Recombinant Ashbya gossypii Protein SEY1 (SEY1)

What is Ashbya gossypii Protein SEY1 and what are its key characteristics?

Ashbya gossypii Protein SEY1 (UniProt ID: Q74ZD5) is a transmembrane protein encoded by the SEY1 gene (locus AGR264C) in the filamentous fungus Ashbya gossypii. The protein consists of 791 amino acids with enzyme classification EC 3.6.5.-, suggesting it functions as a hydrolase acting on acid anhydrides. The full-length protein has characteristic domains including a GTPase domain and transmembrane regions that are critical for its function in membrane dynamics .

The protein sequence begins with MSEDGASKCQDSIQLIDEQKQFNEKTLEY and contains multiple functional domains important for its biological activity. SEY1 is predominantly expressed in the endoplasmic reticulum membrane where it plays roles in membrane fusion processes .

How is Ashbya gossypii used in scientific research and biotechnology?

Ashbya gossypii is a filamentous Saccharomycete fungus with significant biotechnological applications. It is primarily used for:

• Industrial production of riboflavin (vitamin B2)

• Production of other high-value compounds including folic acid, nucleosides, and biolipids

• Host system for recombinant protein production

• Model organism for studying filamentous growth and protein secretion

The fungus has gained attention as a biotechnology platform due to its efficient secretory pathway and the extensive molecular toolbox available for its genetic manipulation. A. gossypii has a relatively small genome size compared to other filamentous fungi, making it amenable to genetic engineering approaches .

What expression systems are typically used for producing recombinant SEY1 protein?

Recombinant Ashbya gossypii Protein SEY1 can be produced using several expression systems, each with distinct advantages depending on research requirements:

For SEY1 specifically, yeast-based expression systems often provide better results due to the protein's transmembrane nature and need for proper folding and post-translational modifications that are similar to its native environment .

What are the optimal storage conditions for recombinant Ashbya gossypii Protein SEY1 to maintain its stability and activity?

Recombinant Ashbya gossypii Protein SEY1 requires specific storage conditions to maintain its stability and functional activity:

The optimal storage buffer typically consists of Tris-based buffer with 50% glycerol, specifically optimized for this protein. For short-term storage (up to one week), store working aliquots at 4°C. For extended storage, conserve at -20°C or -80°C .

Important considerations for maintaining protein stability include:

• Avoiding repeated freeze-thaw cycles, as this can significantly degrade protein quality and activity

• Dividing the protein into small single-use aliquots prior to freezing

• Using appropriate stabilizing additives in the buffer based on specific experimental requirements

• Monitoring protein degradation through analytical methods such as SDS-PAGE before critical experiments

When handling the protein for experiments, it's crucial to maintain cold chain practices and to use fresh aliquots for sensitive applications requiring optimal protein activity.

What experimental approaches are recommended for studying SEY1 protein-protein interactions?

For investigating SEY1 protein interactions, several complementary methodologies are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use anti-SEY1 antibodies to pull down the protein complex

  • Identify interacting partners through mass spectrometry

  • For membrane proteins like SEY1, use mild detergents (0.5-1% NP-40 or Triton X-100) to solubilize membrane components without disrupting protein-protein interactions

• Yeast Two-Hybrid Screening:

  • Particularly useful for identifying novel interacting partners

  • Adapt specifically for membrane proteins by using split-ubiquitin or membrane yeast two-hybrid systems

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse SEY1 to a biotin ligase

  • Proteins in close proximity become biotinylated and can be isolated using streptavidin

  • Particularly valuable for identifying transient or weak interactions in the native cellular context

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag SEY1 and potential interacting partners with appropriate fluorophores

  • Measure energy transfer as indication of protein-protein proximity

  • Useful for validating interactions in living cells

When designing these experiments, it's critical to consider the transmembrane nature of SEY1 and appropriate controls to distinguish specific from non-specific interactions .

What purification strategies yield the highest purity and activity for recombinant SEY1 protein?

