Recombinant Arabidopsis thaliana Oleosin 21.2 kDa (At5g40420)

What are the optimal storage conditions for recombinant Oleosin 21.2 kDa?

Recombinant Oleosin 21.2 kDa is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C . After reconstitution, it is recommended to:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) and aliquot for long-term storage

• Store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles

The shelf life of the lyophilized form is approximately 12 months at -20°C/-80°C, while the reconstituted liquid form typically maintains stability for 6 months at -20°C/-80°C .

How is recombinant Oleosin 21.2 kDa expressed and purified?

Recombinant Oleosin 21.2 kDa is typically expressed in E. coli expression systems using the following methodology:

• The full-length mature protein sequence (amino acids 2-199) is cloned into an expression vector with an N-terminal His tag

• The construct is transformed into an E. coli expression strain

• Protein expression is induced under optimized conditions

• The recombinant protein is purified using affinity chromatography based on the His tag

• The purified protein is subjected to quality control assessments including SDS-PAGE to ensure >90% purity

• The final product is typically lyophilized with a Tris/PBS-based buffer containing 6% trehalose at pH 8.0

This methodology yields functional protein suitable for various research applications including antibody production, protein-protein interaction studies, and structural analyses.

What antibodies are available for detecting Oleosin 21.2 kDa and what are their optimal working conditions?

Polyclonal antibodies against Oleosin 21.2 kDa (OLE2) are commercially available. Based on current research protocols, the following specifications and working conditions are recommended:

For Western blot applications, the following protocol has been validated:

• Separate proteins on 15% SDS-PAGE and transfer to PVDF membrane

• Block with 5% skim milk/TBS-T for 1h at room temperature

• Incubate with primary antibody at 1:2,000 dilution in TBS-T for 1h at room temperature

• Wash 4 times for 10 min in TBS-T at room temperature

• Incubate with anti-rabbit IgG HRP-conjugated secondary antibody at 1:10,000 dilution for 1h at room temperature

• Wash and develop with chemiluminescent detection reagent

The antibody has confirmed reactivity with Arabidopsis thaliana and predicted reactivity with Camelina sativa, Capsella rubella, Eutrema salsugineum, and Raphanus sativus, but is not reactive with Glycine max .

How can researchers effectively extract and analyze oil bodies containing Oleosin 21.2 kDa?

To extract and analyze oil bodies containing Oleosin 21.2 kDa, researchers can follow this methodological approach:

• Seed preparation:

  • Collect Arabidopsis seeds at the appropriate developmental stage

  • Homogenize in extraction buffer (typically containing sucrose and protease inhibitors)

• Oil body isolation:

  • Centrifuge the homogenate at low speed (10,000 × g) for 10 minutes to remove cell debris

  • Float the oil bodies by centrifugation at high speed (100,000 × g) for 1 hour

  • Collect the floating oil body fraction

• Protein extraction:

  • Extract proteins from isolated oil bodies using 2× SDS-sample buffer with 2-mercaptoethanol

  • Denature at 95°C for 5 minutes

• Analysis methods:

  • SDS-PAGE: Use 15% gels for optimal separation; Oleosin 21.2 kDa typically appears at 19-21 kDa

  • Western blot: Follow antibody protocols described in section 2.1

  • Microscopy: Label oil bodies with lipophilic dyes (e.g., Nile Red) for size and morphology analysis

  • Mass spectrometry: For detailed protein characterization and post-translational modification analysis

For studies involving mutants, protocols have been established using the ole1234 quadruple mutant (lacking Ole1, Ole2, Ole3, and Ole4), which displays larger and fewer lipid droplets compared to wild-type plants, as visualized by Nile Red staining .

What experimental approaches can be used to study the function of Oleosin 21.2 kDa in Arabidopsis?

Multiple experimental approaches have been validated for investigating Oleosin 21.2 kDa function in Arabidopsis:

• Genetic approaches:

  • T-DNA insertion mutants (ole2 single mutants or ole1234 quadruple mutants)

  • RNAi or CRISPR-Cas9 for targeted gene silencing or knockout

  • Complementation studies with wild-type or modified oleosin genes

• Imaging techniques:

  • Confocal microscopy with fluorescent markers (e.g., GFP-Q4) to visualize ER structure

  • Electron microscopy to examine ultrastructural details of oil bodies and ER

  • Live cell imaging to track dynamic changes in oil body formation and movement

• Biochemical analyses:

  • Lipid profiling to assess changes in oil composition and content

  • Protein-protein interaction studies (co-immunoprecipitation, yeast two-hybrid)

  • Lipidomics to characterize lipid species associated with oleosins

• Chemical biology approaches:

  • Use of small-molecule probes (e.g., eroonazole photoaffinity probes) for target identification

  • Clickable probe labeling combined with proteomics to identify protein interactions

Research has shown that the ole1234 quadruple mutant displays abnormal ER structure in seeds and seedlings, suggesting oleosins are required for normal ER organization. Chemical perturbation using eroonazole, which targets oleosins, induces ER vesiculation, further supporting the role of oleosins in maintaining ER structure .

