Recombinant Nocardia farcinica 1- (5-phosphoribosyl)-5-[ (5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase (hisA)

What are the optimal expression systems for recombinant Nocardia farcinica hisA protein?

The optimal expression system for recombinant N. farcinica hisA would be similar to those used for other N. farcinica proteins. Based on successful expression strategies for N. farcinica proteins, Escherichia coli BL21(DE3) cells have demonstrated effectiveness when using pET expression vectors. For instance, the recombinant expression of N. farcinica proteins like NFA49590 was achieved using pET30a(+) vectors transformed into E. coli BL21 by electroporation, with subsequent selection on LB agar plates containing kanamycin (50 μg/mL) . This system provides a good balance of protein yield and proper folding for functional studies of N. farcinica proteins.

How should I design primers for PCR amplification of the N. farcinica hisA gene?

When designing primers for PCR amplification of the N. farcinica hisA gene, consider the following methodological approach:

• Obtain the complete genomic sequence of the target gene from databases

• Design forward and reverse primers with appropriate restriction sites for directional cloning

• Include additional nucleotides (typically 3-4) at the 5' end of primers to facilitate restriction enzyme cutting

• Verify primer specificity using in silico PCR tools

• Optimize annealing temperatures and GC content

For example, in the case of NFA49590, researchers used forward primer 5'-ACATGAATTCATGGTCGAGGTCGACTGT-3' and reverse primer 5'-ACATAAGCTTTCAGCCGATGCTGAACGG-3', incorporating EcoRI and HindIII restriction sites for subsequent cloning into the expression vector . A similar approach would be applicable for hisA gene amplification.

What are the optimal induction conditions for maximizing soluble hisA protein expression?

For maximizing soluble recombinant N. farcinica hisA protein expression, optimization of several parameters is critical:

Research with other N. farcinica proteins showed that expression conditions with 0.2 mM IPTG induction at 16°C overnight were effective in producing soluble protein . Temperature optimization is particularly important, as studies have shown that protein expression increases with higher induction temperatures, but solubility may be compromised.

What is the most effective purification strategy for His-tagged recombinant N. farcinica hisA?

The most effective purification strategy for His-tagged recombinant N. farcinica hisA would involve:

• Cell lysis using sonication in appropriate buffer systems

• Centrifugation to separate soluble and insoluble fractions (12,000 rpm, 4°C, 20 min)

• Filtration of supernatant through a 0.45 μm filter

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns

• Gradient elution with increasing imidazole concentrations

• Buffer exchange to remove imidazole using dialysis or desalting columns

For poorly soluble proteins, inclusion body solubilization using 6 M urea followed by on-column refolding during purification has proven effective for other N. farcinica proteins . The purification protocol should be optimized based on the specific characteristics of the hisA protein, considering factors such as isoelectric point, molecular weight, and stability.

How can I verify the enzymatic activity of purified recombinant hisA?

Verification of enzymatic activity for recombinant N. farcinica hisA can be accomplished through:

• Spectrophotometric assays measuring the conversion of ProFAR to PRFAR

• Coupled enzyme assays that link hisA activity to detectable color changes

• Complementation assays using E. coli hisA mutants

• High-performance liquid chromatography (HPLC) to quantify substrate consumption and product formation

When establishing the assay, it is essential to optimize buffer composition, pH, temperature, and metal ion requirements. Additionally, kinetic parameters (Km, Vmax, kcat) should be determined to characterize the enzyme fully. Controls should include heat-inactivated enzyme and reactions lacking substrate to establish baseline values.

What are the challenges in maintaining stability of purified N. farcinica hisA and how can they be addressed?

Maintaining stability of purified N. farcinica hisA presents several challenges that can be addressed through systematic approaches:

Specific buffer formulations should be experimentally determined for hisA, as protein stability requirements may vary. Storage stability studies should be conducted to determine the optimal conditions for maintaining enzymatic activity over time.

How does the structure of N. farcinica hisA compare to homologous enzymes from other bacteria?

