cloning the jip-5 coding sequence into an expression vector with an N-terminal His-tag

• Transforming E. coli BL21(DE3) cells

• Inducing expression with IPTG (typically 0.5-1.0 mM)

• Harvesting cells and purifying using nickel affinity chromatography

• Further purification via ion exchange and/or size exclusion chromatography

What are the optimal conditions for expressing and purifying functional jip-5 protein?

The optimal conditions for expressing and purifying functional jip-5 protein require careful consideration of several parameters to maintain protein stability and activity:

Expression Optimization:

• Temperature: Lower induction temperatures (16-20°C) are often preferred for WD repeat proteins to enhance proper folding

• IPTG concentration: 0.1-0.5 mM typically provides better soluble protein yield than higher concentrations

• Expression time: Extended expression (16-24 hours) at lower temperatures often yields better results than shorter periods at higher temperatures

• Media composition: Enriched media like Terrific Broth supplemented with glucose (0.5-1%) can improve yield

Purification Conditions:

• Buffer composition:

  • 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 300-500 mM NaCl to maintain solubility

  • 5-10% glycerol as a stabilizing agent

  • 1-5 mM β-mercaptoethanol or DTT to prevent oxidation

• Protease inhibitors: PMSF (1 mM) and a commercial protease inhibitor cocktail

• Purification strategy: Multi-step approach involving:

  • Immobilized metal affinity chromatography (IMAC)

  • Ion exchange chromatography

  • Size exclusion chromatography for highest purity

Based on studies of Neurospora crassa protein expression systems, protease activity has been identified as a major limitation . Therefore, incorporating protease inhibitors and using a strain with reduced protease activity can significantly improve yield. The use of a fourfold protease deletion strain has shown success with other recombinant proteins from Neurospora crassa .

How can CRISPR/Cas9 technology be applied to study jip-5 function in Neurospora crassa?

CRISPR/Cas9 technology provides an efficient approach for studying jip-5 function through targeted mutagenesis:

Method for CRISPR/Cas9-mediated editing of jip-5 in N. crassa:

• System preparation:

  • Incorporate the cas9 gene into the N. crassa genome under the control of an optimized promoter (Pccg1nr is recommended based on heterologous protein expression studies)

  • Design guide RNAs (gRNAs) specific to jip-5 targeting sequences

• Delivery method:

  • Use electroporation to introduce naked gRNA directly into N. crassa cells containing genomic cas9

  • This approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process

• Selection strategy:

  • Co-target a selectable marker gene (e.g., cyclosporin-resistant-1, csr-1) alongside jip-5

  • This has been shown to increase editing efficiency from ~10% to nearly 100% under selection conditions

• Verification of mutations:

  • PCR amplification of the target region followed by sequencing

  • Protein expression analysis using Western blotting

  • Phenotypic characterization of mutants

• Phenotypic analysis:

  • Growth rate measurement under various conditions

  • Microscopy to examine hyphal morphology and polarized growth

  • Protein-protein interaction studies to identify binding partners

  • Transcriptomic analysis to determine effects on gene expression networks

Recent studies have demonstrated that this approach yields editing efficiencies of 7.35-11.89% without selection, comparable to traditional homologous recombination methods . When combined with selection markers, the efficiency increases significantly, making it a powerful tool for studying genes like jip-5.

What protein-protein interactions has jip-5 been shown to participate in, and how might these be studied?

While specific protein-protein interactions for jip-5 are not detailed in the search results, its identity as a WD repeat protein suggests it likely functions as a scaffold for protein complex assembly. Methodologies to study these interactions include:

Techniques for studying jip-5 protein-protein interactions:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged jip-5 in Neurospora crassa

  • Purify protein complexes using affinity chromatography

  • Identify interacting partners via mass spectrometry

  • Quantify relative abundance using spectral counting or SILAC approaches

• Yeast two-hybrid (Y2H) screening:

  • Create a fusion of jip-5 with a DNA-binding domain

  • Screen against a Neurospora cDNA library fused to an activation domain

  • Identify positive interactions through reporter gene activation

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against jip-5 or use tagged versions

