Recombinant Coxiella burnetii Elongation factor G (fusA), partial

What is Recombinant Coxiella burnetii Elongation factor G (fusA)?

Recombinant Coxiella burnetii Elongation factor G (fusA) is a protein derived from the genome of Coxiella burnetii, a gram-negative intracellular pathogen responsible for Q fever. The "recombinant" designation indicates that the protein has been artificially produced using molecular biology techniques rather than extracted directly from the pathogen. The specific product referenced is a partial recombinant protein derived from the Coxiella burnetii strain Dugway 5J108-111 . Elongation factor G functions as a critical component in bacterial protein synthesis machinery, specifically during the translocation phase of translation.

Unlike virulence factors such as AnkF that directly interact with host cells, Elongation factor G serves a fundamental role in the bacterium's core cellular processes. While less studied than some virulence factors, this protein represents an important target for understanding basic Coxiella biology and potentially for therapeutic intervention.

What is the functional significance of Elongation factor G in Coxiella burnetii?

Elongation factor G (EF-G) plays an essential role in protein synthesis in Coxiella burnetii, mirroring its function in other bacteria. During the elongation phase of translation, EF-G catalyzes the GTP-dependent translocation of peptidyl-tRNA and mRNA by one codon on the ribosome. This critical step enables the sequential addition of amino acids to the growing peptide chain.

In Coxiella burnetii, an obligate intracellular pathogen that replicates within an acidic phagolysosomal vacuole (the Coxiella burnetii-containing vacuole or CCV), protein synthesis machinery is particularly important for adaptation to the hostile intracellular environment . The bacterium must efficiently translate proteins required for survival and replication within host cells, making translation factors like EF-G essential for pathogenesis.

How is Recombinant Coxiella burnetii Elongation factor G (fusA) produced?

The production of Recombinant Coxiella burnetii Elongation factor G (fusA) employs a baculovirus expression system as indicated in the product specifications . This expression platform offers several advantages for producing complex bacterial proteins, including proper folding and potential post-translational modifications.

The production process typically follows these methodological steps:

• Gene isolation and cloning: The fusA gene sequence is amplified from Coxiella burnetii genomic DNA and inserted into a baculovirus transfer vector containing appropriate regulatory elements.

• Recombinant baculovirus generation: Insect cells are co-transfected with the transfer vector and baculovirus DNA, generating recombinant viral particles expressing the fusA gene.

• Viral amplification: The recombinant baculoviruses are propagated in insect cell culture to achieve high viral titers.

• Protein expression: Large-scale insect cell cultures are infected with the recombinant baculovirus, leading to expression of the target protein.

• Protein purification: The expressed protein is extracted and purified through chromatographic techniques, potentially utilizing affinity tags incorporated during the cloning process.

The final product achieves >85% purity as verified by SDS-PAGE analysis , making it suitable for most research applications.

What are the optimal storage conditions for Recombinant Coxiella burnetii Elongation factor G (fusA)?

Proper storage is critical for maintaining the structural integrity and biological activity of Recombinant Coxiella burnetii Elongation factor G (fusA). According to product specifications, researchers should adhere to the following evidence-based storage protocol:

• Short-term storage (up to one week): Working aliquots can be maintained at 4°C .

• Standard storage: Store at -20°C for routine research applications .

• Extended storage: For long-term preservation, -20°C or preferably -80°C is recommended .

It is critically important to avoid repeated freeze-thaw cycles, as these can lead to protein denaturation, aggregation, and loss of functional activity . This sensitivity to freeze-thaw stress is common among complex proteins with multiple domains, such as elongation factors. To mitigate this risk, researchers should:

• Prepare small working aliquots during initial reconstitution

• Thaw aliquots on ice when needed for experiments

• Avoid rapid temperature changes that could disrupt protein structure

• Consider adding stabilizing agents such as glycerol if extended room-temperature work is required

What methodologies are available for purifying Recombinant Coxiella burnetii Elongation factor G (fusA)?

The purification of Recombinant Coxiella burnetii Elongation factor G (fusA) requires a strategic approach based on the protein's biochemical properties and the expression system employed. For the baculovirus-expressed product referenced in the search results , several complementary purification techniques are applicable:

• Affinity Chromatography:

  • While the specific tag type is determined during manufacturing , common affinity tags include His-tag, GST-tag, or FLAG-tag

  • Tag-specific resins enable selective capture of the fusion protein

  • Elution conditions must be optimized to maintain protein structure and activity

• Ion Exchange Chromatography:

  • Exploits the unique charge distribution of fusA

  • Anion exchange (e.g., Q-Sepharose) or cation exchange (e.g., SP-Sepharose) can be employed depending on the protein's isoelectric point

  • Salt gradient elution allows separation from contaminants with different charge properties

• Size Exclusion Chromatography:

  • Provides further purification based on molecular size

  • Effective for removing aggregates and degradation products

  • Also enables buffer exchange into optimal storage conditions

The multi-step purification process typically achieves >85% purity as verified by SDS-PAGE . Researchers should carefully consider the impact of each purification step on protein activity, as some methods may affect the native conformation of this complex translation factor.

