Recombinant Escherichia fergusonii Bifunctional protein aas (aas)

What is the Escherichia fergusonii Bifunctional protein aas and what are its primary functions?

The aas protein in Escherichia fergusonii is a bifunctional enzyme consisting of 719 amino acids. The protein contains multiple functional domains that enable it to participate in membrane phospholipid turnover and fatty acid activation. Specifically, the bifunctional aas protein possesses both 2-acylglycerophosphoethanolamine acyltransferase and acyl-CoA synthetase activities, allowing it to catalyze the reacylation of lysophospholipids and the activation of fatty acids through their conversion to acyl-CoA . This enzyme plays a crucial role in maintaining membrane integrity and phospholipid homeostasis in E. fergusonii. The protein shares significant homology with the aas protein in E. coli, suggesting evolutionary conservation of this important metabolic function across related bacterial species .

How does the amino acid sequence of E. fergusonii aas protein compare to other bacterial homologs?

The E. fergusonii Bifunctional protein aas comprises 719 amino acids with a distinct sequence that shares high similarity with its E. coli counterpart. A comparative analysis of the amino acid sequences reveals:

Both proteins share the same domain architecture, but several key amino acid substitutions exist, particularly in the N-terminal region where the E. fergusonii sequence begins with MLFGFFRKLCQ compared to the E. coli sequence starting with MLFSFFRNLCR . These subtle differences may influence substrate specificity or regulatory interactions while maintaining the core catalytic functions. The high conservation between these homologs suggests that the bifunctional nature of this protein is essential for bacterial membrane homeostasis across Enterobacteriaceae .

What expression systems are commonly used for producing recombinant E. fergusonii aas protein?

Recombinant E. fergusonii Bifunctional protein aas is most commonly expressed in E. coli expression systems. The heterologous expression typically employs the following methodological approach:

• Gene cloning: The full-length aas gene (encoding all 719 amino acids) is amplified from E. fergusonii genomic DNA using specific primers that incorporate appropriate restriction sites.

• Vector construction: The amplified gene is inserted into expression vectors (commonly pET-series vectors) that provide:

  • An N-terminal His tag for purification purposes

  • Strong inducible promoters (typically T7)

  • Appropriate selection markers

• Expression conditions: Optimal expression is achieved using BL21(DE3) or similar E. coli strains with induction via IPTG at concentrations of 0.5-1.0 mM when cultures reach mid-log phase (OD600 = 0.6-0.8).

• Purification: The recombinant protein is typically purified using nickel affinity chromatography targeting the His-tag, followed by size exclusion chromatography if higher purity is required .

This expression system yields protein in the form of lyophilized powder that requires reconstitution in deionized sterile water to concentrations of 0.1-1.0 mg/mL, with recommended addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

What strategies can be employed for creating point mutations in the E. fergusonii aas protein to study structure-function relationships?

Creating targeted point mutations in the E. fergusonii aas protein requires a systematic approach based on structure-guided predictions. The following methodological workflow is recommended:

• Computational analysis: Begin with sequence alignments and structural modeling to identify conserved catalytic residues or substrate-binding motifs that warrant investigation. This can be accomplished using protein secondary structure prediction tools like those described in the literature with Q3 accuracy exceeding 85% .

• Primer design for site-directed mutagenesis: Design overlapping primers containing the desired nucleotide changes. Critical considerations include:

  • Maintaining primer length between 25-45 nucleotides

  • Positioning the mutation in the middle of the primer

  • Ensuring GC content of 40-60%

  • Calculating melting temperatures (Tm) above 78°C for the mutation site

• SOE-PCR (Splice Overlap Extension PCR): This technique has proven effective for generating point mutations as demonstrated in recombinant plasmid construction studies. The procedure involves:

  • Initial PCR reactions with mutant primers to generate overlapping fragments

  • A second PCR using the overlapping fragments as templates

  • Final amplification using flanking primers to generate the full-length mutant gene

• Validation of mutations: Confirm mutations through DNA sequencing before proceeding to expression and purification.

• Functional characterization: Compare wild-type and mutant proteins through:

  • Enzyme kinetics (Km, Vmax, kcat)

  • Thermal stability assays

  • Structural analysis via circular dichroism or crystallography

This approach has been successfully employed for analyzing key catalytic residues in related bifunctional enzymes, providing insights into how specific amino acids contribute to substrate recognition and catalysis .

How can researchers design optimal linkers when creating fusion constructs with the E. fergusonii aas protein?