  • Precipitate protein complexes from Neurospora cell extracts

  • Identify co-precipitated proteins by Western blotting or mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion proteins of jip-5 and potential partners with split fluorescent protein fragments

  • Express in Neurospora crassa

  • Visualize interactions through restored fluorescence at sites of protein-protein interaction

• Protein crosslinking coupled with mass spectrometry:

  • Use chemical crosslinkers to stabilize transient interactions

  • Digest crosslinked complexes and analyze by MS

  • Identify interaction sites and partners

Based on studies of other WD repeat proteins, jip-5 might participate in complexes involved in signal transduction, cytoskeletal organization, or chromatin remodeling. The exocyst complex in Neurospora crassa, which is crucial for polarized growth and vesicle tethering to the plasma membrane, involves protein-protein interactions that could potentially include WD repeat proteins like jip-5 .

What are the best approaches for designing jip-5 truncation constructs to study domain-specific functions?

Designing effective truncation constructs for jip-5 requires systematic analysis of its domain architecture:

Strategy for jip-5 truncation design:

• Domain prediction and structure analysis:

  • Use bioinformatics tools (SMART, Pfam, InterPro) to identify WD repeat boundaries

  • Perform secondary structure prediction to avoid disrupting structural elements

  • Align with structurally characterized WD repeat proteins like human WDR5 to guide truncation boundaries

• Construct design considerations:

  • Create constructs containing individual WD repeats, pairs of repeats, and the complete β-propeller

  • Include 3-5 amino acid buffer regions at domain boundaries to preserve folding

  • Maintain the "Velcro" closure between first and last repeats if truncating the full propeller

  • Include N-terminal and/or C-terminal tags positioned to minimize interference with folding

• Expression vector selection:

  • Use vectors with inducible promoters (T7, trc) for E. coli expression

  • Include solubility-enhancing fusion partners (MBP, SUMO, TrxA) with cleavable linkers

  • Incorporate TEV or similar protease sites for tag removal

• Validation methods:

  • Circular dichroism to confirm secondary structure formation

  • Size exclusion chromatography to assess folding status

  • Thermal shift assays to evaluate stability of constructs

  • Limited proteolysis to identify stable domains

Example truncation strategy for a typical WD repeat protein:

This approach allows for systematic identification of functional regions within the jip-5 protein and their contribution to protein-protein interactions and cellular functions.

How can RNA-seq be used to study the transcriptional effects of jip-5 knockout or overexpression?

RNA-seq provides a powerful approach to understand the transcriptional network affected by jip-5 manipulation:

RNA-seq workflow for studying jip-5 function:

• Experimental design:

  • Generate jip-5 knockout mutants using CRISPR/Cas9 system and jip-5 overexpression strains

  • Include appropriate wild-type controls and biological replicates (minimum 3)

  • Consider time-course experiments to capture early and late responses

  • Include different growth conditions to identify condition-specific effects

• Sample preparation:

  • Extract high-quality total RNA (RIN > 8) from N. crassa mycelia

  • Enrich for mRNA using poly(A) selection or rRNA depletion

  • Prepare strand-specific libraries to capture antisense transcription

  • Include spike-in controls for quantification accuracy

• Sequencing parameters:

  • Paired-end sequencing (2 × 150 bp) for improved mapping

  • Minimum 20-30 million reads per sample for adequate coverage

  • Sequence depth needs to be adjusted based on expected expression levels

• Data analysis pipeline:

  • Quality control and adapter trimming (FastQC, Trimmomatic)

  • Alignment to N. crassa genome (HISAT2, STAR)

  • Transcript quantification (featureCounts, HTSeq)

  • Differential expression analysis (DESeq2, edgeR)

  • Functional enrichment analysis (GO, KEGG pathway)

  • Co-expression network analysis

Based on RNA-seq approaches used in N. crassa studies, it's important to consider tissue-specific and time-dependent responses. In a study examining N. crassa response to bacterial inoculation, approximately 17% of the genome (1,863 genes) showed differential expression across two time points, with distinct sets of genes up- or down-regulated at each time point .