How can the purity of Recombinant Coxiella burnetii Elongation factor G (fusA) be verified?

Verifying the purity of Recombinant Coxiella burnetii Elongation factor G (fusA) is essential for ensuring experimental reproducibility and valid research outcomes. Multiple analytical techniques should be employed for comprehensive purity assessment:

• SDS-PAGE Analysis:

  • Standard method referenced in the product specifications showing >85% purity

  • Enables visualization of the target protein band and contaminating proteins

  • Densitometric analysis of Coomassie-stained gels provides quantitative purity estimation

• Western Blot Analysis:

  • Confirms protein identity using antibodies specific to fusA or to affinity tags

  • Reveals potential degradation products or truncated forms of the protein

  • Higher sensitivity than Coomassie staining for detecting minor contaminants

• Mass Spectrometry:

  • Provides precise molecular weight determination and sequence coverage

  • Can identify post-translational modifications and sequence variants

  • MALDI-TOF or LC-MS/MS approaches offer complementary information

• Functional Assays:

  • GTPase activity assays confirm biochemical functionality

  • Ribosome binding assays verify structural integrity of functional domains

  • Results should be compared to established reference standards

For most research applications, a combination of SDS-PAGE analysis and at least one orthogonal method is recommended to ensure both purity and identity confirmation before proceeding with experiments.

How does Recombinant Coxiella burnetii Elongation factor G (fusA) compare to other Coxiella burnetii proteins in terms of functionality?

Recombinant Coxiella burnetii Elongation factor G (fusA) serves a fundamentally different role than many of the more extensively studied Coxiella proteins involved in host-pathogen interactions. This distinction has important implications for research applications and potential therapeutic targeting.

Unlike the effector protein AnkF, which is translocated into host cells via the Type IV Secretion System (T4SS) and directly interacts with host vimentin to establish the replicative CCV , fusA functions strictly within the bacterial cytoplasm. The research by Frontiers in Cellular and Infection Microbiology demonstrated that AnkF knockout mutants were severely impaired in intracellular replication despite normal invasion capabilities . This contrasts with fusA, which affects bacterial survival more broadly as part of the essential translation machinery.

Genomic analysis of Coxiella burnetii reveals that proteins like fusA are highly conserved across strains, whereas virulence factors show greater variability. This conservation reflects the essential nature of translation factors compared to the more adaptable virulence mechanisms .

Can Recombinant Coxiella burnetii Elongation factor G (fusA) be used in vaccine development research?

The potential utility of Recombinant Coxiella burnetii Elongation factor G (fusA) in vaccine development presents a complex research question requiring examination of both theoretical considerations and empirical evidence.

Previous immunization experiments with recombinant Coxiella burnetii proteins have yielded mixed results. A systematic study examining eight recombinant proteins (Omp, Pmm, HspB, Fbp, Orf410, Crc, CbMip, and MucZ) administered as a mixture failed to induce protective immunity in a murine challenge model . Only animals vaccinated with the licensed Q-VaxTM vaccine demonstrated milder symptoms and reduced pathology .

Several factors influence the vaccine potential of fusA:

• Antigenicity considerations:

  • As a cytoplasmic protein, fusA may not be naturally exposed to the host immune system during infection

  • Unlike membrane proteins that showed significant mutations in outbreak strains , fusA is likely more conserved

  • The partial recombinant form may not present the full complement of epitopes

• Methodological approaches for evaluation:

  • Expression as a his-tagged fusion protein enables purification but may affect immunogenicity

  • Adjuvant selection critically influences immune response quality

  • Challenge studies must assess multiple parameters (bacterial burden, pathology, symptoms)

• Potential applications:

  • Component in multi-subunit vaccines alongside surface antigens

  • Diagnostic marker to distinguish vaccinated from infected individuals

  • Tool for studying immunodominant epitopes in Coxiella

While fusA alone may not be ideal as a vaccine antigen due to its cytoplasmic localization, incorporating it in comprehensive antigen screening studies remains valuable for developing next-generation Q fever vaccines with improved safety and efficacy profiles.

What are common challenges in working with Recombinant Coxiella burnetii Elongation factor G (fusA) and how can they be addressed?

Researchers working with Recombinant Coxiella burnetii Elongation factor G (fusA) may encounter several technical challenges that require methodological solutions to ensure experimental success. Based on the protein's characteristics and general recombinant protein properties, the following issues and solutions should be considered:

• Stability concerns:

  • Challenge: The product documentation explicitly warns against repeated freeze-thaw cycles , indicating potential stability issues.