How can Oleosin 21.2 kDa be utilized as a fusion partner for recombinant protein expression in plant oil bodies?

Oleosin 21.2 kDa serves as an effective fusion partner for targeting recombinant proteins to oil bodies in plants through the following methodological framework:

• Vector construction:

  • Design a fusion construct with Oleosin 21.2 kDa as the targeting moiety

  • Insert the gene of interest either at the N- or C-terminus of oleosin

  • Include appropriate promoters (e.g., seed-specific promoters) and selection markers

• Transformation methods:

  • Use Agrobacterium-mediated floral dip transformation for Arabidopsis

  • Select transformants using appropriate selection markers

  • Confirm transformation by PCR and Western blot analysis

• Expression validation:

  • Screen T3 homozygous transgenic plants to ensure stable expression

  • Extract oil bodies and verify the presence of the fusion protein by Western blot

  • Assess protein functionality through appropriate bioassays

• Purification strategy:

  • Isolate oil bodies by flotation centrifugation

  • Further purify the fusion protein through affinity chromatography if tagged

  • Consider TEV protease cleavage sites for releasing the protein of interest from oleosin

This approach has been successfully demonstrated with oleosin-hFGF5 fusion proteins in Arabidopsis, where the recombinant oil-body-expressed oleosin-hFGF5 maintained its biological activity of inhibiting hair follicle epithelial cell proliferation .

What is the relationship between Oleosin 21.2 kDa and endoplasmic reticulum structure in Arabidopsis seedlings?

Research using both chemical and genetic approaches has revealed significant insights into the relationship between Oleosin 21.2 kDa and ER structure:

• Chemical probes and oleosin targeting:

  • Eroonazole, an ER-disrupting small molecule, induces extensive ER vesiculation in Arabidopsis seedlings

  • A clickable eroonazole photoaffinity probe (compound 2) selectively labels oleosins, including Oleosin 21.2 kDa

  • This labeling is reduced in oleosin mutants, confirming specificity of the interaction

• ER structural changes in oleosin mutants:

  • Wild-type embryos expressing the ER marker GFP-Q4 show compact ER structure with ER and lipid droplets in an interwoven pattern

  • The ole1234 quadruple mutant shows altered ER morphology that is more homogeneous and less reticulated

  • Wild-type ER develops typical cisternae connected by tubules, while ole1234 mutant ER lacks this distinct organization

• Developmental considerations:

  • The effects on ER structure are most pronounced during seed germination and early seedling development

  • Eroonazole-induced vesiculation is reduced in older seedlings, correlating with decreased oleosin content

  • The ole1234 mutant displays germination and growth defects that worsen after seed harvesting

These findings demonstrate that oleosins, including Oleosin 21.2 kDa, play a critical role in establishing and maintaining normal ER structure during early plant development, particularly during the transition from seed to seedling.

How do post-translational modifications affect Oleosin 21.2 kDa function and stability?

While the search results don't provide direct information about post-translational modifications (PTMs) of Oleosin 21.2 kDa, I can provide a methodological framework for investigating this important aspect based on current research approaches in the field:

• Identification of PTMs:

  • Mass spectrometry-based proteomics to identify phosphorylation, acetylation, ubiquitination, and other modifications

  • Site-directed mutagenesis of potential modification sites to confirm functional relevance

  • Use of modification-specific antibodies for Western blot analysis

• Functional consequences:

  • Generation of phosphomimetic or phospho-null mutants to study the effects of phosphorylation

  • Analysis of protein-protein interactions and oil body targeting efficiency with modified oleosin variants

  • Assessment of protein half-life and degradation pathways for modified versus unmodified oleosins

• Regulation during development:

  • Temporal profiling of PTMs during seed development, germination, and seedling growth

  • Identification of enzymes responsible for adding or removing modifications

  • Analysis of stress responses and their impact on oleosin modification patterns

• Structural implications:

  • Molecular dynamics simulations to predict how PTMs affect protein conformation and membrane interactions

  • Biophysical characterization of modified oleosin variants

  • Assessment of oil body size and stability in plants expressing PTM-mimicking oleosin variants

This research area represents an important frontier for understanding the fine-tuning of oleosin function and oil body dynamics throughout plant development and in response to environmental conditions.

