Recombinant Vibrio cholerae serotype O1 Probable oxaloacetate decarboxylase gamma chain 2 (oadG2)
Product Specs
Note: We will prioritize shipping the format that we have in stock. However, if you have specific format requirements, please indicate them when placing the order, and we will fulfill your needs.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please contact us in advance, as additional fees 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: vch:VC0794
STRING: 243277.VC0794
Q&A
What is the amino acid sequence and structural composition of oadG2?
The full-length Vibrio cholerae serotype O1 Probable oxaloacetate decarboxylase gamma chain 2 (oadG2) protein consists of 90 amino acids with the sequence: MQSTSLFLEGINLLTLGMGFVFIFLIFLVYATRAMSQLIVRFAPPEVPAKTTNKKASANKAKANPNQNQGELLAVLTAAVHHHKTQQKLS. This transmembrane protein contains hydrophobic regions that contribute to its membrane-anchoring properties, which are essential for the proper assembly and function of the oxaloacetate decarboxylase complex .
What are the optimal storage conditions for maintaining oadG2 protein stability?
Recombinant oadG2 protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid degradation. Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they significantly reduce protein stability and activity. The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain structural integrity during storage .
How does oadG2 differ from other components of the oxaloacetate decarboxylase complex?
The oadG2 protein functions as the gamma chain component of the oxaloacetate decarboxylase complex in Vibrio cholerae. Unlike the alpha and beta chains that contain catalytic domains, the gamma chain (oadG2) serves primarily as a membrane anchor for the complex. This 90-amino acid protein contains hydrophobic regions that facilitate membrane integration, distinguishing it from the larger, catalytically active subunits of the complex.
What expression systems are most effective for recombinant oadG2 production?
For optimal expression of recombinant oadG2, E. coli-based expression systems have proven most effective. The recombinant protein referenced in the literature was successfully expressed with an N-terminal His-tag in E. coli, allowing for efficient purification while maintaining protein functionality . When designing expression constructs, researchers should consider that:
| Expression System | Advantages | Considerations |
|---|---|---|
| E. coli BL21(DE3) | High yield, rapid growth | May require optimization of induction conditions |
| E. coli Rosetta™ | Enhanced expression of proteins with rare codons | Higher cost but improved expression for proteins with rare codons |
| Cell-free systems | Avoids toxicity issues | Lower yield but useful for difficult-to-express proteins |
What purification protocol is recommended for His-tagged oadG2?
Purification of His-tagged oadG2 can be achieved using standard immobilized metal affinity chromatography (IMAC) protocols. After cell lysis, the clarified lysate should be applied to Ni-NTA or similar metal affinity resin, followed by washing with increasing concentrations of imidazole to remove non-specifically bound proteins. The protein typically elutes at imidazole concentrations between 250-300 mM. Post-purification, dialysis into a Tris/PBS-based storage buffer with 6% Trehalose at pH 8.0 is recommended to ensure stability .
How should researchers reconstitute lyophilized oadG2 protein for experimental use?
For reconstitution of lyophilized oadG2 protein, briefly centrifuge the vial prior to opening to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of 5-50% glycerol (with 50% being standard) is recommended for long-term storage at -20°C/-80°C. After reconstitution, the solution should be gently mixed to ensure complete solubilization while avoiding protein denaturation through excessive agitation .
How can oadG2 be used to study horizontal gene transfer in Vibrio cholerae?
Researchers can leverage oadG2 as a marker for studying horizontal gene transfer (HGT) in Vibrio cholerae by integrating antibiotic resistance cassettes (such as aph, conferring kanamycin resistance) within or adjacent to the oadG2 gene. This approach enables the tracking of gene transfer events between different V. cholerae strains. In experimental setups, co-culturing potential donor and recipient strains on chitinous surfaces (which induce natural competence) for approximately 30 hours allows for transformation events to occur. Subsequent selection on kanamycin-containing media and whole genome sequencing of transformants can reveal the extent and patterns of horizontal gene transfer .
What techniques are effective for studying oadG2 function in the context of Vibrio cholerae pathogenicity?
To investigate oadG2's potential role in Vibrio cholerae pathogenicity, researchers can employ gene deletion methods based on:
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Counter-selectable suicide plasmids (e.g., pGP704-Sac28)
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Natural transformation combined with FLP recombination (TransFLP method)
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Direct insertion of antibiotic resistance cassettes (aph, cat, or bla) into the target gene
These approaches allow for precise genetic manipulation to create oadG2 knockout strains, which can then be compared to wild-type strains in various virulence assays. Complementation studies, where the deleted gene is reintroduced either on a plasmid or at a neutral chromosomal site, should be performed to confirm that observed phenotypes are specifically due to oadG2 deletion rather than polar effects or secondary mutations .
