Recombinant Escherichia coli Branched-chain-amino-acid aminotransferase (ilvE)

What is the genomic organization of the ilvE gene within the E. coli chromosome?

The ilvE gene is part of the ilvEDA operon in Escherichia coli K-12, which along with the ilvC operon, has been precisely mapped through restriction cleavage analysis and complementation studies . Research has established that the ilvEDA operon occupies approximately 2.4 megadaltons of DNA sequence, while the ilvC operon occupies about 0.9 megadaltons . These operons are separated by a region spanning 0.6-0.75 megadaltons on the E. coli chromosome . Transcriptional studies confirm that both the ilvEDA and ilvC operons are transcribed clockwise on the E. coli K-12 map .

To study this organization experimentally, researchers typically use the following approaches:

• Restriction enzyme mapping with enzymes like EcoRI

• Heteroduplex analysis with hybrid phages

• Complementation analysis with plasmids containing DNA fragments

• Expression studies measuring enzyme activities from hybrid constructs

How can researchers effectively isolate and amplify the ilvE gene for recombinant expression?

Isolation of the ilvE gene requires careful primer design to ensure specificity within the ilvEDA operon context. The experimental approach involves:

• Genomic DNA extraction: Standard protocols using lysozyme treatment followed by phenol-chloroform extraction yield high-quality E. coli genomic DNA.

• PCR amplification: Design primers with the following considerations:

  • Include restriction sites compatible with your expression vector

  • Maintain the reading frame for proper fusion with purification tags

  • Consider codon optimization when moving between different E. coli strains

• Verification of amplified product:

  • Agarose gel electrophoresis to confirm size

  • Restriction digestion to verify internal sites

  • Sequencing to ensure no mutations were introduced during amplification

For controlled experimental evaluation, implement a rigorous design that includes positive controls and verification steps at each stage . This enables clear establishment of cause-effect relationships between your isolation methods and outcomes.

What expression systems yield optimal recombinant ilvE production in E. coli?

The choice of expression system significantly impacts the yield and activity of recombinant ilvE. A methodological approach to expression system selection includes:

Expression vectors comparison:

For rigorous experimental evaluation:

• Test multiple expression systems in parallel using standardized growth conditions

• Implement a controlled trial method comparing expression levels with identical cell densities

• Quantify both total protein expression and enzyme activity to determine functional yield

• Analyze solubility fraction separately from inclusion bodies

What are the critical parameters for optimizing recombinant ilvE expression while maintaining enzymatic activity?

Optimizing recombinant ilvE expression requires balancing maximal yield with enzymatic activity preservation. The methodological approach should include:

To evaluate these parameters systematically, implement a data.table approach to analyze multiple variables simultaneously . This allows efficient comparison of parameters across experimental conditions to identify optimal combinations.

What purification protocols yield the highest purity and specific activity for recombinant ilvE?

Purification of recombinant ilvE requires a multi-step approach to achieve high purity while preserving enzymatic activity:

Standard purification workflow:

• Cell lysis optimization:

  • Sonication (10-15 cycles, 30s on/30s off) in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Alternative: High-pressure homogenization at 15,000-20,000 psi

  • Include protease inhibitors (PMSF or commercial cocktails)

  • Add 10-20 μM pyridoxal phosphate to stabilize the enzyme

• Initial capture:

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) with Ni-NTA

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Washing: Increase imidazole to 20-40 mM to reduce non-specific binding

  • Elution: Step gradient of 100-250 mM imidazole

• Intermediate purification:

  • Ion exchange chromatography (IEX) based on theoretical pI of ilvE

  • Size exclusion chromatography (SEC) for final polishing and buffer exchange

Purification yields and activity assessment:

To evaluate purification efficiency, implement quantitative assessments at each step including SDS-PAGE densitometry, enzymatic activity assays, and total protein determination.

How can researchers address stability and activity loss during purification of recombinant ilvE?

