Recombinant Neurospora crassa Cyanovirin-N homolog (NCU05495)

What is Recombinant Neurospora crassa Cyanovirin-N homolog (NCU05495)?

Recombinant Neurospora crassa Cyanovirin-N homolog (NCU05495) is a purified recombinant protein derived from the filamentous fungus Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987). It belongs to the Cyanovirin-N homolog family of lectins, which are characterized by their ability to bind to high-mannose glycans. The protein is typically expressed in E. coli with an N-terminal 6xHis tag and has a theoretical molecular weight of 16.9 kDa. The expression region spans amino acids 1-111 of the native protein sequence, and the protein demonstrates high purity levels (>90%) when analyzed by SDS-PAGE .

How does NCU05495 compare structurally to other Cyanovirin-N homologs?

Cyanovirin-N homologs (CVNHs) typically share a similar structural architecture despite varying degrees of sequence identity. Like the well-characterized Cyanovirin-N (CV-N), NCU05495 possesses a structure comprising two domains, each containing carbohydrate-binding sites specific for Manα(1–2)Manα epitopes found on high-mannose glycans. While CV-N from cyanobacterium exhibits approximately 43% sequence identity with certain CVNH proteins (such as Cyt-CVNH from Cyanothece 7424), the structural fold is conserved across the family. The crystal structure analysis of CVNH proteins has revealed that they maintain the characteristic domain architecture that facilitates their glycan binding capabilities .

What are the optimal storage conditions for Recombinant NCU05495?

For optimal preservation of protein activity, Recombinant Neurospora crassa Cyanovirin-N homolog (NCU05495) should be stored at -20°C. The protein is typically supplied in one of two forms: (1) as a liquid in Tris/PBS-based buffer containing 5-50% glycerol, or (2) as a lyophilized powder that was originally prepared in Tris/PBS-based buffer with 6% Trehalose at pH 8.0. It is crucial to avoid repeated freeze/thaw cycles, as these can lead to protein denaturation and loss of activity. For research applications requiring frequent use, it is advisable to prepare small working aliquots from the stock solution to minimize freeze/thaw cycles. When working with the lyophilized form, reconstitution should be performed according to the manufacturer's protocol to ensure proper protein folding and activity .

How do the glycan binding properties of NCU05495 differ from those of other Cyanovirin-N homologs?

The binding kinetics also differ between CVNH proteins. For example, Cyt-CVNH exhibits dissociation constants (Kd) of approximately 500 nM for Man-9 binding to both domains, indicating relatively equal affinity across the protein. This balanced affinity profile may explain the lack of cross-linking and precipitation observed with Cyt-CVNH compared to CV-N. These distinctions are crucial for researchers designing experiments to investigate carbohydrate-protein interactions and may have significant implications for potential antiviral applications .

What methodological approaches are most effective for studying NCU05495-glycan interactions?

Based on research with related CVNH proteins, several methodological approaches are effective for studying NCU05495-glycan interactions:

A comprehensive approach combining these techniques would provide the most complete understanding of NCU05495's interaction with high-mannose glycans and potential binding partners .

What is the potential of NCU05495 for antiviral research applications?

Based on research with structurally similar Cyanovirin-N homologs, NCU05495 may hold significant promise for antiviral research, particularly against enveloped viruses that display high-mannose glycans on their surface proteins. The mechanistic basis for antiviral activity in this protein family involves binding to Manα(1–2)Manα epitopes on viral envelope glycoproteins, such as HIV-1 gp120, thereby preventing viral attachment and entry into host cells .

Some CVNH proteins have demonstrated potent anti-HIV activity in the low nanomolar range, with certain homologs (like Cyt-CVNH) exhibiting approximately 4-fold higher potency than the prototype CV-N. The absence of cross-linking and precipitation issues that plague some CVNHs might make NCU05495 particularly valuable for developing stable antiviral formulations .

Researchers exploring NCU05495's antiviral potential should consider:

• Evaluating binding specificity against different glycan structures present on viral envelope proteins

• Assessing inhibitory activity against viral infection in cellular models

• Investigating potential synergistic effects with other antiviral agents

• Exploring structure-function relationships to enhance antiviral efficacy

• Evaluating potential cytotoxicity and immunogenicity issues that might limit therapeutic applications

What expression systems are optimal for producing functional NCU05495?

