Recombinant Escherichia coli Protein FdrA (fdrA)

What is Escherichia coli Protein FdrA (fdrA)?

Escherichia coli Protein FdrA (fdrA) is a full-length bacterial protein consisting of 555 amino acids. It is also known by synonyms including ylbD, b0518, JW0506, and Protein FdrA, with UniProt ID Q47208 . The protein appears to be involved in metabolic processes in E. coli, though its precise functional characterization requires further study by researchers working with purified recombinant forms of the protein.

What expression systems are optimal for FdrA protein production?

For recombinant FdrA protein production, E. coli expression systems have proven effective. The commercially available recombinant FdrA is produced as an N-terminal His-tagged fusion protein expressed in E. coli . When designing your expression system, consider:

• Vector selection: pET-series vectors with T7 promoters provide high-level expression

• Host strain considerations: BL21(DE3) or Rosetta strains may improve expression of potentially rare codons

• Induction conditions: Optimize IPTG concentration and induction temperature (typically lower temperatures of 16-25°C may improve protein solubility)

• Fusion tags: The N-terminal His-tag facilitates purification while minimally impacting structure

Research suggests that expression in the native organism (E. coli) minimizes issues with protein folding and post-translational modifications that might arise in non-native expression systems.

What are the challenges in expressing full-length FdrA protein?

Researchers may encounter several challenges when expressing the full-length 555 amino acid FdrA protein:

• Protein solubility: The full-length protein may form inclusion bodies requiring optimization of expression conditions

• Protein stability: Long proteins can be susceptible to proteolytic degradation during expression

• Purification complexity: Complete removal of host cell proteins while maintaining native conformation

• Activity preservation: Ensuring the recombinant protein maintains functional activity

To address these challenges, researchers should consider:

• Optimizing expression at lower temperatures (16-20°C)

• Including protease inhibitors during purification

• Carefully selecting buffer components to enhance stability

• Performing activity assays at each purification step to monitor functional integrity

What is the recommended storage and handling procedure for purified FdrA protein?

For optimal stability and activity of purified recombinant FdrA protein:

• Storage temperature: Store at -20°C/-80°C for long-term preservation

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Buffer composition: Tris/PBS-based buffer with 6% Trehalose, pH 8.0 maintains stability

• Reconstitution: Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Cryoprotectant: Add glycerol to a final concentration of 5-50% before freezing

• Working stock: Store working aliquots at 4°C for up to one week to avoid degradation

Following these guidelines will help maintain protein integrity and experimental reproducibility across studies.

How can I assess the purity and activity of recombinant FdrA protein?

Assessment of recombinant FdrA protein quality involves multiple analytical techniques:

Standard commercial preparations typically achieve >90% purity as determined by SDS-PAGE . Researchers should validate activity using assays specific to the predicted function of FdrA before proceeding with experimental applications.

How can I design experiments to investigate FdrA protein interactions?

When investigating FdrA protein interactions, consider these methodological approaches:

• Pull-down assays:

  • Immobilize His-tagged FdrA on Ni-NTA resin

  • Incubate with E. coli lysate or purified candidate interacting proteins

  • Wash extensively and elute complexes

  • Analyze eluted fractions by SDS-PAGE and mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize FdrA on SPR chip surface

  • Flow potential binding partners over the surface

  • Measure real-time binding kinetics (kon and koff)

  • Calculate binding affinity (KD)

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine stoichiometry, enthalpy, and entropy of interactions

  • No immobilization required, providing solution-phase measurements

• Crosslinking coupled with mass spectrometry:

  • Use chemical crosslinkers (e.g., DSS, BS3) to capture transient interactions

  • Digest crosslinked complexes and analyze by LC-MS/MS

  • Identify specific residues involved in protein-protein interactions

These methods provide complementary information about binding partners, interaction strength, and structural details of complexes involving FdrA.

What are the methodologies for studying FdrA protein function in vivo?

To investigate FdrA function within living E. coli cells:

• Gene knockout studies:

  • Generate fdrA deletion mutants using CRISPR-Cas9 or λ-Red recombineering

  • Perform phenotypic analysis (growth rate, metabolic profiles, stress responses)

  • Complement with wild-type and mutant FdrA variants

• Protein localization:

  • Create GFP or mCherry fusion constructs with FdrA

  • Express at near-physiological levels

  • Visualize subcellular localization using fluorescence microscopy

  • Correlate localization with different growth conditions or stressors

• Interactome analysis:

  • Perform in vivo crosslinking followed by immunoprecipitation (IP)

  • Analyze IP fractions by mass spectrometry to identify interaction partners

  • Validate interactions with co-IP and reciprocal pull-downs

• Transcriptomics/proteomics:

  • Compare gene expression profiles between wild-type and fdrA mutant strains

  • Identify pathways affected by FdrA perturbation

  • Integrate with metabolomic data for comprehensive functional analysis

These approaches provide multi-dimensional perspectives on FdrA function within its native cellular context.

How can I resolve solubility issues with recombinant FdrA protein?

When encountering solubility challenges with FdrA:

• Expression optimization:

  • Reduce induction temperature to 16-18°C

  • Decrease inducer concentration

  • Extend expression time (overnight at lower temperatures)

  • Test different E. coli strains (e.g., Arctic Express for cold-adapted chaperones)

• Buffer optimization:

  • Screen different pH conditions (7.0-8.5)

  • Test various salt concentrations (100-500 mM NaCl)

  • Add stabilizing agents (glycerol, trehalose, arginine)

  • Include reducing agents if cysteines are present (DTT or TCEP)

• Protein engineering approaches:

  • Test different fusion tags (MBP, SUMO, TRX)

  • Consider domain-based expression if full-length proves challenging

  • Identify and mutate aggregation-prone regions

• Refolding protocols:

  • If inclusion bodies form, develop gradient-based refolding strategies

  • Use slow dialysis to remove denaturants

  • Test additives like arginine and non-detergent sulfobetaines

Commercial preparations typically achieve >90% purity with good solubility , suggesting these approaches can successfully overcome solubility challenges.

How can contradictory results in FdrA functional studies be reconciled?

When faced with contradictory results in FdrA research:

Systematic evaluation using this framework can help identify the source of contradictions and establish consensus regarding FdrA function.