Designing optimal linkers for E. fergusonii aas protein fusion constructs requires careful consideration of multiple factors to ensure proper folding and activity of both functional domains. The methodological approach should include:

• Linker selection criteria:

  • Flexibility: Glycine-serine linkers (G4S)n provide excellent flexibility, as demonstrated in successful bifunctional fusion proteins. The sequence GGGGS repeated 1-3 times (5-15 amino acids) offers sufficient space between domains while minimizing structural interference .

  • Length determination: The optimal length should be determined experimentally, starting with 5-15 amino acids for most applications. Shorter linkers may constrain domain movement, while excessively long linkers can introduce proteolytic susceptibility.

  • Hydrophilicity: The linker should be hydrophilic to ensure solvent exposure and prevent interference with domain folding.

• Experimental validation strategy:

  • Create a library of constructs with varying linker lengths and compositions

  • Express and purify the fusion variants

  • Conduct comparative activity assays to identify constructs that maintain activities comparable to individual domains

  • Perform thermal stability analysis to ensure proper folding

• Advanced design considerations:

  • Domain orientation: Test both N- and C-terminal fusions of aas with its partner protein

  • Structural modeling: Use in silico approaches to predict potential steric clashes

  • Protease resistance: Avoid sequences recognized by common proteases

The successful application of flexible GS linkers has been demonstrated in creating bifunctional enzymes combining chitinase and protease, which provides a methodological template applicable to aas fusion proteins . When designing these constructs, researchers should systematically evaluate multiple variants to identify the optimal configuration that preserves the dual functionalities of the aas protein.

What are the optimal conditions for expressing and purifying active E. fergusonii Bifunctional protein aas?

Optimizing expression and purification of active E. fergusonii Bifunctional protein aas requires careful attention to multiple experimental parameters. The following protocol represents best practices based on current research:

• Expression system optimization:

  • Host strain selection: BL21(DE3) pLysS provides tight control of basal expression and is recommended for potentially toxic proteins like membrane-associated aas

  • Vector selection: pET-28a(+) with T7 promoter and N-terminal His-tag has demonstrated high-yield expression

  • Growth medium: Use auto-induction media (ZYM-5052) for higher yields compared to standard LB with IPTG induction

  • Temperature control: Express at 18-20°C for 16-18 hours post-induction to enhance proper folding

• Cell lysis and protein solubilization:

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF

  • Detergent selection: Include 0.5% CHAPS for optimal solubilization while maintaining activity

  • Sonication parameters: 6 cycles of 30s on/30s off at 40% amplitude on ice

• Purification strategy:

  • IMAC (Immobilized Metal Affinity Chromatography): Use Ni-NTA resin with stepwise imidazole gradient (20, 50, 250 mM)

  • Secondary purification: Size exclusion chromatography using Superdex 200 in buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

  • Quality control: Verify purity via SDS-PAGE (>90%) and activity via enzyme-specific assays

• Storage conditions:

  • Short-term (1 week): 4°C in purification buffer with 5% glycerol

  • Long-term: Add glycerol to 50% final concentration, aliquot, and store at -80°C

  • Avoid repeated freeze-thaw cycles which significantly reduce activity

This optimized protocol typically yields 5-8 mg of purified protein per liter of bacterial culture with >90% purity and preserved bifunctional activity.

How can the E. fergusonii aas protein be utilized in metabolic engineering applications?

The E. fergusonii Bifunctional protein aas presents significant potential for metabolic engineering applications due to its dual functionality in fatty acid activation and phospholipid remodeling. Strategic approaches for leveraging this protein include:

• Engineering enhanced phospholipid production:

  • Overexpression of aas in recombinant systems can increase phospholipid turnover and membrane lipid composition control

  • Integration with fatty acid biosynthesis pathways can create strains with altered membrane properties

  • Systematic monitoring of phospholipid profiles using LC-MS/MS is essential to validate engineering outcomes

• Development of microbial cell factories:

  • The aas protein can be incorporated into metabolic pathways for producing high-value lipids or fatty acid derivatives

  • Co-expression with complementary enzymes can establish novel biosynthetic routes

  • A case study approach demonstrates that recombinant strains harboring both the cai operon (for L-carnitine conversion to γ-butyrobetaine) and the gbu gene cluster (for γ-butyrobetaine conversion to TMA) successfully establish complete metabolic pathways in single organisms

• Creation of specialized recombinant strains:

  • Engineered E. fergusonii strains expressing aas variants can serve as research tools for studying:
    a) Membrane homeostasis under various environmental stresses
    b) Lipid metabolism in response to carbon source changes
    c) Bacterial adaptation mechanisms through membrane remodeling

  • Such strains can be constructed using techniques similar to those used for introducing the E. timonensis gbu gene cluster into E. fergusonii

• Experimental design considerations:

  • Expression levels must be carefully controlled as excessive aas activity may disrupt membrane integrity

  • Growth conditions, particularly temperature and carbon source availability, significantly impact aas activity

  • Complementation with chaperone proteins like GroES/GroEL may be necessary for optimal function, as demonstrated in analogous recombinant systems

These applications demonstrate how aas can be instrumentalized beyond its native role, serving as a versatile tool for metabolic engineering aimed at modifying bacterial lipid metabolism and membrane composition.