The selection of an appropriate expression system is critical for obtaining high-quality, functional Recombinant Neurospora crassa Cyanovirin-N homolog (NCU05495). Current commercial preparations typically utilize E. coli as the expression host, which offers several advantages for protein production :

For researchers working with NCU05495, the E. coli system offers a balance of cost-effectiveness and yield, particularly when the protein is not dependent on host-specific post-translational modifications for its function. The N-terminal 6xHis tag commonly used (as in commercial preparations) facilitates efficient purification through immobilized metal affinity chromatography (IMAC) .

How can researchers effectively characterize the purity and functionality of NCU05495 preparations?

Comprehensive characterization of NCU05495 preparations should include both purity assessment and functional validation. The following methodological approach is recommended:

• Purity Assessment:

  • SDS-PAGE with Coomassie staining (expecting >90% purity)

  • Western blot using anti-His antibodies to confirm tag presence

  • Size exclusion chromatography to evaluate aggregation state

  • Mass spectrometry to confirm molecular weight (theoretical MW: 16.9 kDa) and sequence integrity

• Functional Validation:

  • Glycan binding assays using purified high-mannose oligosaccharides

  • Surface plasmon resonance to determine binding kinetics

  • Circular dichroism spectroscopy to evaluate secondary structure

  • Thermal shift assays to assess protein stability

  • Functional antiviral assays if evaluating inhibitory potential

• Quality Control Parameters:

  • Endotoxin testing (particularly important for E. coli-expressed proteins)

  • Aggregation analysis via dynamic light scattering

  • Freeze-thaw stability assessment

  • pH and buffer composition optimization

A typical validation workflow would begin with purity assessment followed by structural characterization and culminate in functional binding assays. This systematic approach ensures that the protein preparation is suitable for downstream research applications .

What analytical methods are most appropriate for studying NCU05495-glycan binding specificity?

Understanding the glycan binding specificity of NCU05495 requires sophisticated analytical approaches. Based on methods used with similar CVNH proteins, the following techniques are recommended:

• Glycan Array Screening:

  • Provides a high-throughput assessment of binding to diverse glycan structures

  • Allows identification of specific glycan epitopes recognized by NCU05495

  • Requires fluorescently labeled protein or secondary detection methods

• NMR Spectroscopy:

  • 1H-15N HSQC experiments to map chemical shift perturbations upon glycan binding

  • Titration studies to determine binding affinities (Kd values)

  • STD-NMR to identify glycan epitopes in direct contact with the protein

  • Particularly valuable since some CVNH proteins do not exhibit the cross-linking and precipitation issues observed with CV-N

• X-ray Crystallography of Protein-Glycan Complexes:

  • Provides atomic-level details of binding interactions

  • Reveals specific hydrogen bonding and van der Waals contacts

  • Informs structure-based design of modified proteins with altered specificity

• Isothermal Titration Calorimetry (ITC):

  • Determines thermodynamic parameters (ΔH, ΔS, ΔG)

  • Quantifies binding stoichiometry

  • Provides dissociation constants independent of fluorescent labeling

• Bio-Layer Interferometry or Surface Plasmon Resonance:

  • Measures real-time kinetics of association and dissociation

  • Determines kon and koff rates in addition to equilibrium constants

  • Allows for comparison of different glycan structures under identical conditions

Combined application of these methods provides complementary data for a comprehensive understanding of NCU05495's glycan recognition properties .

How can researchers address inconsistent results in glycan binding studies with NCU05495?

Inconsistent results in glycan binding studies with NCU05495 can stem from multiple sources. A systematic troubleshooting approach should consider:

• Protein Quality Issues:

  • Verify protein purity by SDS-PAGE and mass spectrometry

  • Assess protein folding using circular dichroism spectroscopy

  • Evaluate aggregation state with size exclusion chromatography or dynamic light scattering

  • Check for oxidation of critical residues using mass spectrometry

• Experimental Condition Variables:

  • Standardize buffer composition, pH, and ionic strength

  • Control temperature consistently across experiments

  • Validate glycan quality and purity

  • Minimize freeze-thaw cycles of protein samples

• Methodological Considerations:

  • Compare results across different analytical platforms

  • Calibrate instruments properly with appropriate standards

  • Include positive and negative controls in each experiment

  • Consider potential interfering factors specific to each method

• Data Analysis Approaches:

  • Apply consistent mathematical models for binding data

  • Use statistical methods to evaluate reproducibility

  • Implement global fitting approaches when appropriate

  • Consider potential multi-site binding effects

Table of Common Issues and Solutions:

By systematically addressing these factors, researchers can improve the reproducibility and reliability of NCU05495 glycan binding studies .