Maintaining ilvE stability and activity throughout purification requires addressing several critical factors:

• Buffer optimization:

  • Include 10-20% glycerol as a stabilizing agent

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

  • Maintain pyridoxal phosphate (10-50 μM) throughout all purification steps

  • Test different pH ranges (7.0-8.5) for optimal stability

• Temperature management:

  • Conduct all purification steps at 4°C

  • Avoid freeze-thaw cycles by aliquoting purified enzyme

  • Test different storage conditions (-80°C, -20°C, 4°C with glycerol)

• Activity preservation techniques:

  • Add stabilizing ligands (e.g., low concentrations of substrates)

  • Include protease inhibitors throughout purification

  • Minimize exposure to air/oxygen during concentration steps

  • Consider carrier proteins (BSA) for very dilute enzyme solutions

• Validation approaches:

  • Monitor activity throughout purification using standardized assays

  • Assess thermal stability using differential scanning fluorimetry

  • Verify structural integrity via circular dichroism spectroscopy

  • Implement quality control checkpoints at each purification stage

When troubleshooting activity loss, systematic analysis using the controlled trial method allows researchers to identify critical factors affecting enzyme stability .

What are the optimal methods for assessing recombinant ilvE enzyme kinetics and substrate specificity?

Characterizing recombinant ilvE kinetics requires methodology that addresses both the forward and reverse reactions of this aminotransferase:

Spectrophotometric coupled assays:

• Forward reaction (amino acid → keto acid):

  • Couple with glutamate dehydrogenase to monitor NADH oxidation at 340 nm

  • Reaction buffer: 50 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 0.2 mM NADH

  • Include 5 mM α-ketoglutarate as amino group acceptor

  • Vary branched-chain amino acid concentration (0.1-10 mM)

• Reverse reaction (keto acid → amino acid):

  • Couple with leucine dehydrogenase to monitor NADH formation at 340 nm

  • Use glutamate as amino donor (fixed concentration 10-20 mM)

  • Vary branched-chain keto acid concentration (0.1-10 mM)

HPLC-based direct assays:

• Derivatize amino acids with o-phthalaldehyde or DABS-Cl

• Employ reverse-phase HPLC for product quantification

• Use internal standards for accurate quantification

Substrate specificity analysis:

For robust experimental design, implement multi-replicate assays with appropriate controls and standardization using commercial enzyme preparations when available.

How does pH and temperature affect recombinant ilvE enzymatic activity and stability?

Systematic characterization of pH and temperature effects on ilvE activity follows this methodology:

pH profile analysis:

• Prepare a series of buffers covering pH range 5.0-10.0 (0.5 pH unit intervals)

  • pH 5.0-6.0: MES buffer (50 mM)

  • pH 6.5-7.5: HEPES buffer (50 mM)

  • pH 8.0-9.0: Tris buffer (50 mM)

  • pH 9.5-10.0: CAPS buffer (50 mM)

• Standardize ionic strength across all buffers using KCl additions

• Measure enzyme activity under standard conditions, varying only the buffer

• Plot relative activity versus pH to determine optimum pH

Temperature effects:

• Activity profile: Measure initial reaction rates at temperatures from 20-60°C

• Thermal stability:

  • Pre-incubate enzyme at various temperatures (20-60°C) for defined periods

  • Measure residual activity under standard conditions

  • Calculate half-life at each temperature

Temperature-pH interaction matrix:

• Generate a 3D profile by measuring activity across temperature and pH combinations

• Identify optimal combinations for maximum activity and stability

Structure-function correlations:

• CD spectroscopy to monitor secondary structure changes with temperature

• Thermal shift assays to determine melting temperatures at different pH values

Implementing the experimental evaluation design principles ensures consistent data collection and interpretation across multiple experimental conditions .

How can recombinant ilvE be utilized in metabolic engineering of branched-chain amino acid biosynthesis?

Recombinant ilvE plays a crucial role in metabolic engineering strategies targeting branched-chain amino acid (BCAA) biosynthesis. Methodological approaches include:

Overexpression strategies:

• Coordinate expression with pathway enzymes:

  • Co-express ilvE with ilvBN (acetohydroxy acid synthase)

  • Balance expression levels using different promoter strengths

  • Employ polycistronic constructs vs. multiple plasmids

• Feedback regulation circumvention:

  • Co-express with feedback-resistant ilvBN variants

  • Balance pathway flux through careful promoter selection

  • Monitor intermediate accumulation to identify bottlenecks

Flux optimization approaches:

• Precursor supply enhancement:

  • Increase pyruvate and threonine availability through upstream engineering

  • Reduce competing pathways through selective knockdowns

  • Implement dynamic pathway regulation using biosensors

• Cofactor management:

  • Ensure sufficient pyridoxal phosphate through vitamin B6 pathway enhancement

  • Balance NAD(P)H availability for reductive steps

  • Consider α-ketoglutarate/glutamate balance for transamination efficiency

Production improvement metrics:

For integration of live simulation with constructive modeling approaches, researchers should ensure interoperability of systems and composability of models , allowing for accurate prediction of metabolic outcomes before implementation.