Example findings from N. crassa transcriptomics:

Principal component analysis has been shown to effectively separate transcriptional variance based on experimental conditions, with the first two principal components accounting for substantial variance (33% and 15% in one study) . This approach would be valuable for distinguishing the effects of jip-5 manipulation from other variables.

What methods can be used to assess the localization and trafficking of jip-5 in Neurospora crassa?

Understanding the subcellular localization and trafficking of jip-5 requires specialized microscopy and biochemical approaches:

Methods for jip-5 localization studies:

• Fluorescent protein tagging:

  • Generate C- or N-terminal GFP/mCherry fusions of jip-5

  • Express under native promoter to maintain physiological expression levels

  • Validate functionality of tagged proteins through complementation tests

  • Use CRISPR/Cas9 to tag the endogenous locus for most accurate localization

• Confocal microscopy approaches:

  • Live-cell imaging to track protein movement

  • Co-localization with organelle markers (ER, Golgi, nucleus, etc.)

  • Time-lapse imaging to capture dynamic localization during different growth stages

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

• Super-resolution microscopy:

  • Structured illumination microscopy (SIM) for improved resolution

  • Single-molecule localization microscopy for precise localization

  • Expansion microscopy to physically enlarge structures for better visualization

• Biochemical fractionation:

  • Separate cellular compartments (cytosol, nucleus, membranes, etc.)

  • Detect jip-5 in different fractions via Western blotting

  • Combine with protease protection assays to determine topology

• Immunoelectron microscopy:

  • Generate specific antibodies against jip-5 or use anti-tag antibodies

  • Perform immuno-gold labeling for high-resolution localization

  • This approach provides nanometer-scale resolution of protein localization

For studying dynamics in N. crassa specifically, the Spitzenkörper (apical vesicle cluster) is a key structure in hyphal growth. Research on the exocyst complex in N. crassa has revealed how proteins involved in polarized growth localize and function at the hyphal tip . Similar approaches could be applied to jip-5:

• Examine co-localization with markers of the Spitzenkörper

• Track movement along hyphae using time-lapse microscopy

• Correlate localization with growth dynamics

• Examine changes in localization in response to environmental signals

This multi-method approach allows for comprehensive characterization of jip-5 localization and trafficking patterns in the complex cellular architecture of Neurospora crassa.

How does jip-5 contribute to hyphal growth and development in Neurospora crassa?

As a WD repeat-containing protein in Neurospora crassa, jip-5 likely plays a role in fundamental cellular processes related to hyphal growth and development. While specific functions of jip-5 are not detailed in the search results, a methodological approach to studying its role includes:

Approaches to study jip-5 role in hyphal growth:

• Growth phenotype analysis of jip-5 mutants:

  • Measure linear growth rates on different media (standard growth rate for wild-type N. crassa is >5 mm/h at 37°C)

  • Analyze branching patterns and hyphal morphology

  • Examine aerial hyphae formation and conidiation

  • Test growth under various stress conditions (temperature, osmotic stress, cell wall stress)

• Cytoskeletal organization assessment:

  • Visualize actin and microtubule cytoskeleton using fluorescent markers

  • Examine the Spitzenkörper organization and dynamics

  • Analyze cell wall deposition patterns using cell wall stains (Calcofluor White, Congo Red)

  • Study septation patterns and Woronin body distribution

• Vesicle trafficking analysis:

  • Track secretory vesicle movement using fluorescent markers

  • Examine endocytosis using FM4-64 dye

  • Study protein secretion efficiency

  • Analyze cell wall protein incorporation using specific antibodies

• Interaction with polarity factors:

  • Examine genetic interactions with known polarity genes

  • Test physical interactions with components of the exocyst complex

  • Analyze localization in relation to polarity markers

The exocyst complex in N. crassa, which is essential for polarized growth, functions by tethering Spitzenkörper vesicles to the apical plasma membrane . If jip-5 interacts with this complex or similar machinery, disruption would likely affect hyphal extension and morphology.

What role might jip-5 play in protein-protein interactions and cellular signaling?

