Dengue Envelope-1 15kDa

What is the Dengue Envelope-1 15kDa protein?

The Dengue Envelope-1 15kDa is a genetically engineered recombinant protein derived from the C-terminus of Dengue Type-1 envelope protein. It contains neutralizing epitopes and the receptor binding domain critical for virus-host cell interactions. The protein is typically produced with a 6x His-Tag fused at its C-terminus to facilitate purification and detection . This domain plays a significant role in viral pathogenesis and represents a target for neutralizing antibodies in host immune responses.

How does Dengue Envelope-1 15kDa fit within the virus structure?

The 15kDa protein represents Domain III of the envelope protein from Dengue virus serotype 1. In the intact virus, the envelope protein forms part of the outer protein shell that mediates host cell attachment and membrane fusion. According to cryo-EM structural analysis, the envelope proteins form dimers that arrange in a herringbone pattern on the virus surface at neutral pH. During infection, these proteins undergo pH-regulated conformational changes essential for viral entry .

How is Dengue Envelope-1 15kDa relevant to serotype specificity?

Dengue virus exists as four distinct serotypes (DENV 1-4), each sufficiently different that there is no cross-protection between them, enabling epidemics caused by multiple serotypes (hyperendemicity) . The Envelope-1 15kDa represents the serotype-specific Domain III of DENV-1, containing epitopes that can elicit type-specific neutralizing antibodies. Understanding the structural and antigenic differences between equivalent domains from different serotypes is critical for comprehensive vaccine development.

What expression systems are optimal for producing Dengue Envelope-1 15kDa?

The protein is most commonly expressed in Escherichia coli expression systems . For optimal production:

• Use BL21(DE3) or similar strains with T7 RNA polymerase system

• Optimize induction conditions (temperature, IPTG concentration, duration)

• Consider lower expression temperatures (16-20°C) to improve protein solubility

• Supplement media with glucose to reduce basal expression

• Include protease inhibitors during extraction to prevent degradation

While E. coli is the standard system, researchers investigating conformational epitopes might consider eukaryotic expression systems for proper post-translational modifications.

What purification strategy yields the highest purity protein?

The standard purification approach takes advantage of the C-terminal 6x His-Tag :

• Initial capture using immobilized metal affinity chromatography (IMAC)

• Secondary purification via proprietary chromatographic techniques

• Quality assessment using 10% PAGE with Coomassie staining to verify >95% purity

• Buffer exchange into storage buffer (1xPBS pH 7.4 with 0.02% sodium nitrate)

• Endotoxin removal step when the protein is used for immunological studies

This multi-step approach consistently yields protein with >95% purity suitable for research applications .

What are the optimal storage conditions for Dengue Envelope-1 15kDa?

For maximum stability:

The protein should be stored in small aliquots to prevent repeated freeze-thaw cycles that could compromise structural integrity and activity .

How can researchers assess the structural integrity of stored protein?

To verify that the protein maintains its structural integrity after storage:

• SDS-PAGE under reducing and non-reducing conditions to check for aggregation

• Western blot with conformation-sensitive antibodies

• Functional binding assays with known interacting partners

• Circular dichroism to assess secondary structure maintenance

• Thermal shift assays to evaluate protein stability

Regular quality control is essential when using the protein for critical applications like vaccine development or binding studies.

How is Dengue Envelope-1 15kDa utilized in vaccine development research?

The protein has significant applications in vaccine research:

• As an antigen in subunit vaccine formulations

• For mapping neutralizing epitopes critical for protective immunity

• In studying antibody responses to Domain III across different serotypes

• For evaluating cross-protection potential between serotypes

• In structure-based vaccine design approaches

The presence of neutralizing epitopes and the receptor binding domain makes this protein particularly valuable for vaccine development studies .

What methods are most effective for studying receptor binding functions?

To investigate the receptor binding properties:

• Surface plasmon resonance (SPR) with purified potential receptor molecules

• Cell-based binding assays with receptor-expressing cell lines

• Competition assays with known ligands or blocking antibodies

• Site-directed mutagenesis to identify critical binding residues

• Fluorescence-based techniques to quantify binding affinity

Combining multiple approaches provides comprehensive understanding of the protein's interaction with host cell receptors.