The structural comparison between N. farcinica hisA and homologous enzymes from other bacteria would typically reveal:

• Conservation of the (βα)8-barrel fold characteristic of phosphoribosyl isomerases

• Variations in loop regions that may influence substrate specificity

• Differences in surface charge distribution affecting protein-protein interactions

• Species-specific structural features that could be targeted for selective inhibition

Comparative structural analysis using homology modeling based on crystallized hisA proteins from other species, complemented by circular dichroism spectroscopy to verify secondary structure elements, would provide valuable insights into the unique features of N. farcinica hisA. This information could guide structure-based drug design efforts targeting N. farcinica infections.

What cellular signaling pathways are activated by N. farcinica proteins and how might this relate to hisA function?

N. farcinica proteins have been shown to activate several signaling pathways in host cells that could potentially relate to hisA function:

• MAPK signaling pathway activation through phosphorylation of ERK1/2, JNK, and p38

• NF-κB pathway activation through phosphorylation of p65

• Production of proinflammatory cytokines including TNF-α, IL-6, and IL-10

• TLR4-dependent signaling leading to immune response activation

While these pathways have been documented for NFA49590 and Nfa34810 proteins, the potential immunomodulatory effects of hisA remain to be investigated. The enzyme's role in histidine biosynthesis could indirectly affect these pathways through metabolic regulation, particularly in infection microenvironments where histidine availability may be limited.

What techniques are most effective for analyzing protein-protein interactions involving N. farcinica hisA?

Several complementary techniques can effectively analyze protein-protein interactions involving N. farcinica hisA:

• Pull-down assays using His-tagged hisA as bait followed by mass spectrometry

• Yeast two-hybrid screening to identify potential interaction partners

• Surface plasmon resonance (SPR) to determine binding kinetics and affinity

• Biolayer interferometry for real-time interaction analysis

• Co-immunoprecipitation followed by Western blotting for in vivo validation

• Microscale thermophoresis for quantitative interaction analysis in solution

How can I develop an immunoassay to detect antibodies against N. farcinica hisA in clinical samples?

Development of an immunoassay for detecting antibodies against N. farcinica hisA in clinical samples would involve:

• Production of highly purified recombinant hisA protein as the capture antigen

• Optimization of coating conditions (concentration, buffer, pH) for maximal antigen presentation

• Blocking to minimize non-specific binding

• Determination of appropriate dilution factors for clinical samples

• Selection of suitable detection antibodies and visualization systems

• Establishment of positive and negative controls

• Validation using known positive and negative clinical samples

Similar approaches have been successful for other N. farcinica proteins, where Western blot and ELISA methods were used to detect antibodies in sera from infected animals, with specificity confirmed using antisera from animals infected with related Nocardia species . This methodology allows for evaluation of both the antigenicity and specificity of the target protein.

What knockout or knockdown strategies are most effective for studying the role of hisA in N. farcinica pathogenesis?

For studying the role of hisA in N. farcinica pathogenesis, several genetic manipulation approaches can be considered:

• Homologous recombination-based gene deletion, similar to the strategy used for creating Δnfa34810 mutants

• CRISPR-Cas9 genome editing for precise modifications

• Antisense RNA strategies for conditional knockdown

• Transposon mutagenesis for random insertional inactivation

After creating knockout or knockdown strains, functional characterization should include:

• Growth curve analysis in defined media with and without histidine supplementation

• Invasion assays using cell lines such as HeLa or A549 cells

• Intracellular survival assessment in macrophages

• Virulence assessment in appropriate animal models

• Complementation studies to confirm phenotype specificity

These approaches would help determine whether hisA is essential for growth, survival, or virulence of N. farcinica in different environments and infection models.

How can recombinant N. farcinica hisA be used to develop potential vaccine candidates?

The development of vaccine candidates based on recombinant N. farcinica hisA would follow a systematic approach:

• In silico analysis to predict conserved epitopes across Nocardia strains

• Assessment of antigenicity using sera from infected animals or patients

• Evaluation of immune activation properties in vitro using macrophages or dendritic cells

• Determination of cytokine profiles induced by the protein

• Immunization studies in appropriate animal models

• Challenge experiments to assess protective efficacy

How do post-translational modifications affect the function and immunogenicity of N. farcinica hisA?

