Recombinant Shewanella loihica Disulfide bond formation protein B (dsbB)
Description
Introduction to Shewanella loihica and Disulfide Bond Formation
Shewanella loihica is a Gram-negative bacterium originally isolated from an iron-rich microbial mat in the Pacific Ocean near Hawaii . The type strain, designated as PV4 (DSM 17748, ATCC BAA-1088, CIP 109777), was collected in September 1997 . Like other members of the Shewanella genus, S. loihica is noted for its diverse respiratory capabilities, particularly its ability to reduce metals, making it significant for bioremediation applications and understanding extracellular electron transfer processes .
Disulfide bonds, formed by the oxidation of pairs of cysteines, are crucial for the folding and stability of many exported proteins in bacteria . In bacterial systems, particularly the well-studied Escherichia coli, disulfide bond formation is facilitated by a pathway involving the periplasmic protein DsbA and the membrane-bound protein DsbB . This system operates as follows:
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DsbA introduces disulfide bonds into proteins translocated into the periplasm
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The membrane-bound DsbB reoxidizes DsbA, restoring its activity
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DsbB transfers electrons to the electron transport chain via membrane-bound quinones
Table 2: Comparison of DsbB Proteins from Different Shewanella Species
| Feature | S. loihica DsbB | Shewanella sp. DsbB |
|---|---|---|
| UniProt ID | A3QEZ4 | A0KVP7 |
| Length | 171 amino acids | 175 amino acids |
| Active site motif | CXXC present | CXXC present |
| N-terminal region | MSALTRFAQSR | MTAFTRFAHSR |
| Expression system | E. coli | E. coli |
| Tag | N-terminal His | N-terminal His |
Role in Bacterial Adaptation
Members of the Shewanella genus, including S. loihica, are known for their remarkable respiratory versatility and ability to thrive in diverse environments . The proper functioning of the disulfide bond formation system, including DsbB, is likely critical for maintaining the structural integrity and function of extracellular and periplasmic proteins involved in these adaptive processes.
Potential Applications
The recombinant S. loihica DsbB protein has several potential applications:
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Basic Research: Studying bacterial disulfide bond formation mechanisms
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Protein Engineering: Developing improved systems for disulfide bond formation in heterologous proteins
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Biotechnology: Enhancing the production of disulfide-bonded recombinant proteins
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Environmental Applications: Supporting research on Shewanella's metal reduction capabilities, which have implications for bioremediation
Research Tools and Methods
Recent advances in genetic tools for Shewanella species have expanded the possibilities for studying proteins like DsbB in their native context. For instance, the development of efficient electroporation methods and recombineering systems for Shewanella oneidensis may be adaptable to S. loihica, enabling more precise genetic manipulation and functional studies .
Comparative Analysis with Other Disulfide Bond Formation Systems
Bacteria exhibit diversity in their mechanisms of disulfide bond formation. While many Proteobacteria (the phylum containing Shewanella) possess both DsbA and DsbB homologs, suggesting similar redox biology, some other bacterial groups use alternative systems .
Interestingly, some bacteria predicted to catalyze disulfide bond formation lack DsbB homologs despite having DsbA homologs. This observation suggests that alternative mechanisms for reoxidizing DsbA exist in these organisms . This diversity highlights the evolutionary adaptability of disulfide bond formation systems and raises interesting questions about the specific adaptations in the S. loihica system.
Future Research Directions
Several promising avenues for future research on S. loihica DsbB include:
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Detailed structural characterization using crystallography or cryo-EM
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Functional studies to determine substrate specificity and kinetic parameters
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Investigation of its role in S. loihica's metal reduction capabilities
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Exploration of its potential for enhancing heterologous protein production
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Comparative studies with DsbB proteins from other Shewanella species and more distantly related bacteria
Such research would not only enhance our understanding of bacterial disulfide bond formation but could also lead to practical applications in biotechnology and environmental science.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement. We will accommodate your request to the best of our ability.
Note: All protein shipments are standardly packaged with normal blue ice packs. If dry ice packaging is required, please contact us in advance. Additional fees may apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: slo:Shew_2176
STRING: 323850.Shew_2176
Q&A
What is Shewanella loihica dsbB and what are its key characteristics?
Shewanella loihica dsbB (Disulfide bond formation protein B) is a membrane protein that functions as a disulfide oxidoreductase, playing a crucial role in the formation of disulfide bonds in proteins. The protein is encoded by the dsbB gene (locus name Shew_2176) and has a UniProt accession number of A3QEZ4. The full-length protein consists of 171 amino acids and is typically studied as part of the bacterium's extracellular electron transfer mechanisms .
Shewanella loihica PV-4 is classified as an electricigen, meaning it can produce electricity by transferring electrons to external acceptors. This capability makes it valuable for research in bioelectrochemical systems (BES) where the extracellular electron transfer (EET) rate is critical for current output .