What methodologies are recommended for studying interactions between Dengue Envelope-1 15kDa and host proteins?

For protein interaction studies:

• Co-immunoprecipitation with tagged Envelope-1 15kDa

• Pull-down assays using the recombinant protein as bait

• Surface plasmon resonance for quantitative binding kinetics

• ELISA-based binding assays for initial screening

• Structural studies using X-ray crystallography or cryo-EM for complex visualization

Understanding these interactions is crucial for elucidating mechanisms of viral entry and pathogenesis.

How can researchers investigate the interaction between Dengue Envelope protein and cellular actin?

Research has shown that actin interacts with Dengue virus envelope proteins, particularly from serotypes 2 and 4 . To study these interactions:

• Co-immunoprecipitation with anti-actin or anti-envelope antibodies

• Fluorescence microscopy to analyze co-localization patterns

• Western blot analysis of pulled-down complexes

• Mass spectrometry identification of interacting partners

• Functional assays to assess the biological significance of the interaction

Studies have confirmed interaction between actin and DENV E protein even in truncated forms, suggesting an important role in the viral life cycle .

What approaches can researchers use to study pH-dependent conformational changes?

The envelope protein undergoes critical pH-dependent conformational changes during viral entry . To study these with Domain III:

• Circular dichroism spectroscopy at varying pH conditions

• Intrinsic tryptophan fluorescence to detect structural shifts

• Limited proteolysis to identify exposed regions at different pH values

• Size exclusion chromatography to detect oligomerization state changes

• Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

Understanding these changes is crucial for developing inhibitors targeting the fusion process.

How should researchers design studies to evaluate antibody-dependent enhancement (ADE)?

For studying ADE, a phenomenon where sub-neutralizing antibodies can enhance infection:

• Prepare immune complexes using Dengue Envelope-1 15kDa and antibodies

• Use Fc receptor-expressing cell lines (THP-1, U937, K562)

• Compare infection rates between antibody-opsonized and non-opsonized virus

• Evaluate the enhancement effect of Domain III-specific antibodies

• Assess cytokine production profiles in response to enhanced infection

This is critical for vaccine safety assessment, as vaccines must minimize ADE risk while maximizing protection.

What strategies can address protein aggregation issues?

To minimize aggregation problems:

• Optimize buffer conditions (pH, ionic strength, additives)

• Include low concentrations of non-ionic detergents in storage buffer

• Add stabilizing agents like glycerol (5-10%)

• Maintain lower protein concentrations during storage

• Consider fusion partners that enhance solubility

• Use size exclusion chromatography to remove aggregates before experiments

Proper handling significantly impacts experimental reproducibility and reliability.

How can researchers validate the correct folding of recombinant protein?

To ensure proper folding:

• Binding studies with conformation-dependent monoclonal antibodies

• Circular dichroism to assess secondary structure elements

• Thermal shift assays to evaluate structural stability

• Functional binding assays with known receptors

• Comparison with native virus-derived protein where possible

Confirmation of proper folding is essential for meaningful immunological and structural studies.

How does Domain III from DENV-1 compare structurally with other serotypes?

Comparative analysis shows:

• Domain III maintains the immunoglobulin-like fold across serotypes

• Sequence variations occur primarily in surface-exposed loops

• These variations contribute to serotype-specific neutralizing epitopes

• Cross-reactive epitopes exist but vary in accessibility

• Receptor binding residues show different degrees of conservation

These differences explain the limited cross-protection between serotypes and highlight the challenges in developing tetravalent vaccines.

What methodological approaches are best for cross-serotype comparative studies?

For comprehensive cross-serotype analysis:

• Parallel expression and purification of Domain III from all four serotypes

• Consistent experimental conditions for valid comparisons

• Cross-competition binding assays with serotype-specific antibodies

• Comparative epitope mapping using alanine scanning mutagenesis

• Side-by-side evaluation in functional assays

• Structural alignment of domains from different serotypes

This approach provides insights into serotype-specific immunity and potential for cross-protection.