Purification of recombinant SEY1 protein requires specialized strategies due to its transmembrane nature:

Recommended Purification Protocol:

• Cell Lysis and Membrane Fraction Isolation:

  • Use gentle lysis buffers containing protease inhibitors

  • Isolate membrane fractions through differential centrifugation

  • Solubilize membranes using appropriate detergents (n-dodecyl-β-D-maltoside or CHAPS at 1-2%)

• Affinity Chromatography:

  • Utilize appropriate affinity tags (His, GST, or FLAG) for initial capture

  • For His-tagged SEY1: Use Ni-NTA resin with imidazole gradient elution

  • Include detergent in all buffers at concentrations above critical micelle concentration

• Size Exclusion Chromatography:

  • Apply to Superdex 200 column for further purification

  • Assess protein homogeneity and remove aggregates

  • Buffer composition: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% appropriate detergent

• Quality Control:

  • Verify purity by SDS-PAGE (>95%)

  • Confirm identity through Western blotting and mass spectrometry

  • Assess activity through GTPase activity assays with appropriate substrates

This protocol typically yields 1-5 mg of purified protein per liter of culture with >90% purity while maintaining GTPase activity .

How does the unfolded protein response (UPR) in A. gossypii differ from other yeast and fungi when expressing recombinant proteins?

A. gossypii exhibits an unconventional response to protein secretion stress that distinguishes it from other fungi and yeasts:

Unlike Saccharomyces cerevisiae and most filamentous fungi, A. gossypii does not activate a conventional unfolded protein response (UPR) under secretion stress conditions. When exposed to dithiothreitol (DTT) or during recombinant protein expression, well-known UPR target genes (IRE1, KAR2, HAC1, and PDI1 homologs) show no significant upregulation .

Instead, A. gossypii employs alternative mechanisms to cope with secretion stress:

This unique stress response in A. gossypii suggests the evolution of alternative protein quality control mechanisms that may provide advantages for certain types of recombinant protein production. Understanding these mechanisms offers opportunities for engineering improved heterologous protein expression systems .

What are the advantages and limitations of using CRISPR/Cas9 genome editing for modifying SEY1 in A. gossypii?

The CRISPR/Cas9 system for A. gossypii provides powerful capabilities for SEY1 modification but comes with specific considerations:

Advantages:

• Marker-free modifications: Enables precise editing without permanent marker integration, preserving natural gene regulation

• One-vector strategy: All required components (Cas9, sgRNA, dDNA) are delivered in a single vector, increasing efficiency in the multinucleated syncytium of A. gossypii

• High editing efficiency: Approximately 60% success rate in genome editing

• Multiplexing capability: Allows simultaneous editing of multiple genomic targets

• Versatility: Supports various modifications including deletions, insertions, and nucleotide substitutions

Limitations and Considerations:

• PAM sequence requirement: The system requires a 5'-NGG-3' protospacer adjacent motif (PAM) near the target site

• Multinucleated nature challenges: A. gossypii is multinucleated, requiring sporulation steps to obtain homokaryotic clones

• Off-target effects: Potential for unintended modifications at similar sequences

• Plasmid stability: Episomic plasmids are not fully stable in A. gossypii, requiring careful clone selection

Methodological Protocol for SEY1 Modification:

• Design sgRNA targeting the SEY1 gene (AGR264C)

• Design dDNA repair templates containing desired modifications

• Assemble CRISPR/Cas9 vector with specific sgRNA-dDNA using directional cloning

• Transform A. gossypii with the constructed vector

• Select transformants on G418-containing medium

• Induce sporulation of heterokaryotic transformants

• Isolate and verify homokaryotic clones containing the desired modification

This approach has been successfully applied to modify various genes in A. gossypii and represents a significant advancement for genetic engineering of this industrial fungus .

How does the secretome composition of A. gossypii impact recombinant SEY1 production and purification strategies?

The unique characteristics of the A. gossypii secretome have important implications for recombinant SEY1 production and downstream processing:

A. gossypii secretes a relatively limited number of native proteins (1-4% of its proteome) compared to other filamentous fungi. The secretome composition is characterized by proteins primarily having isoelectric points between 4-6 and molecular weights above 25 kDa. Less than 33% of the secreted proteins are hydrolases, which is lower than typically observed in filamentous fungi .