WD repeat proteins typically function as protein-protein interaction platforms in signaling networks. To investigate jip-5's specific role:

Methodological approaches to study jip-5's role in signaling:

• Interactome mapping:

  • Perform immunoprecipitation followed by mass spectrometry (IP-MS)

  • Use BioID or APEX proximity labeling to identify proteins in close proximity to jip-5

  • Conduct yeast two-hybrid screens with jip-5 as bait

  • Analyze changes in the interactome under different growth conditions or stresses

• Signaling pathway analysis:

  • Monitor phosphorylation cascades in wild-type vs. jip-5 mutants

  • Test responses to known signaling molecules (e.g., calcium, cAMP)

  • Examine activation of downstream transcription factors

  • Analyze cross-talk between different signaling pathways

• Functional complementation studies:

  • Express truncated versions of jip-5 in null mutants to identify critical domains

  • Test heterologous WD repeat proteins for functional complementation

  • Create chimeric proteins to map functional domains

• Response to environmental signals:

  • Analyze transcriptional changes in response to environmental cues

  • Test sensitivity to signaling inhibitors

  • Examine cellular responses to nutrient limitation, light, temperature changes

Neurospora crassa possesses sophisticated environmental sensing mechanisms, including two-component systems, G-protein coupled pathways, and MAP kinase cascades . If jip-5 functions in these pathways, disruption would affect the organism's ability to respond appropriately to environmental signals, which could be monitored through phenotypic and molecular analyses.

How does jip-5 compare functionally to homologous proteins in other fungal species?

Comparative analysis of jip-5 with homologs in other fungi provides evolutionary insights and functional context:

Methodological approach to comparative functional analysis:

• Sequence comparison and phylogenetic analysis:

  • Identify jip-5 homologs across fungal species using BLAST and HMM searches

  • Perform multiple sequence alignment to identify conserved residues

  • Construct phylogenetic trees to map evolutionary relationships

  • Identify species-specific variations that might relate to functional specialization

• Domain architecture comparison:

  • Analyze conservation of WD repeat number and spacing

  • Identify additional domains present in homologs

  • Map conservation onto structural models

  • Identify lineage-specific insertions or deletions

• Heterologous expression studies:

  • Express jip-5 homologs from other fungi in N. crassa jip-5 mutants

  • Test functional complementation

  • Identify species-specific functions through domain swapping

  • Compare localization patterns of homologs when expressed in N. crassa

• Cross-species interactome comparison:

  • Compare binding partners of jip-5 homologs across species

  • Identify conserved vs. species-specific interactions

  • Correlate interaction differences with functional divergence

• Comparison in model fungal systems:

  • Saccharomyces cerevisiae and Schizosaccharomyces pombe as tractable models

  • Aspergillus nidulans for comparison to another filamentous fungus

  • Candida albicans for insights into pathogenic fungal biology

  • Magnaporthe oryzae for comparison to a plant pathogen

Neurospora crassa possesses features found in higher eukaryotes but absent in both budding and fission yeast, including DNA methylation and H3K27 methylation . Comparative analysis would reveal whether jip-5 functions in conserved processes or in Neurospora-specific pathways.

How can jip-5 be utilized in heterologous protein expression systems?

WD repeat proteins like jip-5 can potentially enhance heterologous protein expression systems through several applications:

Applications of jip-5 in protein expression systems:

• As a fusion partner for difficult-to-express proteins:

  • WD repeat domains can enhance solubility of fusion partners

  • The β-propeller structure provides stability in various expression systems

  • Can be used with a cleavable linker for subsequent tag removal

• As a scaffold for multi-protein complex assembly:

  • Co-expression of jip-5 with interaction partners to facilitate complex formation

  • Engineering binding interfaces to create novel protein assemblies

  • Use as a platform to organize metabolic enzymes for improved pathway efficiency

• In N. crassa expression systems:

  • Neurospora crassa has been established as a host for heterologous protein production

  • Advantages include rapid growth, non-toxicity, and ability to secrete large amounts of protein

  • Optimization using genetic engineering has yielded promising results

N. crassa expression systems have been successfully optimized through genetic engineering and bioprocess development:

• The ccg1 promoter (Pccg1nr) has proven effective for controlling expression

• A fourfold protease deletion strain significantly improved protein yields

• Expression of fusion proteins with glucoamylase (GLA-1) enhanced secretion

• Scalable cultivation from 1L to 10L bioreactors has been demonstrated

Based on these findings, jip-5 could potentially be incorporated into optimized N. crassa expression systems as a fusion partner or co-expression target to enhance protein production or facilitate complex assembly.

