Recombinant Brucella melitensis biotype 2 NADH-quinone oxidoreductase subunit K (nuoK)
Product Specs
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Note: All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. For the lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
KEGG: bmi:BMEA_A0854
Q&A
How does Brucella melitensis nuoK differ from nuoK proteins in other bacterial species?
The nuoK protein from Brucella melitensis biotype 2 shares significant sequence homology with other members of the alpha-proteobacteria group, particularly within the Brucellaceae family. When compared to the nuoK protein from Brucella ovis (strain ATCC 25840), there is approximately 98% sequence identity, with only minimal variations in non-critical residues .
Both proteins share identical functional domains and a highly conserved core sequence:
| Species | UniProt ID | Sequence Length | Notable Differences |
|---|---|---|---|
| B. melitensis biotype 2 | C0RIE9 | 102 aa | Reference sequence |
| B. ovis (ATCC 25840) | A5VPZ3 | 102 aa | >98% identity to reference |
The high conservation of nuoK across Brucella species suggests its essential role in respiratory chain function and potential as a target for comparative studies in bacterial metabolism and pathogenesis mechanisms.
What expression systems are optimal for producing functional Recombinant Brucella melitensis nuoK protein?
E. coli remains the predominant expression system for recombinant Brucella melitensis nuoK protein production due to its efficiency and scalability. The protein is typically expressed as a His-tagged fusion to facilitate purification using nickel affinity chromatography .
For optimal expression of functional nuoK, researchers should consider the following methodological approaches:
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Vector selection: pET-based expression systems provide high-yield expression under the control of T7 promoters
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E. coli strain selection: BL21(DE3) derivatives are preferred due to reduced protease activity
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Induction parameters: IPTG concentration of 0.5-1.0 mM at mid-log phase (OD600 of 0.6-0.8)
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Expression temperature: Reduced temperature (16-20°C) post-induction can improve protein folding
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Purification approach: Stepwise elution with imidazole from nickel nitrilotriacetate columns under non-denaturing conditions preserves functional integrity
For membrane proteins like nuoK, inclusion of solubilizing agents such as mild detergents during purification is essential for maintaining proper folding and function.
How can researchers analyze the functional activity of recombinant nuoK in experimental settings?
Analysis of nuoK functional activity presents unique challenges due to its role as a subunit of the larger NADH-quinone oxidoreductase complex. Methodological approaches should incorporate:
| Parameter | Wild-type homodimer | Mutant/wild-type heterodimer | Significance |
|---|---|---|---|
| Km(NADPH) | Baseline value | Similar to wild-type with 2e⁻ acceptors | Independent subunit function |
| kcat(NADPH) | 100% | ~50% with 2e⁻ acceptors | Partial activity retention |
| Km(NADH) | Baseline value | Similar to wild-type with 2e⁻ acceptors | Preserved cofactor binding |
| Activity with 4e⁻ acceptors | 100% | Similar to mutant homodimer | Dependent subunit function |
These methodologies enable comprehensive characterization of nuoK's contribution to respiratory function and electron transport.
What are the challenges and solutions for improving stability of purified recombinant nuoK protein?
Stability challenges with nuoK stem from its hydrophobic nature and membrane association. Advanced research approaches to address these include:
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Optimized storage conditions: The protein shows greatest stability when stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . For long-term storage, addition of 50% glycerol and storage at -20°C/-80°C is recommended to prevent activity loss.
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Preventing aggregation: Regular aliquoting prevents repeated freeze-thaw cycles, which can cause protein aggregation and activity loss. Working aliquots should be stored at 4°C for no longer than one week .
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Reconstitution protocols: For lyophilized protein preparations, reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL . This concentration range balances solubility with functional activity.
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Detergent screening: Systematic evaluation of detergent types and concentrations can identify optimal conditions for maintaining nuoK stability without disrupting functional domains.
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Fusion partner strategies: Alternative tag systems beyond the standard His-tag may improve solubility and stability profiles, though these must be evaluated for impact on functional activity.
Researchers reporting purified preparations with >90% purity (as determined by SDS-PAGE) have achieved optimal stability using these methodological refinements .
What purification strategies yield highest purity and activity for recombinant nuoK protein?
A multi-step purification protocol optimized for membrane proteins like nuoK typically includes:
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Initial capture: Immobilized metal affinity chromatography (IMAC) using nickel nitrilotriacetate resin is the primary capture step for His-tagged nuoK, with stepwise imidazole elution (20-250 mM) to separate heterodimers from homodimers .
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Secondary purification: Size exclusion chromatography separates oligomeric forms and removes aggregates.
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Quality assessment: Purity >90% by SDS-PAGE is the standard threshold for experimental applications . Both denaturing and non-denaturing PAGE techniques should be employed to confirm oligomeric state.
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Activity preservation: Throughout purification, inclusion of stabilizing agents (trehalose 6%, glycerol 50%) maintains functional integrity .
Researchers have successfully applied this methodology to related NADH-quinone oxidoreductase complexes, demonstrating protein composition verification through immunoblot analysis following both SDS and non-denaturing polyacrylamide gel electrophoresis .
How can researchers effectively design structure-function studies for nuoK using site-directed mutagenesis?
Structure-function analysis of nuoK requires systematic mutagenesis approaches:
This methodological framework provides a comprehensive approach to deciphering structure-function relationships in nuoK and its contribution to the NADH-quinone oxidoreductase complex.
How can recombinant nuoK protein be utilized in developing diagnostic tools for Brucella detection?
The application of recombinant nuoK in diagnostics builds upon established methodologies for recombinant protein antigens:
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Serological test development: Similar to approaches used for other bacterial pathogens, nuoK can serve as a target antigen in ELISA-based detection systems . The high conservation across Brucella species makes it a potentially valuable diagnostic target.
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Specificity assessment: Comparative studies with nuoK proteins from different Brucella species (e.g., B. melitensis vs B. ovis) can determine cross-reactivity profiles and establish species-specific detection parameters .
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Sensitivity optimization: The use of His-tagged full-length nuoK enables standardized purification and quantification, ensuring consistent diagnostic performance .
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Validation methodology: Following the approach used for other recombinant antigens, researchers should verify immunoreactivity using:
This research direction represents a promising avenue for expanding the diagnostic toolkit for Brucella infections, potentially enabling improved species-specific detection.
What research gaps remain in understanding nuoK's role in Brucella pathogenesis and metabolism?
Critical research gaps that warrant investigation include:
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Structural characterization: Unlike other NADH-quinone oxidoreductase components, detailed structural information for nuoK remains limited. X-ray crystallography or cryo-EM studies of the complete complex would provide valuable insights into subunit interactions.
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Host-pathogen interactions: The potential role of nuoK in Brucella virulence and intracellular survival has not been fully characterized. Investigation of nuoK mutants in infection models could elucidate its contribution to pathogenesis.
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Metabolic adaptation: How nuoK function may be modulated under different environmental conditions (pH, oxygen limitation, nutrient restriction) remains unclear. Studies examining expression and activity under host-relevant conditions would address this gap.
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Comparative analysis across species: While sequence homology between B. melitensis and B. ovis nuoK is high , functional comparisons across species with different host preferences and virulence profiles could reveal adaptations relevant to pathogenesis.
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Protein-protein interaction network: A comprehensive interactome analysis would clarify nuoK's interactions beyond the NADH-quinone oxidoreductase complex and potentially reveal novel functional roles.
Addressing these research gaps would significantly advance understanding of respiratory metabolism in Brucella and potentially identify new targets for therapeutic intervention.
