Recombinant Protein AaeX (aaeX)

What is Recombinant Protein AaeX (aaeX) and what is its structure?

Recombinant Protein AaeX (aaeX) is a full-length protein (67 amino acids) derived from Escherichia coli. The protein has a UniProt ID of B1XHL4 and is characterized by its hydrophobic amino acid sequence: MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRVLVPTGIYDFVWHPALFNTALYCCLFYLISRLFV . This sequence suggests AaeX contains transmembrane domains, which aligns with challenges typically associated with membrane protein expression and purification as noted in recent literature .

Research methodologies for structural analysis typically include X-ray crystallography or NMR spectroscopy following successful purification using affinity chromatography, particularly with His-tagged versions of the protein. When working with AaeX, researchers should consider its hydrophobic nature when designing extraction and purification protocols.

What expression systems are most suitable for producing Recombinant Protein AaeX?

Escherichia coli remains the predominant expression system for Recombinant Protein AaeX due to its cost-effectiveness, rapid growth, and established genetic tools . When working with AaeX, consider these methodological approaches:

• Temperature optimization: Lowering culture temperature after induction can increase soluble protein expression by slowing protein synthesis and allowing proper folding .

• Fusion tag selection: His-tagging is commonly used for AaeX , but other solubility-enhancing tags such as SUMO, TRX, or MBP might improve expression yields of functional protein .

• Codon optimization: Since AaeX is a bacterial protein expressed in E. coli, codon bias is less problematic than with heterologous proteins, but optimization can still improve expression by 3-5 fold in some cases .

• Expression strain selection: Consider specialized strains designed to handle membrane proteins, particularly those with enhanced membrane protein folding capacity or modified secretion pathways like the ESETEC secretion technology or TatExpress strain .

What are the optimal storage conditions for Recombinant Protein AaeX?

For long-term stability of Recombinant Protein AaeX, implement the following evidence-based storage protocol:

• Store lyophilized powder at -20°C/-80°C upon receipt .

• After reconstitution, add 5-50% glycerol (final concentration) with 50% being the recommended default concentration .

• Aliquot the protein solution to avoid repeated freeze-thaw cycles, as these significantly reduce protein activity .

• For short-term use, working aliquots may be stored at 4°C for up to one week .

This storage methodology maintains protein integrity by preventing aggregation and denaturation that commonly affect recombinant proteins during freeze-thaw cycles.

How should Recombinant Protein AaeX be reconstituted for experimental use?

Follow this methodological approach for optimal reconstitution of AaeX:

• Briefly centrifuge the vial before opening to bring contents to the bottom .

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Allow complete dissolution by gentle mixing rather than vortexing to prevent protein denaturation.

• For long-term storage, add glycerol to a final concentration of 5-50% before aliquoting and freezing .

The reconstitution buffer (Tris/PBS-based buffer, 6% Trehalose, pH 8.0) is designed to maintain protein stability. If changing buffer conditions for specific experimental requirements, use dialysis or buffer exchange columns to gradually transition to the new buffer while monitoring protein stability.

What purification methods are most effective for Recombinant Protein AaeX?

For His-tagged Recombinant Protein AaeX, implement this methodological purification workflow:

• Primary capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ resins to capture the His-tagged protein .

• Intermediate purification: Ion-exchange chromatography, selecting the appropriate resin based on the protein's calculated isoelectric point. For AaeX, which likely has a calculated pI near neutral pH, both cation and anion exchange options should be tested at pilot scale to determine optimal binding .

• Polishing step: Size exclusion chromatography to remove aggregates and achieve >90% purity as determined by SDS-PAGE .

To minimize endotoxin contamination, consider incorporating an anion-exchange step specifically designed to remove DNA and lipopolysaccharide impurities from the feed .

How does the His-tagging of Recombinant Protein AaeX affect its structure and function?

The N-terminal His-tag used in commercially available Recombinant Protein AaeX can impact structural and functional properties through several mechanisms:

• Solubility effects: His-tags can increase solubility of membrane proteins like AaeX by masking hydrophobic regions, but may interfere with native membrane insertion.

• Metal ion interference: The His-tag's affinity for divalent metal ions (such as Zn2+, Co2+, Ni2+) may interfere with functional assays, particularly if AaeX interacts with metal ions as part of its native function.