Post-translational modifications (PTMs) of N. farcinica hisA may significantly impact both function and immunogenicity:

Research methodologies should include mass spectrometry-based proteomic analysis to identify and characterize PTMs in native versus recombinant hisA. Comparison of E. coli-expressed hisA with protein purified from N. farcinica would be valuable for understanding the impact of prokaryotic versus native PTMs on protein function and immunogenicity.

What are the challenges in crystallizing N. farcinica hisA and how can they be overcome?

Crystallization of N. farcinica hisA presents several challenges that can be addressed through systematic approaches:

• Protein heterogeneity: Implement additional purification steps such as ion exchange and size exclusion chromatography to achieve monodisperse protein preparations

• Protein flexibility: Consider ligand binding to stabilize protein conformation or design truncated constructs removing flexible regions

• Buffer optimization: Screen extensive buffer conditions varying pH, ionic strength, and additives

• Crystallization techniques: Explore vapor diffusion, microbatch, and microfluidic approaches

• Crystal quality: Implement seeding techniques for improving crystal size and diffraction quality

• Construct engineering: Design surface entropy reduction mutations to promote crystal contacts

For challenging proteins, alternative structural approaches such as cryo-electron microscopy or small-angle X-ray scattering could provide valuable structural information even in the absence of crystals suitable for X-ray diffraction studies.

How can systems biology approaches be used to understand the role of hisA in N. farcinica metabolism and pathogenesis?

Systems biology approaches to understand the role of hisA in N. farcinica metabolism and pathogenesis would integrate:

• Transcriptomic profiling comparing wild-type and hisA mutant strains under various conditions

• Metabolomic analysis to identify changes in metabolite profiles associated with histidine biosynthesis

• Proteomic studies to identify interaction networks and expression changes

• Flux balance analysis to model metabolic adaptations in response to hisA perturbation

• Integration with host-response data to create host-pathogen interaction models

These approaches would help clarify how hisA contributes to N. farcinica fitness and virulence in different microenvironments encountered during infection. Of particular interest would be understanding metabolic adaptations that occur when histidine availability is limited, as might be the case in certain host tissues or during antibiotic treatment.

How does the enzymatic activity of N. farcinica hisA compare with homologous enzymes from other bacterial pathogens?

Comparative analysis of N. farcinica hisA with homologous enzymes from other bacterial pathogens would examine:

• Substrate specificity differences through detailed kinetic analysis

• Catalytic efficiency (kcat/Km) comparisons under standardized conditions

• pH and temperature optima variations reflecting adaptation to different niches

• Inhibitor sensitivity profiles relevant to potential drug development

• Allosteric regulation mechanisms that may differ between species

Such comparative studies are valuable for identifying unique features of N. farcinica hisA that might be exploited for selective targeting. The methodology would include parallel expression, purification, and characterization of hisA enzymes from multiple species using identical protocols to enable direct comparisons.

What evolutionary insights can be gained from comparing hisA sequences across different Nocardia species?

Evolutionary analysis of hisA sequences across different Nocardia species can provide several insights:

• Identification of conserved catalytic residues essential for function

• Detection of species-specific variations that may correlate with pathogenicity

• Evidence of selective pressure indicating functional importance

• Potential horizontal gene transfer events in the evolutionary history

• Correlation between sequence conservation and structural elements

Methodologically, this would involve multiple sequence alignment, phylogenetic tree construction, calculation of selection parameters (dN/dS ratios), and mapping of sequence conservation onto structural models. Similar approaches with other N. farcinica proteins have revealed species-specific variations that correlate with antigenic specificity , suggesting that such analysis of hisA could yield valuable insights into species-specific adaptations.

How do environmental factors affect the expression and activity of hisA in N. farcinica compared to other bacterial species?

The impact of environmental factors on hisA expression and activity in N. farcinica compared to other bacterial species could be assessed through:

• qRT-PCR analysis of hisA expression under varying conditions (pH, temperature, nutrient limitation)

• Reporter gene assays to monitor promoter activity in different environments

• Enzyme activity assays under varying conditions to establish environmental optima

• Protein stability assessments across different physiological conditions

• In vivo expression studies during infection using transcriptomics

This research would help understand how N. farcinica adapts its histidine biosynthesis pathway to different environments, particularly during infection. Research with other N. farcinica proteins has demonstrated that environmental conditions significantly impact protein expression and localization , suggesting that similar effects may be observed with hisA.