How can researchers overcome expression and purification challenges with recombinant Oleosin 21.2 kDa?

Researchers working with recombinant Oleosin 21.2 kDa often encounter technical challenges due to its highly hydrophobic nature. The following methodological approaches can help overcome these issues:

• Expression optimization:

  • Use specialized E. coli strains designed for membrane/hydrophobic proteins (e.g., C41(DE3), C43(DE3))

  • Reduce expression temperature (16-20°C) to slow protein synthesis and allow proper folding

  • Consider codon optimization for the expression host

  • Test different induction conditions (IPTG concentration, induction time)

  • Supplement media with lipids or detergents to mimic the natural environment

• Solubilization strategies:

  • Test various detergents (e.g., n-dodecyl-β-D-maltoside, CHAPS, Triton X-100) for extraction

  • Consider urea or guanidine hydrochloride for initial extraction followed by refolding

  • Explore nanodiscs or liposomes as membrane mimetics for functional studies

  • Add small amounts of lipids during purification to stabilize the hydrophobic domain

• Purification troubleshooting:

  • Implement stepwise detergent exchange during purification

  • Include glycerol (10-20%) in all purification buffers to enhance stability

  • Test different immobilized metal ions (Ni²⁺, Co²⁺, Cu²⁺) for optimal His-tag binding

  • Consider on-column refolding for proteins purified under denaturing conditions

• Storage and stability:

  • Evaluate different lyophilization excipients beyond trehalose (e.g., sucrose, mannitol)

  • Test the addition of antioxidants to prevent oxidation of methionine residues

  • Consider flash-freezing small aliquots rather than repeated freeze-thaw cycles

These strategies can significantly improve yield and quality of recombinant Oleosin 21.2 kDa for downstream applications.

What are the common pitfalls in oleosin mutant analysis and how can they be addressed?

Analysis of oleosin mutants presents several challenges that can affect experimental outcomes. These pitfalls and their solutions include:

• Genetic redundancy issues:

  • Pitfall: Single oleosin mutants often show subtle or no phenotypes due to functional redundancy among the 16 oleosin genes in Arabidopsis

  • Solution: Generate higher-order mutants (e.g., ole1234 quadruple mutant) to overcome redundancy; use CRISPR-Cas9 for multiple gene targeting

• Developmental timing considerations:

  • Pitfall: Oleosin phenotypes may be transient or stage-specific, particularly during seed germination

  • Solution: Perform detailed time-course experiments; analyze multiple developmental stages; use inducible expression systems for temporal control

• Seed storage effects:

  • Pitfall: Phenotypes of oleosin mutants can worsen after seed harvesting and storage

  • Solution: Standardize seed storage conditions; use freshly harvested seeds for critical experiments; document seed age in publications

• Pleiotropic effects:

  • Pitfall: Changes in oil body structure can indirectly affect multiple cellular processes

  • Solution: Use complementation studies with wild-type and mutated versions of oleosins; perform rescue experiments with specific oleosin genes

• Microscopy artifacts:

  • Pitfall: Sample preparation for microscopy can disrupt the native organization of oil bodies and ER

  • Solution: Compare multiple fixation and sample preparation methods; use live cell imaging when possible; complement with biochemical approaches

Addressing these challenges through careful experimental design and appropriate controls is essential for accurate interpretation of oleosin mutant phenotypes.

How can researchers effectively analyze data from oleosin-targeting chemical probes?

Chemical biology approaches using probes like eroonazole provide valuable insights into oleosin function but require specialized data analysis methods:

• Target validation strategies:

  • Compare labeling patterns between active probes and inactive analogs

  • Conduct competition experiments with unmodified compounds

  • Verify labeling specificity in oleosin mutants (e.g., ole1234)

  • Perform dose-dependent labeling to establish binding kinetics

• Proteomics data analysis:

  • Use stringent enrichment criteria (typically >2-fold) when comparing probe-labeled vs. control samples

  • Account for non-specific binding of probes to abundant proteins

  • Consider both the number of peptides identified and the percentage of protein coverage

  • Validate mass spectrometry hits through independent methods (Western blot, immunoprecipitation)

• Probe-phenotype correlations:

  • Establish clear structure-activity relationships between probe binding and biological effects

  • Use concentration-response curves to distinguish on-target from off-target effects

  • Compare cellular phenotypes between chemical and genetic perturbations

  • Consider the potential for multiple targets contributing to observed phenotypes

• Visualization and quantification:

  • Develop quantitative metrics for ER morphology changes (e.g., vesiculation index)

  • Use appropriate image analysis software for objective quantification

  • Employ statistical methods suitable for phenotypic data (e.g., non-parametric tests for morphological data)

  • Consider machine learning approaches for complex phenotypic classification

What are the emerging applications of Oleosin 21.2 kDa in biotechnology and synthetic biology?