How can contradiction detection methods be applied to genomic studies involving oadG2?
When studying genomic aspects of oadG2 across different Vibrio cholerae strains, researchers may encounter contradicting information in the scientific literature or genomic databases. To address this, systematic contradiction detection approaches can be employed:
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Self-contradictory analysis: Examine single documents or datasets for internally inconsistent information about oadG2 structure or function
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Contradicting document pairs analysis: Identify and resolve conflicts between different published sources regarding oadG2
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Conditional contradictions analysis: Investigate complex scenarios where information in one source creates contradictions between other sources
These methods are particularly important when integrating information from multiple genomic databases or published studies, as they help identify and resolve conflicting data to ensure research accuracy .
What are common issues in recombinant oadG2 expression and how can they be addressed?
Recombinant expression of membrane proteins like oadG2 often presents challenges including:
| Challenge | Potential Solution | Implementation Notes |
|---|---|---|
| Low expression yield | Optimize codon usage for E. coli | Consider strain-specific codon bias |
| Protein aggregation | Express at lower temperatures (16-20°C) | Reduce induction concentration and extend expression time |
| Toxicity to host cells | Use tightly regulated expression systems | Consider IPTG-inducible systems with minimal leaky expression |
| Poor solubility | Include solubilizing agents/detergents | Try various detergents at concentrations above CMC |
For oadG2 specifically, expression as a fusion protein with tags that enhance solubility (e.g., MBP, SUMO) in addition to the His-tag may improve yield and solubility of the recombinant protein .
How can researchers verify the structural integrity of purified oadG2?
Verification of oadG2 structural integrity is essential before functional studies. SDS-PAGE analysis can confirm the expected molecular weight and purity (>90% is typically considered acceptable for most applications) . For more detailed structural analysis, circular dichroism (CD) spectroscopy can assess secondary structure content, particularly important for confirming proper folding of transmembrane regions. Mass spectrometry techniques can verify the exact mass and detect any post-translational modifications or truncations that might affect function.
What controls should be included in experiments investigating oadG2 function?
Robust experimental design for oadG2 functional studies should include:
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Positive controls: Wild-type V. cholerae strains expressing native oadG2
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Negative controls: oadG2 deletion mutants (ΔoadG2)
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Complementation controls: ΔoadG2 strains with oadG2 reintroduced on a plasmid or at a neutral chromosomal location
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Technical controls: Heat-inactivated protein samples to distinguish between enzymatic and non-enzymatic effects
For studies examining oadG2's role in horizontal gene transfer, using transformation-deficient strains (e.g., comEC mutants) as controls can help distinguish between transformation-dependent and transformation-independent effects .
How is oadG2 being used in current approaches to study Vibrio cholerae pathogenicity islands?
Current research is exploring oadG2's potential associations with Vibrio pathogenicity islands (VPIs). The protein may serve as a marker for studying the transfer of pathogenicity islands between Vibrio strains. Researchers are investigating whether oadG2 or its genetic locus contributes to the transmission dynamics of VPI-1, VPI-2, VSP-I, VSP-II, or the cholera toxin prophage CTX. By tracking oadG2 alongside these mobile genetic elements during horizontal gene transfer experiments, insights into co-transfer patterns and genetic linkage can be gained .
What role might oadG2 play in adaptation to environmental conditions?
The oxaloacetate decarboxylase complex, of which oadG2 is a component, plays roles in bacterial metabolism that may contribute to environmental adaptation. Future research directions include investigating how oadG2 expression and function change under various environmental conditions relevant to V. cholerae's lifecycle, such as:
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Marine vs. host environments
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Varying salinity and pH conditions
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Presence of specific carbon sources
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Biofilm vs. planktonic growth states
Understanding these adaptations could provide insights into V. cholerae's environmental persistence and transition between environmental reservoirs and human hosts.
How can genomic analysis methods be applied to study oadG2 variation across Vibrio strains?
Advanced genomic analysis methods can reveal important variations in oadG2 across different Vibrio cholerae strains. When analyzing whole genome sequencing data, researchers should be aware of challenges such as rRNA operon assembly errors that have led to underestimation of rRNA clusters in reference genomes. Similarly, regions with high SNP frequency (approximately 1 in 55 nucleotides for conserved genes between some strains) require careful analysis. Long-read sequencing technologies like PacBio can provide more accurate assemblies of repetitive regions, while Sanger sequencing remains valuable for confirming specific mutations or polymorphisms in the oadG2 gene .
