Recombinant Haemophilus somnus GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the optimal expression system for recombinant H. somnus guaA production?

Based on comparative studies with other H. somnus recombinant proteins, the E. coli C41(DE3) strain has demonstrated superior results for membrane-associated and potentially toxic proteins from H. somnus. This strain is specifically designed for expressing toxic transmembrane proteins that might otherwise inhibit cell growth in conventional expression systems . When expressing H. somnus guaA, consider the following optimization strategies:

• Autoinduction system: This approach has shown the highest yield for H. somnus recombinant proteins compared to IPTG induction, likely because it eliminates the need to monitor culture density and manually add inducers .

• Temperature considerations: Extended expression periods (16h) at lower temperatures (room temperature rather than 37°C) may increase the proportion of soluble protein .

• Cell fraction analysis: Check both soluble and insoluble cytoplasmic fractions, as H. somnus recombinant proteins have been successfully purified from both compartments .

What cloning strategies are most effective for H. somnus guaA gene amplification?

For successful cloning of H. somnus guaA, the following approach has proven effective with similar H. somnus genes:

• Primer design: Design specific primers that incorporate restriction enzyme sites (such as NcoI and XhoI) for directional cloning into expression vectors .

• PCR optimization: Include 2% DMSO in PCR reactions to improve amplification of GC-rich regions in H. somnus genes .

• PCR parameters: Use a touchdown PCR protocol with initial denaturation at 94°C for 10 minutes, followed by 30 cycles consisting of 15 seconds at 94°C, 30 seconds annealing at 52°C, and 90 seconds extension at 68°C, with a final extension of 10 minutes at 68°C .

• Vector selection: The pET expression system (particularly pET22b+) has been successfully used for H. somnus recombinant proteins, providing tight regulation and high-level expression .

How can I optimize buffer conditions to improve H. somnus guaA solubility?

H. somnus recombinant proteins often exhibit solubility challenges, particularly those with membrane associations. Based on research with other H. somnus proteins:

• Addition of 10% glycerol to purification buffers significantly improves the solubility of recombinant H. somnus proteins .

• Detergents may be necessary if the protein remains insoluble, with mild non-ionic detergents (0.1% Triton X-100) being a good starting point.

• For guaA specifically, phosphate buffers (50mM) with moderate salt concentration (150-300mM NaCl) at pH 7.5-8.0 typically provide a good environment for maintaining enzyme activity.

• Inclusion of reducing agents (1-5mM DTT or β-mercaptoethanol) can prevent unwanted disulfide bond formation and improve stability.

How do H. somnus guaA expression levels compare between cytoplasmic and periplasmic targeting strategies?

Based on studies with other H. somnus recombinant proteins:

The periplasmic targeting strategy using the pET system with a signal sequence has shown limited success with H. somnus proteins. Despite theoretical advantages for proper folding in the oxidizing environment of the periplasm, experimental data with H. somnus OMP40 showed that:

• The majority of the recombinant protein was found in both soluble and insoluble cytoplasmic fractions despite periplasmic targeting .

• The periplasmic fraction contained only minimal amounts of the recombinant protein .

• The insoluble cytoplasmic fraction (inclusion bodies) often contained the highest yield, particularly when using the autoinduction process .

For guaA expression specifically, cytoplasmic expression would likely be more efficient, as enzymatic proteins typically fold properly in the cytoplasm, especially when expressed at lower temperatures.

What analytical methods are most reliable for assessing the purity and activity of recombinant H. somnus guaA?

A comprehensive analytical approach should include:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (expect >95% purity after optimized purification)

  • Western blotting using anti-His antibodies if a His-tag is incorporated

  • Size exclusion chromatography to confirm monodispersity and absence of aggregates

• Activity assessment:

  • Spectrophotometric assay measuring the conversion of XMP to GMP with glutamine as the amino group donor

  • Coupled enzyme assays monitoring either glutamate production or NADPH oxidation

  • Isothermal titration calorimetry (ITC) to determine binding constants for substrates

• Structural integrity:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Thermal shift assays to assess protein stability under various buffer conditions

What are the sequence homology considerations when working with H. somnus guaA?

When working with H. somnus guaA, sequence homology considerations are critical for experimental design and interpretation:

• H. somnus proteins often show high sequence similarity with homologous proteins in other gram-negative bacteria, particularly within the Pasteurellaceae family .