What are the recommended storage conditions for recombinant dsbB?
Proper storage is crucial for maintaining protein stability and activity. For recombinant Shewanella loihica dsbB, the following storage conditions are recommended:
| Storage Purpose | Temperature | Buffer Composition | Additional Notes |
|---|---|---|---|
| Long-term storage | -20°C to -80°C | Tris-based buffer with 50% glycerol or Tris/PBS-based buffer with 6% Trehalose, pH 8.0 | Aliquoting is necessary for multiple use |
| Working stock | 4°C | Same as storage buffer | Stable for up to one week |
| Reconstitution | - | Deionized sterile water | Reconstitute to 0.1-1.0 mg/mL |
Repeated freezing and thawing is not recommended as it can compromise protein integrity and activity. For extended storage, it's advisable to add glycerol (final concentration 30-50%) and store aliquots at -20°C or -80°C .
How does dsbB function in the context of Shewanella loihica's electron transfer capabilities?
In Shewanella loihica, dsbB is involved in the proper folding of proteins by catalyzing disulfide bond formation, which is particularly important for the structure and function of outer membrane cytochromes (OMCs) that participate in extracellular electron transfer (EET).
Shewanella loihica PV-4 employs two primary mechanisms for EET:
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Direct Electron Transfer (DET): Involves direct contact between cell-surface proteins (primarily cytochromes) and the electrode surface
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Mediated Electron Transfer (MET): Utilizes soluble mediators, particularly flavins, to shuttle electrons between cells and electrodes
Research has shown that at lower electrode potentials, Shewanella loihica primarily utilizes flavin-mediated EET, while at higher potentials, both DET and MET contribute to the current output. This dual capability makes Shewanella loihica an interesting model organism for studying bacterial electricity production .
What expression systems are optimal for recombinant dsbB production?
Based on established protocols, E. coli expression systems have been successfully employed for recombinant Shewanella loihica dsbB production. When designing an expression system, researchers should consider:
| Expression System | Advantages | Considerations |
|---|---|---|
| E. coli with His-tag | Easy purification, high yield | Potential interference with membrane insertion |
| E. coli with native sequence | More natural protein folding | More complex purification process |
| Homologous expression in Shewanella | Native post-translational modifications | Lower yield, technically more challenging |
For most research applications, recombinant dsbB with an N-terminal His-tag expressed in E. coli provides a good balance between yield and functionality. This approach allows for effective purification using immobilized metal affinity chromatography (IMAC) .
How can I verify the purity and activity of recombinant dsbB?
Verifying the purity and activity of recombinant dsbB is essential before proceeding with experiments. The following methods are recommended:
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Purity assessment: SDS-PAGE analysis should show greater than 90% purity, with dsbB appearing at the expected molecular weight .
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Western blotting: Using antibodies against the tag (e.g., His-tag) or dsbB itself to confirm identity.
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Activity assay: While specific activity assays for dsbB may vary depending on research context, functional assays typically involve:
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Measuring disulfide bond formation in substrate proteins
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Assessing the protein's ability to complement dsbB-deficient strains
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Evaluating electron transfer capabilities in bioelectrochemical systems
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What are key considerations for designing biofilm experiments with Shewanella loihica?
When designing biofilm experiments with Shewanella loihica for studying dsbB function and electron transfer mechanisms, consider the following factors:
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Electrode material selection: Reticulated vitreous carbon (RVC) electrodes have been shown to increase surface area available for biofilm formation compared to conventional flat electrodes .
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Electrode potential: Research has demonstrated that electrode potential significantly affects cell attachment, biofilm formation, and the balance between DET and MET mechanisms:
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Biofilm age: The maturity of the biofilm affects the dominant electron transfer mechanisms, with older biofilms showing increased reliance on mediator-based electron transfer .
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Environmental conditions: Oxygen availability significantly impacts protein expression profiles, with proteins involved in anaerobic energy metabolism showing up to 10-fold increases when transitioning from aerobic to suboxic conditions .
How do electrode potential and biofilm age affect extracellular electron transfer mechanisms?
Research on Shewanella loihica PV-4 has revealed complex relationships between electrode potential, biofilm age, and extracellular electron transfer (EET) mechanisms:
| Condition | Dominant EET Mechanism | Observations |
|---|---|---|
| Low electrode potential | Flavin-mediated MET | Lower current output, less biofilm formation |
| High electrode potential | Combined DET and MET | Enhanced cell attachment, higher current output |
| Early biofilm | Primarily DET | Direct contact between cells and electrode |
| Mature biofilm | Increased MET contribution | Mediators may become confined within biofilm |
This dynamic relationship demonstrates that experimental conditions must be carefully controlled and reported when studying Shewanella loihica electron transfer capabilities. Electrode potential should be considered a critical variable that influences both biofilm formation and the underlying molecular mechanisms of electron transfer .
What proteomics approaches are most effective for studying dsbB and related proteins?