What role does the E. fergusonii Bifunctional protein aas play in bacterial membrane homeostasis and how can this be experimentally investigated?

The E. fergusonii Bifunctional protein aas plays a critical role in bacterial membrane homeostasis through its dual enzymatic activities. Understanding this role requires sophisticated experimental approaches:

These methodological approaches collectively provide a comprehensive framework for investigating the multifaceted roles of aas in bacterial membrane biology.

How can recombinant E. fergusonii aas protein be used to study pathways involving L-carnitine metabolism?

Recombinant E. fergusonii Bifunctional protein aas can serve as a valuable tool for investigating L-carnitine metabolism pathways, particularly in conjunction with other engineered components. The methodological approach includes:

• Pathway reconstruction and analysis:

  • E. fergusonii naturally harbors the cai operon responsible for converting L-carnitine into γ-butyrobetaine (γBB), making it an ideal chassis for studying complete metabolic pathways

  • By engineering E. fergusonii to express both native pathways and recombinant components like the E. timonensis gbu gene cluster, researchers can reconstruct the entire L-carnitine→γBB→TMA transformation pathway in a single organism

  • This system allows for controlled studies of each step in the pathway and identification of rate-limiting factors

• In vitro reconstitution studies:

  • Purified recombinant aas protein can be combined with other pathway enzymes to:
    a) Determine kinetic parameters for each reaction step
    b) Identify substrate competition or inhibition patterns
    c) Test potential inhibitors for therapeutic development

  • Reaction progress can be monitored using LC-MS/MS for metabolite quantification

• In vivo experimental approaches:

  • Germ-free mouse models can be inoculated with engineered E. fergusonii strains to study:
    a) Serum TMAO levels in response to dietary L-carnitine supplementation
    b) Metabolic consequences of pathway manipulation
    c) Host-microbe interactions related to carnitine metabolism

  • Time-course experiments utilizing these models have demonstrated that recombinant E. fergusonii strains can raise serum TMAO to pathophysiological levels when mice are supplemented with L-carnitine

• Experimental design considerations:

  • Temperature control is critical as the proper functioning of recombinant pathways often requires specific chaperone proteins like GroES/GroEL, particularly for operation at 37°C

  • Expression levels of pathway components must be balanced to prevent metabolic bottlenecks

  • Careful validation of each pathway step is necessary using metabolomic approaches

• Applications in metabolic disease research:

  • Engineered E. fergusonii strains serve as valuable tools for investigating links between gut microbial L-carnitine metabolism and cardiovascular or metabolic diseases

  • These systems enable testing of intervention strategies aimed at blocking specific steps in TMA production

This integrated approach demonstrates how recombinant protein engineering can facilitate sophisticated metabolic pathway studies with implications for both basic science and therapeutic development.

What analytical methods are most effective for characterizing the dual enzymatic activities of the E. fergusonii Bifunctional protein aas?

Comprehensive characterization of the dual enzymatic activities of E. fergusonii Bifunctional protein aas requires specialized analytical methods that can distinguish and quantify each function independently:

• Acyl-CoA synthetase activity analysis:

  • Spectrophotometric coupled assay: Measure ATP consumption or AMP production coupled to NADH oxidation

  • Direct product quantification: Use HPLC or LC-MS to detect and quantify acyl-CoA formation

  • Substrate specificity profiling methodology:

  Fatty Acid Chain Length	Analytical Method	Kinetic Parameters to Determine
  Short-chain (C2-C6)	Radiometric assay with 14C-labeled substrates	Km, Vmax, kcat, kcat/Km
  Medium-chain (C8-C12)	HPLC with UV detection of thioester bond	Substrate preference ratios
  Long-chain (C14-C20)	LC-MS/MS with multiple reaction monitoring	Catalytic efficiency comparison

• 2-acylglycerophosphoethanolamine acyltransferase activity analysis:

  • Thin-layer chromatography (TLC): Separate lysophospholipids and phospholipids followed by phosphate staining

  • Mass spectrometry: Monitor conversion of lysophospholipids to phospholipids using precursor ion scanning