• Based on studies with other H. somnus proteins, you can expect ~70-90% similarity with homologous proteins from related species. For example, the 15K ribosomal protein from H. somnus shows 89% similarity to the E. coli ribosomal protein S9 .

• This high sequence conservation may enable functional complementation approaches, where the H. somnus guaA gene could potentially complement E. coli guaA mutations, providing a functional assay system .

• When designing antibodies or performing immunological studies, consider potential cross-reactivity with homologous proteins from other bacterial species .

How do single amino acid substitutions in the glutamine amidotransferase domain affect the catalytic efficiency of H. somnus guaA?

The glutamine amidotransferase (GAT) domain of H. somnus guaA contains the catalytic triad essential for glutamine hydrolysis and subsequent transfer of the amino group to XMP. Based on structural and functional studies of GMP synthases from other bacterial species:

• Catalytic triad mutations:

  • Cys-His-Glu residues form the catalytic triad in the GAT domain

  • Substitution of the catalytic cysteine with serine typically reduces activity by >95%

  • Conservative substitutions in the His residue (H→N) generally result in 80-90% reduction in glutaminase activity while maintaining some synthase activity with ammonia as substrate

• Substrate binding pocket mutations:

  • Mutations in glutamine-binding residues often lead to higher Km values without necessarily affecting kcat

  • Experimental data from similar enzymes suggest that residues located 4-8Å from the glutamine binding site can affect substrate channeling between domains

• Interdomain communication residues:

  • Key residues at the interface between GAT and synthase domains regulate allosteric communication

  • Point mutations in these regions may produce enzymes that can utilize ammonia but not glutamine as the amino donor

When designing mutagenesis experiments, consider using a complementation system similar to that described for other H. somnus proteins, where the recombinant protein is tested for its ability to complement corresponding E. coli mutations .

How can structural biology approaches be applied to resolve mechanistic questions about H. somnus guaA function?

Advanced structural biology approaches can provide critical insights into H. somnus guaA function:

• X-ray crystallography:

  • Co-crystallization with substrates, products, or substrate analogs can capture different catalytic states

  • Resolution of 2.0Å or better is typically required to visualize the precise positioning of catalytic residues

  • Consider designing truncated constructs (separate domains) if full-length protein crystallization proves challenging

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful if guaA forms higher-order complexes or if crystallization proves difficult

  • Can reveal dynamic conformational changes during catalysis not captured by crystallography

  • Recent advances allow near-atomic resolution for proteins >150 kDa

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Can identify regions of conformational change upon substrate binding

  • Useful for mapping allosteric communication pathways between domains

  • Requires approximately 50-100 μg of purified protein per experiment

• Molecular dynamics simulations:

  • Can model substrate channeling between domains

  • Useful for predicting effects of mutations before experimental validation

  • Combine with experimental data (HDX-MS, SAXS) for validated models

What strategies can address inconsistent kinetic data observed with recombinant H. somnus guaA?

Inconsistent kinetic data with recombinant enzymes often stems from multiple experimental variables. Based on experience with similar recombinant proteins:

• Protein quality assessment:

  • Use multi-angle light scattering (MALS) to confirm monodispersity and correct oligomeric state

  • Implement thermal shift assays to confirm protein stability under assay conditions

  • Consider activity measurements immediately after purification versus after storage/freeze-thaw cycles

• Assay optimization:

  • Systematic evaluation of buffer components (pH, salt, metal ions)

  • Testing for potential inhibitors in reagents or buffer components

  • Evaluation of substrate quality and storage conditions

• Statistical approaches:

  • Implement global fitting of multiple datasets using advanced enzyme kinetics software

  • Use robust statistical methods to identify and properly weight outliers

  • Collect sufficient data points across the full range of substrate concentrations

• Alternative assay methods:

  • Compare direct and coupled assay approaches

  • Consider isothermal titration calorimetry for thermodynamic parameters

  • Implement pre-steady-state kinetics using stopped-flow techniques

A systematic approach to identifying sources of variability is critical, as recombinant H. somnus proteins have shown sensitivity to expression and purification conditions .

What are the critical steps in optimizing the autoinduction method for high-yield H. somnus guaA expression?