Impact on Recombinant SEY1 Production:

• Co-purification challenges: When designing purification strategies for recombinant SEY1, researchers must account for the specific native proteins secreted by A. gossypii that may co-purify:

  • Approximately 18 protein spots are consistently detected at high abundance in culture supernatants

  • Both minimal and rich media yield similar core secretome proteins, though rich media shows slightly more protein diversity (~182 vs ~157 spots)

• Purification strategy considerations:

  • Ion exchange chromatography can be particularly effective given the narrow pI range of most contaminants

  • Size exclusion methods should be optimized to separate SEY1 from the >25 kDa native proteins

  • Hydrophobic interaction chromatography may provide good selectivity due to the transmembrane nature of SEY1

• Expression strategy optimization:

  • The lack of conventional UPR in A. gossypii may be advantageous for producing proteins that are sensitive to UPR-induced modifications

  • Consider the downregulation of glycosylation pathways under stress when designing expression constructs and protocols

These characteristics make A. gossypii's secretome more similar to yeast than to other filamentous fungi, which must be considered when designing recombinant protein production processes .

What experimental approaches are most effective for determining the GTPase activity of recombinant SEY1 protein?

Several complementary methods can be employed to accurately measure the GTPase activity of recombinant SEY1 protein:

• Colorimetric Phosphate Release Assay:

  • Principle: Measures inorganic phosphate released during GTP hydrolysis

  • Protocol:

    • Incubate purified SEY1 (0.5-2 μM) with GTP (50-200 μM) in reaction buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂)

    • Stop reaction at various timepoints with malachite green reagent

    • Measure absorbance at 620-650 nm

    • Calculate activity using phosphate standard curve

  • Advantage: Simple, high-throughput compatible

  • Limitation: Indirect measurement, potential interference from buffer components

• HPLC-Based Nucleotide Analysis:

  • Principle: Direct quantification of GTP consumption and GDP production

  • Protocol:

    • Separate GTP from GDP using reverse-phase HPLC after the enzyme reaction

    • Monitor nucleotide levels at 254 nm

    • Calculate conversion rates based on peak areas

  • Advantage: Direct measurement, highly accurate

  • Limitation: Lower throughput, requires specialized equipment

• Coupled Enzyme Assay:

  • Principle: Links GTP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Protocol:

    • Monitor decrease in NADH absorbance at 340 nm in real-time

    • Calculate GTPase activity based on NADH consumption rate

  • Advantage: Continuous monitoring, high sensitivity

  • Limitation: Potential interference from coupling enzymes

For SEY1 specifically, the colorimetric phosphate release assay is often preferred for initial characterization, while HPLC methods provide more definitive measurements for detailed kinetic analyses. Include appropriate controls such as heat-inactivated SEY1 and no-enzyme controls .

What is known about the role of SEY1 in membrane dynamics and how can researchers study these functions?

SEY1 plays important roles in membrane dynamics, particularly in the endoplasmic reticulum (ER) network:

Key Functions of SEY1:

• Mediates homotypic fusion of ER membranes

• Contributes to ER morphology maintenance

• Involved in ER stress responses independent of conventional UPR

• Participates in membrane trafficking pathways

Methodological Approaches for Studying SEY1 Membrane Functions:

• Live Cell Imaging Techniques:

  • Fluorescent protein tagging of SEY1 (C-terminal tags preferred to preserve function)

  • Time-lapse confocal microscopy to visualize ER dynamics

  • Photobleaching recovery assays (FRAP) to measure membrane continuity and protein mobility

  • Protocol considerations: Use markers for ER (Sec63-GFP) alongside SEY1 fusions

• Electron Microscopy Analysis:

  • Transmission electron microscopy of SEY1 knockout vs. wild-type cells

  • Immunogold labeling to localize SEY1 at membrane fusion sites

  • Critical parameters: Proper fixation techniques to preserve membrane structures

• In vitro Membrane Fusion Assays:

  • Reconstitution of SEY1 into liposomes

  • Monitoring fusion using fluorescent lipid mixing assays

  • Requirements: Purified SEY1 in detergent micelles, synthetic liposomes with appropriate lipid composition

  • Key controls: GTPase-deficient SEY1 mutants

• Genetic Interaction Studies:

  • CRISPR/Cas9-mediated SEY1 deletion or mutation

  • Analyze genetic interactions with other membrane-shaping proteins

  • Phenotypic analysis focusing on ER morphology and stress responses

  • Notable: A. gossypii SEY1 knockouts may reveal unique phenotypes different from S. cerevisiae due to the different UPR mechanisms

These approaches can be combined to comprehensively characterize SEY1's roles in membrane dynamics, with special attention to A. gossypii's unique stress response mechanisms that differ from conventional UPR pathways .

How can researchers effectively analyze post-translational modifications of SEY1 in A. gossypii?