  • Fluorescence-based assays: Utilize fluorescent lysophospholipid analogs to track reacylation in real-time

• Integrated bifunctional activity assessment:

  • Sequential reaction analysis: Supply free fatty acids and lysophospholipids to track complete conversion pathway

  • Competition experiments: Determine preferential activity when both substrates are present

  • Inhibition studies: Use domain-specific inhibitors to dissect the relative contribution of each activity

• Structural-functional correlation approaches:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify substrate-binding regions

  • Site-directed mutagenesis targeting putative catalytic residues followed by activity assays to confirm their roles

  • Limited proteolysis combined with activity assays to identify stable functional domains

• Advanced biophysical characterization:

  • Isothermal titration calorimetry (ITC) for thermodynamic binding parameters

  • Surface plasmon resonance (SPR) for real-time interaction kinetics

  • Circular dichroism spectroscopy to monitor structural changes upon substrate binding

What are common challenges in the expression and purification of recombinant E. fergusonii aas protein and how can they be addressed?

The expression and purification of recombinant E. fergusonii Bifunctional protein aas presents several challenges due to its membrane association and dual enzymatic nature. Common issues and their solutions include:

• Protein solubility problems:

  • Challenge: The aas protein often forms inclusion bodies when overexpressed in E. coli

  • Solution approaches:
    a) Reduce expression temperature to 16-18°C and induce with lower IPTG concentrations (0.1-0.2 mM)
    b) Use specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane protein expression
    c) Co-express with chaperones like GroES/GroEL, which have been shown to assist proper folding at 37°C
    d) Add solubility-enhancing fusion tags like SUMO or MBP at the N-terminus

• Protein stability issues:

  • Challenge: The bifunctional nature can lead to domain misfolding or aggregation

  • Solution approaches:
    a) Include stabilizing agents in purification buffers (5-10% glycerol, 1 mM DTT or TCEP)
    b) Optimize buffer pH and ionic strength through systematic screening (pH 7.0-8.5, NaCl 100-500 mM)
    c) Add specific lipids or detergents that mimic the native membrane environment
    d) Implement a step-wise purification protocol to remove partially folded intermediates

• Low protein yield:

  • Challenge: Expression levels may be insufficient for structural or biochemical studies

  • Solution approaches:
    a) Optimize codon usage for E. coli expression
    b) Use high cell-density fermentation with fed-batch protocols
    c) Screen multiple expression vectors with different promoter strengths
    d) Determine optimal induction timing through time-course experiments

• Activity loss during purification:

  • Challenge: The dual enzymatic activities may be differentially sensitive to purification conditions

  • Solution approaches:
    a) Monitor both enzymatic activities separately throughout purification
    b) Test different detergents for solubilization (CHAPS, DDM, LDAO)
    c) Minimize exposure to potentially denaturing conditions
    d) Include substrate analogs or product mimics in purification buffers to stabilize active conformations

• Protein heterogeneity:

  • Challenge: Multiple conformational states or partial proteolysis can result in heterogeneous preparations

  • Solution approaches:
    a) Add protease inhibitors (PMSF, EDTA, protease inhibitor cocktail) during cell lysis
    b) Perform SEC-MALS analysis to identify and separate different oligomeric states
    c) Implement ion exchange chromatography as a polishing step to resolve conformational variants
    d) Use limited proteolysis followed by mass spectrometry to identify stable domains if full-length protein proves recalcitrant

Implementation of these strategies has been shown to significantly improve the yield and quality of recombinant bifunctional proteins, including those with complex membrane associations .

How can protein secondary structure prediction tools improve our understanding of E. fergusonii aas protein function?

Protein secondary structure prediction tools provide valuable insights into the structural organization of the E. fergusonii Bifunctional protein aas, guiding functional studies and rational engineering approaches. The methodological implementation includes:

• Selection of appropriate prediction algorithms:

  • State-of-the-art methods like PSRSM have demonstrated Q3 accuracy exceeding 86% on benchmark datasets, outperforming traditional approaches like SPINE-X, PSIPRED, and JPRED by 1-3%

  • Ensemble approaches combining multiple prediction methods often provide more reliable results for complex multidomain proteins like aas

• Structure-function correlation methodology:

  • Predict secondary structure elements across the entire 719-amino acid sequence

  • Map predicted α-helices, β-sheets, and loops to putative functional domains

  • Identify conserved structural motifs associated with each enzymatic activity:

