Recombinant Saccharum hybrid Cytochrome b559 subunit alpha (psbE)

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Description

Production and Characterization

Recombinant PsbE is produced via heterologous expression, often in E. coli, followed by affinity chromatography purification . Key features include:

  • Sequence: Full-length (2–83 residues) with a conserved heme-binding domain .

  • Reconstitution: Requires solubilization in Tris/PBS-based buffers and avoidance of repeated freeze-thaw cycles .

  • Stability: Mutations in heme-coordinating residues (e.g., His-22) destabilize the protein, necessitating overexpression for functional PSII assembly .

3.1. PSII Assembly Studies

  • PsbE is indispensable for forming the D2 module during PSII assembly . Mutations in PsbE disrupt PSII accumulation, but tandem amplification of psbEFLJ operons can restore function in cyanobacteria .

  • In sugarcane, recombinant PsbE enables investigations into PSII biogenesis and repair mechanisms under stress .

3.2. Photoprotection Mechanisms

  • Cyt b559 participates in cyclic electron flow within PSII, protecting against photoinhibition . Recombinant PsbE facilitates studies on redox potential variations (e.g., high-potential vs. low-potential forms) and their roles in oxidative stress response .

Comparative Insights from Other Species

  • Cyanobacteria: In Synechocystis sp. PCC 6803, PsbE/F mutants require gene amplification to compensate for destabilized subunits .

  • Thermosynechococcus elongatus: Heme coordination in PsbE is less critical for PSII stability compared to Synechocystis, suggesting species-specific assembly mechanisms .

  • Tobacco: His-tagged PsbE enables efficient PSII core complex isolation, highlighting its utility in structural studies .

Limitations and Future Directions

  • Knowledge Gaps: Direct structural data for sugarcane PsbE is limited; most models derive from cyanobacterial or plant homologs .

  • Technical Challenges: Recombinant PsbE requires precise heme incorporation for functional studies, which remains technically demanding .

Product Specs

Form
Lyophilized powder
Note: We will ship the format currently in stock unless you specify a preference during order placement. Please indicate any format requirements in your order notes.
Lead Time
Delivery times vary depending on the purchase method and location. Please contact your local distributor for precise delivery estimates.
Note: All proteins are shipped with standard blue ice packs unless dry ice is specifically requested in advance. Dry ice shipments incur additional charges.
Notes
Avoid repeated freeze-thaw cycles. Store working aliquots at 4°C for up to one week.
Reconstitution
Centrifuge the vial briefly before opening to collect the contents. Reconstitute the protein in sterile, deionized water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard glycerol concentration is 50% and can serve as a guideline.
Shelf Life
Shelf life depends on various factors, including storage conditions, buffer composition, temperature, and protein stability. Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life at -20°C/-80°C.
Storage Condition
Store at -20°C/-80°C upon receipt. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
The tag type is determined during the manufacturing process.
Tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Synonyms
psbE; PS140; Cytochrome b559 subunit alpha; PSII reaction center subunit V
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
2-83
Protein Length
Full Length of Mature Protein
Species
Saccharum hybrid (Sugarcane)
Target Names
psbE
Target Protein Sequence
SGSTGERSFADIITSIRYWVIHSITIPSLFIAGWLFVSTGLAYDVFGSPRPNEYFTESRQ GIPLITDRFDSLEQLDEFSRSF
Uniprot No.

Target Background

Function

This b-type cytochrome is tightly associated with the photosystem II (PSII) reaction center. PSII, a light-driven water:plastoquinone oxidoreductase, utilizes light energy to extract electrons from H₂O, generating O₂ and a proton gradient for ATP synthesis. It comprises a core antenna complex for photon capture and an electron transfer chain converting photonic excitation into charge separation.

Protein Families
PsbE/PsbF family
Subcellular Location
Plastid, chloroplast thylakoid membrane; Single-pass membrane protein.

Q&A

Basic Research Questions

  • What is Cytochrome b559 and what role does it play in photosystem II of Saccharum hybrids?