Analyzing post-translational modifications (PTMs) of SEY1 in A. gossypii requires specialized approaches to capture the unique characteristics of this fungal system:

Recommended Analytical Workflow:

• Mass Spectrometry-Based PTM Mapping:

  • Sample preparation:

    • Immunoprecipitate SEY1 using specific antibodies or epitope tags

    • Perform in-gel or in-solution digestion with multiple proteases (trypsin, chymotrypsin)

    • Enrich for specific PTMs using appropriate techniques

  • Analysis methods:

    • LC-MS/MS with higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD)

    • Data analysis using search engines capable of identifying multiple PTMs

  • Expected modifications: Phosphorylation, glycosylation, lipidation

• N-Glycosylation Analysis:

  • A. gossypii produces distinct N-glycan structures that differ from S. cerevisiae

  • Methods:

    • PNGase F or Endo H treatment followed by SDS-PAGE mobility shift analysis

    • Glycopeptide enrichment using hydrophilic interaction chromatography

    • Glycan release and profiling by MALDI-TOF or LC-MS

  • Expected findings: Potentially unique N-glycan structures recently characterized in A. gossypii

• Site-Directed Mutagenesis for Functional Verification:

  • Identify putative modification sites through bioinformatics and MS analysis

  • Generate site-specific mutants using CRISPR/Cas9 genome editing

  • Assess functional consequences through:

    • Growth phenotyping

    • Membrane dynamics assays

    • Protein-protein interaction studies

    • GTPase activity measurements

• In vivo Labeling Approaches:

  • Metabolic labeling with isotope-coded or clickable precursors

  • Pulse-chase experiments to track PTM dynamics

  • Visualization techniques:

    • Phospho-specific antibodies if available

    • Glycan-specific lectins for detecting glycosylation

When analyzing SEY1 PTMs, researchers should be aware that A. gossypii has a unique N-glycosylation profile and protein quality control mechanisms that may result in modification patterns distinct from model yeasts like S. cerevisiae .

What are common challenges in expressing recombinant SEY1 in heterologous systems and how can they be addressed?

Expressing recombinant SEY1 presents several challenges due to its transmembrane nature and functional complexity:

When expressing SEY1 specifically in A. gossypii, researchers should note that the conventional UPR is not activated under secretion stress. Instead, alternative stress response pathways are engaged, including upregulation of genes involved in protein unfolding, ERAD, proteolysis, and vesicle trafficking, while glycosylation pathway components are downregulated .

How can researchers accurately compare SEY1 function across different fungal species to understand evolutionary conservation?

To conduct rigorous comparative analyses of SEY1 function across fungal species:

Methodological Framework:

• Sequence and Structural Comparison:

  • Perform multiple sequence alignment of SEY1 homologs across fungal species

  • Identify conserved domains, particularly GTPase domains and transmembrane regions

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Use homology modeling to predict structural conservation

  • Tools: MUSCLE/CLUSTAL for alignment, RAxML for phylogeny, SWISS-MODEL for structure prediction

• Complementation Studies:

  • Delete endogenous SEY1 in various fungal species

  • Express SEY1 orthologs from different species in these deletion backgrounds

  • Assess rescue of phenotypes through:

    • ER morphology analysis using fluorescence microscopy

    • Growth under ER stress conditions

    • Membrane fusion assays

  • Critical control: Expression level normalization across complementation strains

• Domain Swap Experiments:

  • Generate chimeric proteins with domains from different species' SEY1 orthologs

  • Express in SEY1-null backgrounds

  • Map functional domains through phenotypic rescue

  • Analysis: Quantitative assessment of ER network parameters

• Comparative Transcriptomics:

  • Analyze transcriptional responses to SEY1 deletion across species

  • Compare stress response pathways, noting A. gossypii's unique non-UPR response

  • Methods: RNA-seq followed by differential expression analysis

  • Data integration: Cross-species pathway comparison using orthology mapping

• Biochemical Activity Comparison:

  • Purify recombinant SEY1 from multiple species under identical conditions

  • Compare GTPase activity, membrane binding, and oligomerization properties

  • Standardize assay conditions to enable direct comparison

  • Controls: Ensure protein quality and concentration normalization

When conducting these comparisons, researchers should particularly note the unique secretion stress response of A. gossypii compared to other fungi, as it does not activate the conventional UPR pathway that is conserved in most eukaryotes .

What are the critical factors affecting reproducibility in studies involving recombinant A. gossypii SEY1 protein?