What are the critical factors in designing valid control experiments for NCU05495 functional studies?

Designing rigorous control experiments is essential for validating the specificity and biological relevance of observed NCU05495 functions. Critical control considerations include:

• Protein-Specific Controls:

  • Heat-denatured NCU05495 to confirm structure-dependent activity

  • Site-directed mutants with altered binding sites to establish structure-function relationships

  • Related CVNH proteins (e.g., CV-N) to compare family-wide vs. protein-specific effects

  • Non-binding protein controls with similar size/charge properties

• Glycan-Specific Controls:

  • Structurally related glycans lacking the Manα(1–2)Manα epitope

  • Competitive inhibition with free mannose or mannobiose

  • Enzymatically modified glycans with specific linkages removed

  • Synthetic glycan analogs with defined structural features

• Experimental System Controls:

  • Vehicle controls for all buffers and solvents

  • Cell line authentication for cellular assays

  • Time-matched sampling to account for temporal variations

  • Concentration gradients to establish dose-dependency

• Technical Controls:

  • Multiple detection methods to confirm observations

  • Internal standards for quantitative measurements

  • Randomization of sample order to minimize bias

  • Blinded analysis where appropriate

Implementation of these control strategies ensures that observed effects are specifically attributable to NCU05495-glycan interactions rather than experimental artifacts or non-specific effects .

How does NCU05495 compare functionally to other lectins used in glycobiology research?

NCU05495 belongs to the Cyanovirin-N homolog family of lectins, which possess distinctive characteristics compared to other lectin families used in glycobiology research. Understanding these comparative differences is crucial for selecting the appropriate lectin for specific research applications:

The unique advantage of NCU05495 and other CVNH proteins lies in their exquisite specificity for Manα(1–2)Manα epitopes, which are particularly relevant for HIV envelope glycoprotein recognition. Additionally, certain CVNH proteins (like Cyt-CVNH) demonstrate advantages over CV-N, including lack of aggregation upon Man-9 binding and potentially higher antiviral potency. These characteristics make NCU05495 particularly valuable for specialized glycobiology applications focused on high-mannose structures .

What future research directions are most promising for NCU05495 applications?

Based on current understanding of CVNH proteins and their properties, several promising research directions for NCU05495 warrant exploration:

• Structure-Function Engineering:

  • Rational design of NCU05495 variants with enhanced specificity

  • Domain-swapping experiments with other CVNH proteins

  • Engineering multivalent constructs for increased avidity

  • Computational modeling to predict binding modifications

• Expanded Antiviral Applications:

  • Evaluation against diverse enveloped viruses beyond HIV

  • Development of NCU05495-based capture systems for viral diagnostics

  • Investigation of synergistic effects with other antiviral agents

  • Exploration of delivery systems for potential therapeutic applications

• Glycobiology Tool Development:

  • Creation of NCU05495-based affinity reagents for high-mannose glycan enrichment

  • Development of biosensors incorporating NCU05495 for glycan detection

  • Application in glycoproteomic workflows for selective capture

  • Generation of fluorescently labeled variants for imaging applications

• Structural Biology Investigations:

  • High-resolution structures of NCU05495-glycan complexes

  • Molecular dynamics simulations of binding events

  • NMR studies of protein-glycan interactions in solution

  • Comparative analysis with other CVNH family members

• Biological Function Exploration:

  • Investigation of natural role in Neurospora crassa

  • Comparative genomics across fungal species

  • Evolutionary analysis of CVNH protein family

  • Ecological significance of glycan recognition

These research directions would significantly advance understanding of NCU05495 while potentially yielding valuable tools and applications for glycobiology, virology, and structural biology .