Recombinant Pseudomonas putida Disulfide bond formation protein B 1 (dsbB1)
Description
Introduction to Recombinant Pseudomonas putida Disulfide Bond Formation Protein B 1 (dsbB1)
Recombinant Pseudomonas putida Disulfide bond formation protein B 1 (dsbB1) is a bacterial oxidoreductase enzyme critical for maintaining proper protein folding through catalysis of disulfide bond formation. This protein belongs to the Dsb (Disulfide Bond) system, which is essential for bacterial survival under oxidative stress. DsbB1 facilitates the transfer of electrons from cytoplasmic proteins to periplasmic disulfide bonds, enabling the maturation of extracellular or membrane-associated proteins .
Protein Structure
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Amino Acid Sequence:
The full-length dsbB1 from P. putida KT2440 spans 168 residues (Uniprot ID: P59345) with the sequence:
MNEQTSRLNRERRFLVLLGLICLSLIGGALYMQVVLGEAPCPLCILQRYALLFIAVFAFI...(partial sequence) . -
Key Motifs:
Functional Role
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Disulfide Bond Formation: Acts as an oxidoreductase, transferring electrons from reduced substrates (e.g., thioredoxin) to periplasmic proteins, enabling proper folding of virulence factors, enzymes, and transporters .
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Pathway Involvement:
Pathway Role in Pathway Protein Export Maturation of secreted proteins Oxidative Stress Response Maintenance of redox balance Virulence Factor Activation Folding of adhesins, toxins
Production Methodology
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Host Organism: Expressed in Escherichia coli with an N-terminal His-tag for purification .
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Expression Region: Full-length protein (1–168 amino acids) .
Genomic Localization
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Locus: PP_0809 (chromosomal location: 1007946–1008455 bp in P. putida UW4) .
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Orthologs: Part of the POG015207 ortholog group, with homologs in 181 Pseudomonas species .
Taxonomic Distribution
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Core Genome: Conserved across P. putida group strains, reflecting its essential role in bacterial physiology .
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Genomic Variability: While the core genome includes dsbB1, accessory genes (e.g., plasmid-encoded traits) vary, influencing strain-specific adaptation .
Biotechnological Relevance
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Protein Engineering: Used in heterologous systems to improve folding of recombinant proteins (e.g., rhamnolipids, polyketides) in P. putida .
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Mechanistic Studies: Investigated for its role in electron transfer pathways and membrane protein biogenesis .
Pathogenicity and Clinical Relevance
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Nosocomial Infections: While P. putida is generally non-pathogenic, clinical isolates (e.g., HB3267) exhibit antibiotic resistance and virulence traits linked to plasmid-borne genes . DsbB1’s role in pathogenicity remains indirect, as it supports folding of efflux pumps or adhesins .
Challenges and Future Directions
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Production Limitations:
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Research Gaps:
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format preference, please indicate it in your order notes. We will fulfill your request as much as possible.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional charges will 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: ppu:PP_0809
STRING: 160488.PP_0809
Q&A
What role does DsbB1 play in recombinant protein folding in P. putida, and how does its activity compare to other Dsb proteins?
DsbB1 is a periplasmic disulfide bond oxidoreductase essential for the oxidative folding of secreted proteins in Pseudomonas putida. Unlike DsbA, which primarily catalyzes thiol-disulfide exchange, DsbB1 functions as an electron donor, transferring electrons to the periplasmic oxidoreductase DsbC during catalysis . This activity is critical for proteins requiring complex disulfide networks, such as cellulosomes or scaffoldins .
Methodological Insight: To compare DsbB1 and DsbA activities, use a redox-sensitive fluorescent reporter protein (e.g., roGFP2) co-expressed with each protein. Quantify fluorescence shifts under reducing/oxidizing conditions to assess folding efficiency .
How do experimental parameters such as growth conditions and inducer concentration influence DsbB1-mediated folding?
DsbB1 activity is sensitive to environmental factors. For example:
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Temperature: Optimal activity occurs at 30°C (growth temperature of P. putida), with reduced efficiency at 37°C .
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Inducer concentration: Overexpression of DsbB1 via strong promoters (e.g., P<sub>lac</sub>) can overwhelm cellular redox systems, leading to oxidative stress. Use tunable promoters (e.g., P<sub>tac</sub> with IPTG) to balance expression .
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Carbon source: Glycerol-based media (e.g., King’s medium) enhance protein secretion, indirectly improving DsbB1 substrate availability .