Ensuring reproducibility in recombinant SEY1 research requires careful attention to several critical factors:

1. Expression System Considerations:

• Consistent use of host strain and expression vectors

• Standardized induction protocols with defined parameters:

  • Induction timing (cell density at induction)

  • Inducer concentration

  • Post-induction temperature and duration

• Documentation of expression construct design including:

  • Codon optimization strategy

  • Tag position and type

  • Promoter and terminator sequences

2. Purification Variables:

• Detergent selection and concentration critical for membrane protein stability

• Buffer composition standardization:

  • pH (typically 7.0-7.5 for SEY1)

  • Ionic strength (150-300 mM NaCl)

  • Presence of stabilizing additives (glycerol, specific lipids)

• Consistent protein concentration methods

• Standardized storage conditions and freeze-thaw cycles

3. Protein Quality Assessment:

• Routine verification of:

  • Purity (>95% by SDS-PAGE)

  • Identity (mass spectrometry confirmation)

  • Integrity (absence of degradation)

  • Oligomeric state (size exclusion chromatography)

  • Activity (standardized GTPase assays)

• Batch-to-batch variation tracking

4. A. gossypii Culture Conditions:

• Growth phase standardization (exponential vs. stationary)

• Media composition consistency

• Temperature and aeration parameters

• Spore preparation and inoculation procedures

5. Data Reporting Standards:

• Complete methods documentation including:

  • Detailed protocols with all parameters

  • Source and catalog numbers for critical reagents

  • Equipment specifications and settings

  • Data processing methods and software versions

• Raw data availability

• Statistical analysis approach

A particularly critical factor for A. gossypii SEY1 studies is recognizing that the organism lacks conventional UPR activation under secretion stress. This unique characteristic means that standard UPR markers used in other systems may not be appropriate controls, and alternative stress response indicators should be monitored .

What are the most promising research directions for understanding SEY1 function in A. gossypii's unique protein quality control pathways?

Given A. gossypii's unconventional response to secretion stress, several research directions hold particular promise:

• Characterization of Alternative Stress Response Pathways:

  • Comprehensive transcriptomic and proteomic profiling of A. gossypii under various stress conditions

  • Identification of novel stress response regulators that operate independently of canonical UPR

  • Comparative analysis with conventional UPR systems in other fungi

  • Research question: What molecular mechanisms compensate for the absence of conventional UPR activation?

• SEY1's Role in ER Morphology and Stress Adaptation:

  • High-resolution imaging of ER dynamics in wild-type vs. SEY1-deleted strains

  • Analysis of SEY1 interactions with other membrane-shaping proteins under stress

  • Investigation of SEY1 post-translational modifications during stress response

  • Key experiment: Time-course analysis of SEY1 localization, modification, and interactome during DTT-induced stress

• Engineering Improved Recombinant Protein Production:

  • Exploitation of A. gossypii's unique stress response for difficult-to-express proteins

  • Development of SEY1-based genetic modules to enhance secretory capacity

  • Testing whether SEY1 overexpression/modification can improve heterologous protein yields

  • Applied goal: Creation of A. gossypii strains with enhanced secretory capabilities for biotechnology applications

• Structure-Function Analysis of SEY1:

  • Cryo-EM determination of SEY1 structure in membrane environment

  • Mapping of functional domains through targeted mutagenesis

  • Identification of regulatory interaction partners

  • Molecular dynamics simulations of membrane interactions

  • Technical innovation: Development of nanodiscs or SMALPs containing SEY1 for structural studies

• Evolution of Stress Response Mechanisms:

  • Comparative genomics and experimental validation across related fungi

  • Reconstruction of the evolutionary trajectory leading to A. gossypii's unique stress response

  • Identification of genomic changes that enabled alternative stress adaptation

  • Theoretical question: Does the absence of conventional UPR represent a derived or ancestral trait?

These research directions take advantage of A. gossypii's unique biology while addressing fundamental questions about membrane dynamics, protein quality control, and biotechnological applications .

How might the study of A. gossypii SEY1 contribute to improving heterologous protein production systems?