Several promising research directions are emerging for Oleosin 21.2 kDa applications:

• Recombinant protein production platforms:

  • Development of plant-based expression systems using oleosin fusion proteins

  • Design of self-assembling oil body structures for protein display and delivery

  • Creation of oil body-based bioreactors for continuous protein production

• Drug delivery systems:

  • Engineering oleosin-based nanoparticles for targeted drug delivery

  • Development of oil bodies as carriers for hydrophobic pharmaceutical compounds

  • Design of stimuli-responsive oil body systems for controlled release applications

• Biofuel optimization:

  • Manipulation of oleosin expression to increase oil content in biofuel crops

  • Engineering of oleosin variants to alter oil body size and stability

  • Development of synthetic oleosin chimeras with enhanced properties for biofuel applications

• Bioremediation applications:

  • Design of oleosin-enzyme fusions for environmental contaminant degradation

  • Development of plant oil bodies as biosorbents for heavy metal removal

  • Creation of oleosin-based biosensors for environmental monitoring

• Therapeutic protein production:

  • The successful expression of human growth factors as oleosin fusions (e.g., oleosin-hFGF5) demonstrates the potential for producing bioactive therapeutic proteins

  • This approach could be extended to other therapeutic proteins, particularly those that are challenging to express in conventional systems

These emerging applications leverage the unique properties of Oleosin 21.2 kDa to address challenges in protein production, drug delivery, and environmental remediation.

How might advanced imaging techniques further our understanding of oleosin dynamics and interactions?

Advanced imaging methodologies offer new opportunities to explore oleosin dynamics and interactions:

• Super-resolution microscopy:

  • Single-molecule localization microscopy (PALM/STORM) to visualize individual oleosins within oil bodies

  • Stimulated emission depletion (STED) microscopy to examine oleosin clustering and organization

  • Structured illumination microscopy (SIM) for improved visualization of ER-oil body connections

• Live cell imaging advancements:

  • Lattice light-sheet microscopy for extended live imaging with minimal photodamage

  • Single-particle tracking of fluorescently tagged oleosins to monitor mobility and interactions

  • FRET/FLIM approaches to detect protein-protein interactions involving oleosins in living cells

• Correlative microscopy:

  • Correlative light and electron microscopy (CLEM) to bridge high-resolution structural data with functional information

  • Focused ion beam scanning electron microscopy (FIB-SEM) for 3D reconstruction of oil body-ER interfaces

  • Cryo-electron tomography of oil bodies to visualize native oleosin arrangement in the phospholipid monolayer

• Molecular probes and biosensors:

  • Development of conformation-sensitive fluorescent oleosin fusions to detect structural changes

  • Photoconvertible fluorescent protein fusions for pulse-chase imaging of oleosin trafficking

  • Split fluorescent protein systems to visualize oleosin dimerization or protein-protein interactions

These advanced imaging approaches will provide unprecedented insights into oleosin dynamics during oil body formation, maturation, and mobilization.

What computational approaches can enhance our understanding of Oleosin 21.2 kDa structure-function relationships?

Computational methods offer powerful tools for investigating oleosin structure-function relationships:

• Structural prediction and modeling:

  • Ab initio protein structure prediction using AlphaFold2 or RoseTTAFold

  • Molecular dynamics simulations of oleosin integration into phospholipid monolayers

  • Coarse-grained simulations of oil body assembly and stability

  • Modeling of conformational changes during oil body formation and mobilization

• Systems biology approaches:

  • Gene regulatory network analysis of oleosin expression patterns

  • Metabolic modeling of lipid metabolism in wild-type versus oleosin mutants

  • Multi-omics data integration to understand oleosin function in the context of seed development

• Machine learning applications:

  • Development of algorithms to predict oleosin-lipid interactions

  • Pattern recognition in phenotypic data from oleosin mutants

  • Natural language processing to mine oleosin-related literature for novel hypotheses

• Protein engineering tools:

  • Computational design of oleosin variants with enhanced stability or targeting capabilities

  • In silico screening of chemical compounds that modulate oleosin function

  • Prediction of optimal fusion sites for recombinant protein expression

These computational approaches, combined with experimental validation, will accelerate our understanding of oleosin structure-function relationships and facilitate rational design of oleosin-based biotechnological applications.