Based on research with Shewanella species, the following proteomics approaches have proven effective:
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LC-MS without stable isotope labeling: This approach has been successfully applied for differential quantitative proteomic analysis of Shewanella whole cell lysates under varying conditions (aerobic vs. suboxic) .
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LC-MS/MS for peptide identification: This technique enables identification of peptide sequences with high confidence .
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LC-FTICR for quantification: Fourier transform ion cyclotron resonance mass spectrometry provides both confirmation of identifications and precise measurement of relative peptide abundances .
Using these techniques, researchers have identified and quantified thousands of peptides covering hundreds of proteins in Shewanella species. This approach revealed significant changes in protein abundance (up to 10-fold) when transitioning between aerobic and anaerobic conditions, particularly for proteins involved in energy metabolism .
How do aerobic versus anaerobic conditions affect protein expression in Shewanella loihica?
Proteomic analysis has revealed substantial differences in protein expression between aerobic and suboxic/anaerobic conditions in Shewanella species:
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Proteins involved in anaerobic energy metabolism: Show up to 10-fold increases in relative abundance when transitioning from aerobic to suboxic conditions .
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Housekeeping proteins: Remain relatively unchanged in abundance regardless of oxygen availability .
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Electron transfer proteins: Cytochromes and associated proteins show significant upregulation under anaerobic conditions to facilitate alternative respiratory pathways .
These findings highlight the importance of carefully controlling and reporting oxygen levels in experiments involving Shewanella loihica, as oxygen availability dramatically affects the expression of proteins involved in electron transfer pathways.
What are common issues in dsbB purification and how can they be resolved?
Several challenges may arise during the purification and handling of recombinant dsbB:
| Issue | Possible Causes | Solutions |
|---|---|---|
| Low protein yield | Suboptimal expression conditions, protein aggregation | Optimize induction parameters, use detergents for membrane protein extraction |
| Protein inactivity | Improper folding, loss of cofactors | Include appropriate cofactors in buffers, optimize purification conditions |
| Precipitation during storage | Protein instability, buffer incompatibility | Add glycerol or trehalose, adjust buffer composition |
| Inconsistent experimental results | Protein batch variation, degradation | Implement rigorous quality control, prepare fresh aliquots |
For recombinant dsbB specifically, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL with added glycerol (30-50% final concentration) is recommended to maintain protein stability .
How should contradictory data in dsbB research be approached?
When facing contradictory results in dsbB research, consider the following methodological approach:
What statistical approaches are appropriate for analyzing dsbB-related experimental data?
When designing experiments and analyzing data related to dsbB function in Shewanella loihica, consider these statistical approaches:
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For comparing treatment groups: Analysis of variance (ANOVA) for multiple comparisons or t-tests for paired comparisons between specific conditions.
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For time-series data: Repeated measures ANOVA or mixed-effects models to account for temporal correlations.
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For biofilm analyses: Consider spatial statistics to account for the heterogeneous distribution of bacteria observed in biofilms, particularly their preference for flat areas of electrode surfaces .
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For proteomics data: Statistical approaches such as statistical analysis of microarrays have been effectively used to identify proteins with significantly changed abundance levels in Shewanella species .
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Sample size determination: Conduct power analyses before experiments to ensure sufficient statistical power for detecting meaningful differences between conditions.
What are emerging applications of dsbB research in bioelectrochemical systems?
The study of dsbB and related proteins in Shewanella loihica has significant implications for advancing bioelectrochemical systems (BES) with several promising research directions:
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Enhancing current output: Understanding how dsbB affects the formation and function of electron transfer proteins could lead to engineered strains with improved electricity generation capabilities.
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Biofilm optimization: Knowledge of how electrode potential affects biofilm formation and EET mechanisms can inform the design of more efficient microbial fuel cells.
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Mediator engineering: Insights into the transition between DET and MET mechanisms could enable the development of artificial mediators or engineered cells with enhanced electron transfer capabilities.
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Environmental applications: Optimized Shewanella loihica systems could be applied to waste treatment, bioremediation, and sustainable energy production.
The continued study of proteins like dsbB is essential for bridging the gap between fundamental molecular mechanisms and practical applications of electricigens in bioelectrochemical systems.
How might systems biology approaches advance our understanding of dsbB function?
Integrating multiple omics approaches could provide a more comprehensive understanding of dsbB function within the broader context of Shewanella loihica physiology:
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Multi-omics integration: Combining proteomics, transcriptomics, and metabolomics data to understand how dsbB expression correlates with other cellular processes.
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Protein interaction networks: Identifying protein-protein interactions involving dsbB to elucidate its role in electron transfer pathways.
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Comparative genomics: Analyzing dsbB homologs across different Shewanella species and other electricigens to identify conserved features and specialized adaptations.
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Mathematical modeling: Developing quantitative models of electron transfer that incorporate dsbB function to predict system behavior under various conditions.