The unique characteristics of A. gossypii SEY1 and its cellular context offer several opportunities for enhancing heterologous protein production:

• Engineering Alternative Quality Control Systems:

  • A. gossypii's unconventional secretion stress response (non-UPR) provides a novel framework for protein production

  • Potential approach: Transfer components of A. gossypii's alternative stress response pathway to conventional host systems

  • Expected benefit: Reduced ER-associated degradation of complex proteins through alternative folding pathways

  • Implementation strategy: Identify and overexpress key regulators from A. gossypii's stress response in production hosts

• SEY1-Based ER Engineering:

  • SEY1's role in ER membrane dynamics can be exploited to redesign secretory organelles

  • Concept: Modulate SEY1 expression or activity to expand ER capacity

  • Experimental approach: Create SEY1 variants with enhanced membrane fusion activity

  • Predicted outcome: Increased ER volume and secretory capacity in production hosts

• Hybrid Secretory Pathway Construction:

  • Combine elements of A. gossypii's secretory pathway with those from high-secreting organisms

  • Focus areas:

    • SEY1-mediated membrane dynamics

    • A. gossypii's alternative stress response elements

    • Glycosylation pathway components

  • Testing platform: Stepwise reconstruction of secretory pathways in model organisms

• Stress-Resistant Production Strains:

  • Leverage A. gossypii's ability to maintain productivity under stress conditions

  • Strategy: Identify key genes upregulated during DTT stress that enable continued protein secretion

  • Application: Create production strains with enhanced resistance to secretion stress

  • Advantage: More robust production processes with higher protein yields under suboptimal conditions

• Specialized Expression Systems for Difficult Proteins:

  • Develop A. gossypii as a host for proteins that fail in conventional systems

  • Target proteins: Those sensitive to conventional UPR-induced modifications

  • Approach: Engineer A. gossypii strains with enhanced SEY1 function and optimized secretory capacity

  • Validation: Comparative expression trials of problematic proteins in conventional hosts versus modified A. gossypii

These approaches could lead to significant improvements in production systems for biopharmaceuticals, industrial enzymes, and other valuable proteins that are challenging to produce in current systems .

What emerging technologies might accelerate research on SEY1 and other A. gossypii membrane proteins?

Several cutting-edge technologies show particular promise for advancing research on SEY1 and other A. gossypii membrane proteins:

• Advanced Structural Biology Approaches:

  • Cryo-electron tomography: Visualize SEY1 in its native membrane environment without protein extraction

  • Microcrystal electron diffraction (MicroED): Determine structure from nano-sized crystals of membrane proteins

  • Single-particle cryo-EM with improved detectors: Achieve higher resolution of membrane protein complexes

  • Integrative structural biology: Combine multiple techniques (NMR, SAXS, XL-MS) for comprehensive structural understanding

• Genome Engineering Technologies:

  • CRISPR base editors and prime editors: Create precise modifications in SEY1 without double-strand breaks

  • CRISPR activation/interference (CRISPRa/CRISPRi): Modulate SEY1 expression without genetic modification

  • Multiplexed genome engineering: Simultaneously modify SEY1 and interacting partners

  • In vivo DNA assembly: Create libraries of SEY1 variants directly in A. gossypii

• Advanced Imaging Techniques:

  • Super-resolution microscopy: Visualize SEY1 dynamics below the diffraction limit

  • Correlative light and electron microscopy (CLEM): Connect SEY1 function to ultrastructural changes

  • Lattice light-sheet microscopy: Capture SEY1 dynamics with minimal phototoxicity

  • Expansion microscopy: Physically enlarge specimens for improved resolution of membrane structures

• Single-Cell and Spatial Technologies:

  • Single-cell proteomics: Analyze SEY1 expression variation across individual A. gossypii cells

  • Spatial transcriptomics: Map gene expression changes around SEY1-containing regions

  • Mass spectrometry imaging: Visualize lipid distributions around SEY1-enriched membranes

  • Protein correlation profiling: Map SEY1 to specific membrane subdomains

• Membrane Mimetic Systems:

  • Native nanodiscs: Extract SEY1 in native lipid environment

  • Styrene-maleic acid lipid particles (SMALPs): Preserve native membrane context during purification

  • Cell-free membrane protein expression systems: Directly integrate SEY1 into artificial membranes

  • Droplet interface bilayers: Reconstitute SEY1 function in defined membrane systems

• Artificial Intelligence Applications:

  • AlphaFold and RoseTTAFold: Predict SEY1 structure with high accuracy

  • Machine learning for image analysis: Automatically quantify SEY1-dependent membrane phenotypes

  • Neural networks for PTM prediction: Identify likely modification sites on SEY1

  • AI-assisted experimental design: Optimize conditions for SEY1 expression and purification