  • Solution: Prepare small single-use aliquots during initial reconstitution and strictly maintain the cold chain during handling. Consider adding stabilizing agents such as glycerol (10-20%) for preparations requiring extended handling times.

• Reconstitution difficulties:

  • Challenge: Incomplete solubilization leading to protein aggregation or loss of activity.

  • Solution: Follow the recommended protocol of brief centrifugation before opening, and reconstitution in deionized sterile water to 0.1-1.0 mg/mL . Allow sufficient time for complete dissolution, avoiding vigorous vortexing that may denature the protein.

• Activity verification:

  • Challenge: Confirming functional activity of the recombinant protein.

  • Solution: Implement GTP hydrolysis assays using purified ribosomes to verify translocation activity. Ribosome-binding assays using analytical ultracentrifugation or surface plasmon resonance provide additional functional confirmation.

• Tag interference:

  • Challenge: The manufacturing process determines tag type , which may interfere with certain applications.

  • Solution: If tag interference is suspected, consider tag removal using appropriate proteases (e.g., TEV protease for His-tagged proteins) followed by additional purification steps.

• Experimental controls:

  • Challenge: Distinguishing specific effects from artifacts.

  • Solution: Include appropriate negative controls (buffer only, irrelevant protein) and positive controls (commercial E. coli EF-G) in all experiments to validate results.

How can expression of Recombinant Coxiella burnetii Elongation factor G (fusA) be optimized?

Optimizing the expression of Recombinant Coxiella burnetii Elongation factor G (fusA) requires a systematic approach addressing multiple parameters of the baculovirus expression system indicated in the product specifications . The following evidence-based strategies can enhance expression yield and protein quality:

• Baculovirus optimization:

  • Multiplicity of infection (MOI): Titrate between 0.1-10 to identify optimal viral load balancing protein expression and cell viability

  • Harvest timing: Conduct time-course experiments (typically 48-96 hours post-infection) to determine peak expression before cytopathic effects compromise quality

  • Viral passage: Minimize serial passages of baculovirus stocks to prevent genetic instability

• Insect cell culture parameters:

  • Cell line selection: Compare expression in Sf9, Sf21, and High Five™ cells, with the latter often yielding higher expression of complex proteins

  • Culture medium: Evaluate serum-free formulations optimized for high-density growth

  • Temperature: Lowering incubation temperature to 27°C may enhance proper folding of complex proteins

• Sequence optimization:

  • Codon optimization: Adjust codon usage to match insect cell preference while preserving critical structural elements

  • Signal sequence: Consider adding an insect-specific secretion signal for improved processing

  • Fusion partners: Evaluate solubility-enhancing tags like MBP or SUMO

• Post-translational considerations:

  • Add protease inhibitors during extraction to prevent degradation

  • Optimize lysis conditions to maximize recovery of soluble protein

  • Consider mild detergents if membrane association occurs

Drawing from the approach used for other recombinant Coxiella proteins expressed as his-tagged fusion proteins , a parallel optimization in bacterial systems might provide an alternative production platform if baculovirus yields are suboptimal.

What are the best approaches for reconstitution of Recombinant Coxiella burnetii Elongation factor G (fusA)?

Proper reconstitution of Recombinant Coxiella burnetii Elongation factor G (fusA) is critical for maintaining structural integrity and functional activity. The product documentation recommends reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, preceded by brief centrifugation of the vial . To ensure optimal results, researchers should implement the following detailed protocol:

• Pre-reconstitution preparation:

  • Equilibrate the lyophilized protein to room temperature (15-20 minutes) while keeping the vial closed

  • Centrifuge at 10,000 × g for 1 minute to collect all material at the bottom of the vial

  • Prepare sterile materials and work in a laminar flow hood if possible

• Reconstitution procedure:

  • Add the calculated volume of ice-cold deionized sterile water slowly down the side of the vial

  • Gently rotate or invert the vial to dissolve the protein completely (avoid vortexing)

  • Allow reconstitution to proceed for 10-15 minutes on ice

  • Inspect visually for complete dissolution and absence of particulates

• Post-reconstitution handling:

  • Prepare single-use aliquots in low-binding microcentrifuge tubes

  • Flash-freeze aliquots in liquid nitrogen if storing at -80°C

  • Document the reconstitution date, concentration, and storage conditions

• Buffer considerations:

  • For applications requiring specific buffers, consider reconstituting at 2× the final concentration and diluting with 2× buffer

  • Compatible buffers typically include PBS, TBS, or HEPES-based formulations at physiological pH

  • Verify protein stability in the chosen buffer through activity assays before proceeding with experiments

Implementing this methodical approach will maximize protein recovery and maintain the functional integrity of the recombinant fusA protein, ensuring reliable and reproducible experimental results.