Data Table: Optimal Conditions for DsbB1 Activity
Troubleshooting Tip: Use in vivo redox probes (e.g., roGFP2) to monitor periplasmic redox potential during DsbB1 co-expression. Deviations from optimal ranges indicate suboptimal folding environments .
What strategies can address discrepancies in DsbB1 folding efficiency across studies?
Discrepancies often arise from strain-specific differences, secretion pathway compatibility, or substrate-protein interactions. Key approaches include:
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Strain engineering:
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Secretion system optimization:
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Substrate-specific analysis:
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In vitro assays: Purify DsbB1 and test activity on model substrates (e.g., insulin) using HPLC or mass spectrometry.
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In vivo pulse-chase: Track disulfide bond formation kinetics via radiolabeled cysteine incorporation.
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How can DsbB1 be integrated with surface-display systems for recombinant protein production?
DsbB1 co-expression with scaffoldins or autotransporters enhances folding of surface-bound proteins. For example:
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Scaffoldin-DsbB1 fusion: Engineer scaffoldins to include DsbB1 domains, ensuring proximity to secreted proteins .
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Split-ubiquitin systems: Use DsbB1 to oxidize disulfide bonds in split-ubiquitin tags for protein-protein interaction studies.
Case Study: In genome-reduced P. putida EM371, surface-displayed scaffoldins required DsbB1 co-expression to maintain β-glucosidase activity. Without DsbB1, misfolded proteins accumulated in inclusion bodies .
What advanced methodologies can validate DsbB1’s role in multiplex genome-edited strains?
To study DsbB1 in CRISPR-edited P. putida, employ:
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Multiplex CRISPR editing:
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Proteomic analysis:
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Flux analysis:
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Track electron transfer from DsbB1 to DsbC using electron paramagnetic resonance (EPR) spectroscopy.
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How can codon optimization and heterologous gene expression improve DsbB1 activity in P. putida?
Codon optimization is critical due to P. putida’s high GC content (62%). For example:
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Synthetic dsbB1: Replace low-CAI codons (e.g., TAA → TAG for Tyr) to match P. putida’s tRNA abundance .
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Promoter selection: Use P<sub>tac</sub> or P<sub>J23119</sub> for inducible expression, avoiding leaky promoters that exhaust cellular resources .
Data Table: Codon Optimization Impact
| Gene | CAI (Pre-Optimization) | CAI (Post-Optimization) | Expression Fold Increase |
|---|---|---|---|
| dsbB1 | 0.10 | 0.85 | 4–6x |
| Chimeric scaffoldin | 0.15 | 0.80 | 3–5x |
What challenges arise when scaling DsbB1-dependent protein production, and how can they be mitigated?
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Oxidative stress: High DsbB1 activity depletes reducing equivalents (e.g., glutathione). Mitigate by co-expressing glutathione synthase or using fed-batch fermentation to balance redox states .
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Protein aggregation: Use chaperones (e.g., GroEL) or reduce growth rates to slow secretion and allow proper folding.
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Strain instability: Maintain selection pressure (e.g., gentamicin) to prevent plasmid loss during large-scale cultures .
How does DsbB1 interact with other periplasmic redox systems, and what are the implications for synthetic biology?
DsbB1 operates in a redox cascade:
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DsbB1 → DsbC (oxidizes disulfide bonds)
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DsbC → DsbD (transfers electrons to DsbA)
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DsbA → DsbD (reduces misfolded proteins)
Synthetic applications:
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Redox circuit design: Engineer feedback loops (e.g., DsbB1 activation linked to substrate detection) for adaptive folding.
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Modular systems: Use DsbB1 as a "redox switch" to control protein activity (e.g., disulfide-regulated enzymes).
What statistical approaches are needed to validate DsbB1’s impact in high-throughput screens?
For large-scale experiments (e.g., PDX line studies ):
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Mixed-effects models: Account for intra-strain variability by treating strain as a random effect.
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Empirical power analysis: Simulate data using permutations of PDX lines to estimate required sample sizes (e.g., 10–15 PDX lines per group for 80% power) .
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Multi-omics integration: Combine proteomic (DsbB1 levels) and transcriptomic (redox gene expression) data to identify biomarkers of optimal folding.
What emerging tools (e.g., CRISPR, surface engineering) could advance DsbB1 research?
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CRISPR-Cas9 base editing: Precisely mutate dsbB1 to study active-site residues or enhance electron transfer efficiency .
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Surface proteomics: Use click chemistry to label surface-exposed DsbB1 and map its interactions with secreted proteins .
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In silico modeling: Predict DsbB1-substrate binding using molecular dynamics simulations to guide rational engineering.