    Cytochrome b559 (cyt b559) is an intrinsic membrane protein that serves as a critical component of photosystem II (PSII), the membrane-protein complex responsible for photosynthetic oxygen evolution. In Saccharum hybrids, as in other photosynthetic organisms, cyt b559 consists of two subunits—alpha and beta—encoded by the psbE and psbF genes, respectively. Research using deletion mutants in cyanobacteria has conclusively demonstrated that cyt b559 is essential for PSII function; when the psbE and psbF genes are deleted, PSII complexes become completely inactivated . While the precise electron transport role of cyt b559 continues to be investigated, its structural importance for maintaining functional PSII architecture is well-established. The protein's high degree of conservation between cyanobacterial and green plant chloroplastidic forms indicates its fundamental importance in photosynthetic processes across diverse taxonomic groups .

  • How do the genetic characteristics of Saccharum species influence recombinant psbE expression?

    The genetic complexity of Saccharum species significantly impacts recombinant protein expression, including psbE. Saccharum hybrids possess considerable genetic diversity due to their complex polyploid nature and interspecific hybridization history. Studies on introgressed Saccharum hybrids have demonstrated varied levels of genetic diversity across different introgression groups, with polymorphic alleles ranging from 118 to 193 among different hybrid groups . This genetic diversity affects promoter functionality, codon usage bias, and post-translational modification patterns. For example, research on recombinant protein expression in sugarcane has shown that promoter choice significantly influences protein accumulation, with constitutive promoters like maize ubiquitin 1 (Ubi) resulting in different expression levels in leaves versus culms . When expressing recombinant psbE, researchers must consider these genetic factors to optimize expression and ensure proper protein folding and function.

  • What are the advantages of using Saccharum hybrids as expression systems for recombinant photosystem proteins?

    Saccharum hybrids (sugarcane and energy cane) offer several distinct advantages for recombinant photosystem protein production. These species demonstrate high resource-use efficiency, rapid growth, efficient photosynthesis, and exceptional biomass production capacity—potentially yielding up to 49 tons of dry biomass per hectare annually . This makes them ideal platforms for large-scale protein production. Additionally, Saccharum hybrids provide excellent transgene containment since they primarily propagate vegetatively, with limited natural reproductive propagation in many temperate and subtropical regions due to photoperiod sensitivity . Many commercial varieties do not produce viable pollen or seeds under typical field conditions, further restricting transgene flow. For photosystem proteins specifically, Saccharum provides the appropriate cellular machinery for proper folding and post-translational modifications needed for functional membrane proteins. Previous studies have demonstrated successful recombinant protein expression in sugarcane, with accumulation levels ranging from 0.02% to 2.0% of total soluble protein in leaves .

Methodological Considerations

  • What transformation and selection methods work best for generating stable Saccharum lines expressing recombinant psbE?

    Generating stable Saccharum lines expressing recombinant psbE requires optimized transformation and selection protocols. Based on successful approaches with other recombinant proteins in Saccharum, the following methodological considerations are critical:

    1. Transformation method: Biolistic bombardment of embryogenic callus using a particle inflow gun has proven effective. Optimized parameters include:

      • Using 0.5 μg DNA per bombardment

      • Employing 1 M calcium chloride and 14 mM spermidine for DNA coating

      • Maintaining a 7-cm target distance with a 26-inch Hg vacuum

    2. Selection strategy: Co-transformation with a selectable marker (e.g., BAR gene conferring bialaphos resistance) on separate plasmids has proven effective. Selection concentrations should be optimized by species:

      • 3 mg/L bialaphos for sugarcane

      • 1.5 mg/L bialaphos for energy cane

    3. Recovery and regeneration protocol:

      • Allow bombarded callus 10 days recovery on appropriate medium (e.g., MS3) in dark conditions at 28°C

      • Conduct shoot regeneration and root initiation under selection pressure

      • Transfer rooted plantlets to soil under controlled greenhouse conditions (25-30°C day/15-24°C night, 1,200-1,600 μmol m-1 s-1 light intensity)

    4. Screening approach:

      • PCR verification of transgene integration

      • RT-PCR and Western blot analysis for expression confirmation

      • Spectroscopic analysis for functional protein verification

    This comprehensive approach maximizes transformation efficiency while ensuring stable transgene integration and expression.