• Structural perturbation: For small proteins like AaeX (67 amino acids), the His-tag (typically 6-10 amino acids) represents a significant proportion of the total sequence and may affect tertiary structure.

Methodological approach to address these concerns:

• Compare tagged and tag-cleaved versions using functional assays relevant to AaeX

• Employ circular dichroism spectroscopy to assess secondary structure differences

• Use tag cleavage systems (TEV protease, Factor Xa) if the tag interferes with function

• Consider C-terminal tagging as an alternative if N-terminal tagging disrupts function

What are the challenges in optimizing expression yields of Recombinant Protein AaeX in E. coli?

Optimizing AaeX expression in E. coli requires addressing several challenges typical of membrane proteins:

• Toxicity and growth inhibition: Overexpression of membrane proteins like AaeX can lead to cell toxicity and reduced yields .

  Methodological solution: Implement controlled expression systems with tunable promoters rather than strong constitutive promoters. The use of strains with enhanced membrane protein folding capacity can mitigate toxicity.

• Inclusion body formation: Hydrophobic regions in AaeX may promote aggregation.

  Methodological solution: Lower induction temperature (16-25°C), co-express with molecular chaperones, or use fusion tags that enhance solubility (SUMO, MBP, TRX) .

• Carbon metabolism optimization:

  Methodological solution: Implement diverse carbon and nitrogen sources and consider acetate metabolism knockout strains that redirect E. coli carbon fluxes, which has shown up to 5-fold increase in protein production in recent studies .

• Secretion pathway optimization:

  Methodological solution: Leverage specialized secretion systems like ESETEC technology or the TatExpress strain, which has demonstrated delivery of up to 5.4 g/L of recombinant proteins to the periplasm .

How can design of experiments (DOE) be applied to optimize purification of Recombinant Protein AaeX?

Design of experiments (DOE) provides a systematic framework for optimizing multiple parameters simultaneously in protein purification. For AaeX purification, implement this methodological approach:

DOE has been shown to effectively define all aspects of chromatographic separation with greater confidence, including optimal purification conditions for high throughput and yield, best resin combinations, and cost-effective process parameters .

What downstream processing strategies minimize endotoxin contamination in Recombinant Protein AaeX preparations?

Endotoxin contamination is a critical concern when producing recombinant proteins in E. coli for research applications. For AaeX, implement this comprehensive downstream processing strategy:

• Primary recovery optimization: The E. coli X-press strain has demonstrated significant advantages over traditional BL21(DE3), including 3.5-fold lower endotoxin load after primary recovery .

• Anion exchange chromatography: Implement anion exchange chromatography specifically to remove endotoxins, which are negatively charged. Studies have shown this can reduce endotoxin levels significantly while maintaining protein yield .

• Two-phase extraction: Consider aqueous two-phase partitioning as an alternative or complementary technique to chromatography for endotoxin removal .

• Membrane adsorbers: High-performance tangential flow filtration combined with membrane chromatography offers an efficient approach for endotoxin removal, particularly for larger scale preparations .

Comparative analysis of downstream processing strategies has shown that implementing these approaches can result in endotoxin reduction to levels below 0.5 EU/mg while maintaining protein activity and yield .

How do post-translational modifications affect the functionality of Recombinant Protein AaeX?

E. coli-expressed Recombinant Protein AaeX lacks eukaryotic post-translational modifications (PTMs), which may impact its functionality in certain research applications. Consider these methodological approaches:

• Assessment of required PTMs: Determine if AaeX requires specific PTMs for your research application by comparing E. coli-expressed protein with versions expressed in eukaryotic systems.

• Engineering E. coli for PTMs: Recent advances have enabled E. coli to perform simple glycosylation and other PTMs:

  • Engineered strains with transferred N-glycosylation systems from Campylobacter jejuni

  • Modified strains with enhanced disulfide bond formation capability

• Alternative expression systems if PTMs are critical:

  • Yeast systems for simple glycosylation patterns

  • Mammalian or insect cell systems for complex PTMs

• PTM simulation strategies:

  • Chemical modification methods for adding phosphate groups or glycans

  • In vitro enzymatic modification using purified PTM enzymes

Recent studies have shown that PTM machinery in E. coli can be modified through genetic engineering to produce proteins with specific modifications, though further research is needed to establish these systems for industrial production of therapeutic proteins .