What structural and functional insights can be gained from crystallization of jip-5?

Crystallization of jip-5 would provide valuable structural and functional insights:

Methodological approach to jip-5 crystallization and analysis:

• Protein preparation for crystallization:

  • Express jip-5 with removable affinity tags in E. coli or insect cells

  • Perform multi-step purification to achieve >95% purity

  • Conduct stability assays to identify optimal buffer conditions

  • Use limited proteolysis to identify stable domains if full-length protein resists crystallization

• Crystallization screening:

  • Employ sparse matrix and systematic screening approaches

  • Test various protein concentrations (5-20 mg/ml)

  • Explore additives and precipitants

  • Consider surface entropy reduction mutations to enhance crystallization

  • Test co-crystallization with binding partners or ligands

• Structure determination:

  • Collect X-ray diffraction data at synchrotron sources

  • Solve structure by molecular replacement using related WD repeat structures

  • Use heavy atom derivatives or selenomethionine labeling if molecular replacement fails

  • Refine structure to highest possible resolution

• Structural analysis:

  • Characterize the β-propeller architecture and blade organization

  • Map conservation onto the structure to identify functional surfaces

  • Identify potential binding pockets and interaction interfaces

  • Compare with structures of other WD repeat proteins like human WDR5

• Functional validation:

  • Design structure-guided mutations to test functional hypotheses

  • Perform binding studies with predicted interaction partners

  • Use surface plasmon resonance or isothermal titration calorimetry to measure binding affinities

  • Validate in vivo using mutant complementation

Structural analysis would reveal whether jip-5 contains the canonical seven-bladed β-propeller fold typical of many WD repeat proteins, or whether it has evolved specialized features. The structure would facilitate understanding of how jip-5 participates in protein-protein interactions and potentially guide the design of molecules to modulate its function.

How might jip-5 be involved in epigenetic regulation in Neurospora crassa?

Neurospora crassa has rich epigenetic mechanisms, and WD repeat proteins often participate in chromatin regulation:

Approaches to investigate jip-5's potential role in epigenetics:

• Chromatin association analysis:

  • Perform chromatin immunoprecipitation followed by sequencing (ChIP-seq)

  • Use tagged jip-5 to identify genomic binding sites

  • Compare binding patterns with known chromatin marks

  • Analyze co-localization with transcription start sites or gene bodies

• Histone modification analysis:

  • Compare histone modification patterns between wild-type and jip-5 mutants

  • Focus on H3K4 methylation and H4 acetylation, which are regulated by complexes containing WD repeat proteins

  • Use ChIP-seq or mass spectrometry to quantify changes

  • Correlate changes with gene expression alterations

• Protein complex identification:

  • Purify jip-5-containing complexes and identify components

  • Look for associations with known chromatin modifiers

  • Test direct interactions with histones and histone-modifying enzymes

  • Analyze complex composition under different growth conditions

• DNA methylation analysis:

  • Assess DNA methylation patterns in jip-5 mutants using bisulfite sequencing

  • Examine relationships to genome defense mechanisms like RIP (Repeat-Induced Point mutation)

  • Analyze methylation at repetitive elements and transposons

  • Test genetic interactions with known DNA methylation machinery

Neurospora crassa has served as a model system for epigenetics research, revealing mechanisms of DNA methylation and histone modification that would have been difficult to discover in other systems . If jip-5 functions similarly to other WD repeat proteins like WDR5, it might participate in histone methyltransferase complexes that regulate gene expression through H3K4 methylation or other modifications .