  • What purification strategies provide the highest yield and purity of recombinant psbE from Saccharum tissues?

    Purifying recombinant psbE from Saccharum tissues presents challenges due to its membrane-associated nature. Based on methodologies used for other recombinant proteins and photosystem components, an effective purification strategy should include:

    1. Tissue selection and preparation:

      • Target photosynthetically active leaf tissue with highest expression

      • Flash-freeze harvested tissue in liquid nitrogen

      • Grind to fine powder under liquid nitrogen to prevent proteolysis

    2. Membrane protein extraction:

      • Use buffers containing mild detergents (e.g., n-dodecyl-β-D-maltoside or Triton X-100)

      • Include protease inhibitors to prevent degradation

      • Employ differential centrifugation to isolate membrane fractions

    3. Chromatographic purification sequence:

      • Immobilized metal affinity chromatography (IMAC) if using histidine-tagged constructs

      • Ion exchange chromatography to separate based on charge characteristics

      • Size exclusion chromatography for final polishing and to verify oligomeric state

    4. Quality assessment:

      • SDS-PAGE analysis with target purity >85%

      • Western blot confirmation

      • Spectroscopic analysis of heme content

      • Mass spectrometry for protein identification and integrity verification

    5. Storage considerations:

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

      • Long-term storage: -20°C/-80°C with 5-50% glycerol as a cryoprotectant

      • Avoid repeated freeze-thaw cycles

    Following this systematic approach should yield functional recombinant psbE with sufficient purity for subsequent biochemical and functional studies.

  • How can researchers assess the integration of recombinant psbE into functional photosystem II complexes?

    Assessing the integration of recombinant psbE into functional PSII complexes requires multiple complementary approaches:

    1. Biochemical fractionation and analysis:

      • Blue-native PAGE to visualize intact PSII complexes

      • Immunoblotting with antibodies against multiple PSII subunits

      • Co-immunoprecipitation using anti-psbE antibodies to identify interacting partners

    2. Functional assays:

      • Oxygen evolution measurements to assess PSII activity

      • Chlorophyll fluorescence analysis (OJIP test, PAM fluorometry) to evaluate electron transport efficiency

      • P680+ re-reduction kinetics to assess donor-side electron transfer

      • Thermoluminescence to characterize charge recombination events

    3. Structural characterization:

      • Freeze-fracture electron microscopy to visualize PSII complex distribution

      • Single-particle cryo-EM to assess structural integrity of PSII complexes

      • Cross-linking mass spectrometry to map protein-protein interactions

    4. Genetic complementation:

      • Expression of recombinant psbE in psbE-deletion mutants (similar to approaches in Synechocystis 6803)

      • Assessment of rescued phenotypes and PSII function

    5. Comparative analysis:

      • Side-by-side comparison with wild-type PSII complexes

      • Quantitative proteomics to assess stoichiometry of PSII components

    These approaches collectively provide comprehensive evidence for successful integration and functional contribution of recombinant psbE to PSII complexes.

  • What are the critical parameters for optimizing recombinant psbE expression in different Saccharum genotypes?