  Domain	Predicted Secondary Structure	Associated Function	Conservation Analysis
  N-terminal	α-helical bundle (aa 1-240)	Membrane association	Highly conserved
  Central	Mixed α/β (aa 241-500)	Acyl-CoA synthetase	Conserved catalytic motifs
  C-terminal	Predominantly β-sheet (aa 501-719)	Acyltransferase	Variable regions surrounding conserved catalytic residues

• Application to experimental design:

  • Target surface-exposed loops for epitope tagging without disrupting core structure

  • Design domain truncation constructs guided by predicted domain boundaries

  • Identify flexible linker regions suitable for engineering bifunctional fusion proteins

  • Predict potential disordered regions that may impact crystallization

• Integration with evolutionary analysis:

  • Compare predicted secondary structures across homologs from E. coli and other related species

  • Identify structurally conserved regions despite sequence divergence

  • Map sequence variations between E. coli and E. fergusonii aas onto predicted structural elements to understand functional adaptation

• Advanced structural bioinformatics approaches:

  • Combine secondary structure predictions with coevolutionary analysis to predict tertiary contacts

  • Use predicted secondary structure as constraints for molecular modeling

  • Validate predictions experimentally through techniques like circular dichroism spectroscopy and limited proteolysis

• Machine learning enhancement:

  • Train specialized prediction models using datasets enriched for bifunctional enzymes

  • Incorporate protein datasets like ASTRAL, CullPDB, CASP, and CB513 that have been validated for high-quality structure prediction

This integrated approach enables researchers to develop testable hypotheses about structure-function relationships in the aas protein and guides the rational design of experiments to probe specific structural elements contributing to its bifunctional nature.

What strategies can be employed to enhance the stability and activity of recombinant E. fergusonii aas protein for in vitro studies?

Enhancing the stability and activity of recombinant E. fergusonii Bifunctional protein aas for in vitro studies requires a multifaceted approach addressing protein folding, environmental conditions, and potential cofactors:

• Buffer optimization through systematic screening:

  • pH screening: Test stability and activity across pH range 6.0-9.0 in 0.5 unit increments

  • Ionic strength: Optimize NaCl concentration (50-500 mM) and test various buffer systems (Tris, HEPES, Phosphate)

  • Additives: Screen stabilizing agents including:
    a) Polyols: Glycerol (5-20%), sucrose (5-10%), trehalose (5-10%)
    b) Reducing agents: DTT, TCEP, β-mercaptoethanol (0.5-5 mM)
    c) Metal ions: Mg2+, Mn2+, Zn2+ (0.1-2 mM)

  • Develop a stability index by measuring activity retention after defined time intervals at various temperatures

• Protein engineering approaches:

  • Surface entropy reduction: Identify and mutate surface exposed lysine/glutamate patches to alanine

  • Disulfide engineering: Introduce carefully positioned disulfide bonds to stabilize domains

  • Glycine scanning: Replace selected residues with glycine to increase backbone flexibility at domain interfaces

  • N- and C-terminal modifications: Truncate disordered termini that may promote aggregation

• Lipid and detergent supplementation:

  • Reconstitution with native E. fergusonii lipid extracts

  • Screening detergent types and concentrations:

  Detergent Class	Examples to Test	Optimal Concentration Range
  Non-ionic	DDM, LDAO, Triton X-100	1-3x CMC
  Zwitterionic	CHAPS, Fos-Choline	0.5-1% w/v
  Steroid-based	Digitonin, Cholate	0.1-0.5% w/v

  • Lipid nanodisc incorporation: Reconstruct aas protein in nanodiscs with defined lipid composition

• Co-expression and co-purification with stabilizing partners:

  • Chaperone co-expression: GroES/GroEL systems have demonstrated efficacy for related proteins

  • Binding partners: Identify and co-purify with natural protein interactors

  • Stabilizing antibody fragments: Develop and co-purify with conformation-specific nanobodies

• Advanced formulation strategies:

  • Spray-drying with trehalose for room-temperature stable formulations

  • Encapsulation in amphipathic polymers to create artificial membrane environments

  • PEGylation of non-essential surface residues to enhance solubility

  • Development of enzyme-MOF (Metal-Organic Framework) composites for enhanced stability

• Biophysical characterization to guide stabilization:

  • Differential scanning fluorimetry (DSF) to identify stabilizing conditions

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • Limited proteolysis to identify and eliminate protease-sensitive regions

Implementation of these methodologies has demonstrated success in enhancing the stability of complex membrane-associated enzymes, with some approaches yielding 2-5 fold improvements in half-life and retained activity under laboratory conditions . The optimal approach often involves combining multiple strategies tailored to the specific characteristics of the E. fergusonii aas protein.