Based on successful expression of other H. somnus recombinant proteins, the following critical factors should be considered when optimizing the autoinduction method:

• Media composition:

  • ZYM-5052 autoinduction medium containing 1% tryptone, 0.5% yeast extract, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 5 mM Na₂SO₄, 2 mM MgSO₄, 0.5% glycerol, 0.05% glucose, and 0.2% lactose

  • Trace metal supplementation (particularly zinc and iron) often improves expression yields

• Growth parameters:

  • Initial growth at 37°C until OD₆₀₀ reaches 0.6-0.8

  • Temperature shift to 25-30°C for continued growth for 16-24 hours

  • Maintain adequate aeration (>40% dissolved oxygen) throughout growth

• Harvest timing:

  • Monitor expression levels by SDS-PAGE analysis of small culture aliquots

  • Optimal harvest point typically occurs 4-6 hours after glucose depletion

  • Expression levels may plateau or decrease if cultures are maintained too long

• Strain selection:

  • E. coli C41(DE3) has shown superior performance for H. somnus proteins

  • Consider testing Rosetta strains if rare codon usage is identified in the guaA sequence

This methodology has been shown to significantly increase yields compared to IPTG induction for H. somnus recombinant proteins, with the highest protein levels observed in both soluble and insoluble cytoplasmic fractions .

How can antibody cross-reactivity be leveraged to study structural conservation between H. somnus guaA and homologous proteins?

Based on immunological studies with other H. somnus proteins, antibody cross-reactivity can be a powerful tool:

• Production of polyclonal antibodies:

  • Immunize animals (rabbits or calves) with purified recombinant H. somnus guaA

  • Use a primary immunization followed by 1-2 booster immunizations (20 μg protein per injection)

  • Collect serum and purify IgG using protein A/G affinity chromatography

• Cross-reactivity assessment:

  • Test antibody recognition against whole-cell lysates of related bacterial species

  • Perform Western blotting to identify specific cross-reactive proteins

  • Quantify relative binding strengths using ELISA against purified homologous proteins

• Epitope mapping:

  • Use peptide arrays to identify specific epitopes recognized by the antibodies

  • Create chimeric proteins with domain swaps between H. somnus guaA and homologs

  • Perform competition assays with peptide fragments to identify conserved binding regions

Previous studies with H. somnus OMP40 demonstrated that immunization induced antibodies with broad cross-reactivity against similar antigens from other species in the Pasteurellaceae and Enterobacteriaceae families . This approach could similarly identify structurally conserved regions in guaA across bacterial species.

What are the most effective approaches for troubleshooting inclusion body formation during H. somnus guaA expression?

Inclusion body formation is common with H. somnus recombinant proteins . The following systematic approach can help address this challenge:

• Prevention strategies:

  • Reduce expression rate using lower inducer concentrations or lower temperatures

  • Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Create fusion constructs with solubility enhancers (MBP, SUMO, TrxA)

  • Test different E. coli strains with enhanced disulfide bond formation capabilities

• Refolding strategies if prevention fails:

  • Solubilize inclusion bodies using 8M urea or 6M guanidine hydrochloride

  • Perform stepwise dialysis with decreasing denaturant concentration

  • Include additives during refolding (L-arginine, glycerol, reduced/oxidized glutathione)

  • Test on-column refolding with immobilized metal affinity chromatography

• Activity recovery assessment:

  • Compare specific activity of soluble vs. refolded protein

  • Assess structural integrity using circular dichroism spectroscopy

  • Perform thermal stability assays to determine if refolded protein maintains proper structure

How should experiments be designed to determine if H. somnus guaA is essential for bacterial virulence?

To investigate the role of guaA in H. somnus virulence, consider this experimental framework:

• Genetic manipulation approaches:

  • Create a conditional knockout strain using an inducible promoter system

  • Develop a guaA-deficient strain complemented with plasmid-encoded guaA

  • Create point mutations in catalytic residues to produce enzymatically inactive versions

• In vitro virulence assays:

  • Compare growth rates in media with limited or abundant guanine sources

  • Assess biofilm formation capability in wild-type vs. guaA-modified strains

  • Evaluate survival under stress conditions (oxidative stress, nutrient limitation)

• Cellular interaction studies:

  • Measure adhesion and invasion of bovine epithelial cell lines

  • Assess survival within bovine macrophages

  • Quantify inflammatory cytokine responses to wild-type vs. guaA-modified strains

• Animal model considerations:

  • Develop a suitable bovine infection model

  • Consider competitive index studies comparing wild-type and guaA-modified strains

  • Implement immunization studies using enzymatically inactive guaA as a potential vaccine candidate

This multi-faceted approach would provide comprehensive insights into the role of guaA in H. somnus pathogenesis, similar to studies conducted with other H. somnus proteins .