    Optimizing recombinant psbE expression across different Saccharum genotypes requires attention to several critical parameters:

    ParameterOptimization StrategyConsiderations
    Promoter selectionTest multiple promoters: constitutive (e.g., maize Ubi), tissue-specific, and stress-inducibleExpression levels vary between tissues; promoter stacking can increase yields 42.3-fold
    Codon optimizationAnalyze codon usage bias in target genotype; synthesize codon-optimized geneGenetic diversity affects translation efficiency
    Signal peptidesTest chloroplast-targeting vs. ER-targeting sequencesProper subcellular localization is essential for function
    Vector designInclude 5' and 3' UTRs from highly expressed Saccharum genesUTRs affect mRNA stability and translation efficiency
    Transformation protocolOptimize biolistic parameters for each genotypeDifferent genotypes may require adjusted bombardment conditions
    Selection pressureAdjust bialaphos concentration (1.5-3.0 mg/L) based on genotype sensitivityEnergy cane typically requires lower concentrations than sugarcane
    Growth conditionsModulate light intensity, photoperiod, and nutritionThese factors influence photosynthetic protein expression
    Expression inductionApply appropriate hormonal treatments if using inducible promotersCan increase protein accumulation to 2.7% TSP
    Genetic backgroundSelect genotypes with high transferability of gene regulatory elementsEST-SSR marker transferability varies from 84.1-93.4% among Saccharum species

    Researchers should implement a factorial experimental design to identify optimal combinations of these parameters for their specific Saccharum genotype and research objectives. Preliminary small-scale expression studies should precede large-scale production efforts.

Current Challenges and Future Directions

  • What are the major technical challenges in expressing membrane proteins like psbE in Saccharum systems?

    Expressing membrane proteins like psbE in Saccharum systems presents several significant technical challenges:

    1. Membrane integration: Ensuring proper integration into thylakoid membranes requires appropriate targeting sequences and membrane insertion machinery. Unlike soluble proteins, cytochrome b559 subunit alpha must correctly incorporate into the membrane lipid bilayer while maintaining proper topology.

    2. Protein complex assembly: Cytochrome b559 functions as a heterodimer of alpha (psbE) and beta (psbF) subunits . Expressing recombinant psbE alone may result in misfolded protein unless the native psbF is available in sufficient quantities or is co-expressed. The challenge extends to ensuring proper association with other PSII components.

    3. Heme incorporation: As a cytochrome, psbE requires proper incorporation of heme prosthetic groups. The efficiency of heme incorporation machinery in heterologous expression systems can be limiting.

    4. Toxicity considerations: Overexpression of membrane proteins can disrupt membrane integrity and cellular homeostasis, potentially causing toxicity to the host cells.

    5. Extraction and purification difficulties: Membrane proteins require detergents for solubilization, which complicates purification procedures and may affect protein stability and function.

    Future research should focus on developing specialized expression vectors with appropriate targeting sequences, optimizing co-expression strategies for psbE and psbF, and refining membrane protein extraction protocols specifically for Saccharum systems.

  • How can researchers leverage genetic diversity in Saccharum for improved recombinant protein expression?

    Leveraging genetic diversity in Saccharum for improved recombinant protein expression requires systematic characterization and utilization of germplasm resources:

    1. Germplasm screening: Evaluate diverse Saccharum genotypes for natural variation in protein expression capacity. Research has identified significant genetic diversity across different introgression groups (SSH, SRH, SBH, and EIH) with varying numbers of polymorphic alleles (174, 193, 166, 147, and 118, respectively) .

    2. Identification of superior genetic backgrounds: Select genotypes with optimal characteristics for protein expression, such as:

      • Enhanced transcriptional machinery

      • Efficient translation apparatus

      • Appropriate post-translational modification capabilities

      • Higher photosynthetic efficiency for photosystem proteins

    3. Exploitation of rare alleles: Target species-specific rare alleles that may confer advantageous protein expression traits. Studies have identified rare alleles specific to different Saccharum species that could influence gene expression regulation .

    4. Cross-species promoter utilization: Assess the transferability of regulatory elements across Saccharum species. Research has shown high transferability of EST-SSR markers (84.1-93.4%) within Saccharum species , suggesting similar potential for promoter elements.

    5. Hybridization approaches: Develop new Saccharum hybrids with optimized characteristics for recombinant protein production, potentially incorporating traits from species like S. spontaneum that confer stress resistance and adaptability .

    This strategic utilization of genetic diversity could significantly enhance the efficiency and yield of recombinant psbE production in Saccharum expression systems.

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