What experimental designs best evaluate the impact of ilvE modifications on metabolic flux?

Evaluating the impact of ilvE modifications on metabolic flux requires systematic experimental approaches:

• 13C metabolic flux analysis (13C-MFA):

  • Feed cultures with 13C-labeled glucose or other carbon sources

  • Sample at steady state growth conditions

  • Analyze isotopomer distributions by GC-MS or LC-MS/MS

  • Calculate flux distributions using computational modeling

  • Compare wild-type vs. ilvE-modified strains

• Metabolite profiling:

  • Target analysis of pathway intermediates:

    • α-keto acids (α-ketoisocaproate, α-keto-β-methylvalerate, α-ketoisovalerate)

    • Amino acids (leucine, isoleucine, valine)

    • Upstream precursors (pyruvate, threonine)

  • Use LC-MS/MS or GC-MS with appropriate internal standards

  • Implement time-course analysis to capture dynamic changes

• Enzyme activity assays in vivo:

  • Develop cell-free extract assays to measure activities of entire pathways

  • Use permeabilized cells to assess in situ activities

  • Compare activities under different growth conditions

• Growth-based phenotypic analysis:

  • Growth rate measurements in defined media

  • Auxotrophy complementation studies

  • Competitive growth experiments with labeled strains

For controlled experimental evaluation, implement a rigorous design that includes technical and biological replicates, appropriate controls, and statistical analysis to establish clear cause-effect relationships .

What methods are most effective for analyzing structure-function relationships in recombinant ilvE?

Understanding structure-function relationships in ilvE requires integrating various methodological approaches:

• Site-directed mutagenesis strategy:

  • Target conserved active site residues (based on sequence alignment)

  • Create systematic alanine scanning libraries

  • Design mutations based on computational predictions

  • Generate varying side-chain substitutions at key positions

• Structural analysis techniques:

  • X-ray crystallography of ilvE with different ligands

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

  • Cryo-electron microscopy for larger complexes

  • Small-angle X-ray scattering (SAXS) for solution behavior

• Functional correlation approaches:

  • Kinetic analysis of mutant enzymes (Km, kcat, substrate specificity)

  • Thermal stability comparisons using DSF

  • Conformational dynamics via NMR spectroscopy

  • Computational modeling with molecular dynamics simulations

Structure-function correlation table:

For rigorous experimental design, implement controlled comparisons between wild-type and mutant enzymes under identical conditions while systematically varying relevant parameters .

How can researchers effectively develop inhibitors or activators for ilvE through structure-based design?

Developing modulators of ilvE activity through structure-based design involves these methodological approaches:

• Initial screening strategies:

  • Virtual screening of compound libraries against ilvE crystal structure

  • Fragment-based screening using NMR or X-ray crystallography

  • High-throughput enzymatic assays with diverse chemical libraries

  • Rational design based on substrate analogs

• Structure-activity relationship (SAR) development:

  • Synthesize focused libraries around initial hits

  • Evaluate inhibition/activation kinetics (Ki, IC50, activation constants)

  • Determine binding modes through co-crystallization

  • Use computational docking to prioritize compounds

• Mode of action characterization:

  • Determine inhibition type (competitive, uncompetitive, non-competitive)

  • Analyze enzyme-inhibitor complex stability

  • Assess specificity against related aminotransferases

  • Evaluate effects on enzyme conformational dynamics

• In vivo validation:

  • Cell-based assays to confirm target engagement

  • Metabolite profiling to verify pathway modulation

  • Growth phenotype analysis under various conditions

  • Genetic approaches (target overexpression, resistant mutants)

Inhibitor development workflow:

The experimental evaluation design should include appropriate controls and standardization methods to ensure reliable and reproducible results .