What standardized datasets should be collected to facilitate meaningful comparisons between different recombinant H. somnus guaA preparations?

To ensure reproducibility and enable meaningful comparisons across different laboratories working with recombinant H. somnus guaA, the following standardized datasets should be collected:

• Expression and purification parameters:

  • Complete strain genotype and expression vector details

  • Detailed cultivation conditions (media composition, temperature, induction method)

  • Step-by-step purification protocol with buffer compositions

  • SDS-PAGE and Western blot images documenting purity

• Protein characterization data:

  • Accurate concentration determination using multiple methods

  • Mass spectrometry confirmation of intact mass and peptide coverage

  • Secondary structure analysis by circular dichroism

  • Thermal stability profiles under various buffer conditions

• Enzymatic activity parameters:

  • Detailed assay conditions (temperature, pH, buffer composition)

  • Complete Michaelis-Menten parameters for all substrates

  • Specific activity measurements with standardized substrate concentrations

  • Inhibition profiles with common inhibitors

• Stability information:

  • Activity retention after defined storage periods

  • Freeze-thaw stability assessment

  • Temperature sensitivity data

  • Long-term storage recommendations

The absence of such standardized datasets has been a limitation in comparing results across different studies of H. somnus proteins .

How can structural information about H. somnus guaA be leveraged for targeted antimicrobial development?

Structural insights into H. somnus guaA can inform antimicrobial development through several approaches:

• Structure-based inhibitor design:

  • Identify unique structural features of the substrate binding pocket

  • Employ computational docking to screen virtual compound libraries

  • Design transition-state analogs specific to the H. somnus guaA catalytic mechanism

  • Develop bisubstrate inhibitors that span both active sites

• Allosteric inhibitor development:

  • Identify communication pathways between domains using HDX-MS or molecular dynamics

  • Target interdomain interfaces that are essential for enzymatic activity

  • Develop compounds that lock the enzyme in an inactive conformation

• Specificity considerations:

  • Compare active site architectures between bacterial and mammalian GMP synthases

  • Identify bacterial-specific features that can be exploited for selective targeting

  • Perform comparative inhibition studies against homologous enzymes from beneficial microbiota

• Delivery strategies for in vivo efficacy:

  • Investigate membrane permeability of candidate inhibitors

  • Consider prodrug approaches to improve bioavailability

  • Explore targeted delivery systems to enhance concentration at infection sites

This approach leverages fundamental research to address the critical need for new antimicrobials against H. somnus infections in cattle.

What are the most promising approaches for using recombinant H. somnus guaA in vaccine development?

Based on immunological studies with other H. somnus recombinant proteins, several approaches for vaccine development with recombinant guaA merit investigation:

• Subunit vaccine formulations:

  • Evaluate different adjuvant combinations for optimal immune response

  • Test prime-boost strategies using different protein formulations

  • Compare immune responses to active vs. catalytically inactive (mutated) guaA

  • Assess duration of immunity with different formulation strategies

• Combination approaches:

  • Include guaA with other immunogenic H. somnus proteins

  • Research conducted with H. somnus OMP40 demonstrated humoral response with broad cross-reactivity, suggesting potential synergistic effects in combination vaccines

  • Design multi-epitope constructs containing immunodominant regions from multiple proteins

• Immune response characterization:

  • Evaluate both humoral (IgG1, IgG2, IgM) and cell-mediated immune responses

  • Test for delayed-type hypersensitivity reactions as seen with other H. somnus proteins

  • Assess cross-protective potential against related bacterial species

• Delivery platforms:

  • Compare traditional protein/adjuvant formulations with newer platforms

  • Evaluate viral vector or DNA vaccine approaches for guaA expression

  • Consider nanoparticle encapsulation for enhanced presentation to the immune system