Recombinant Gloydius halys Coagulation factor IX-binding protein subunit A

How does the protein's calcium-binding property influence its function?

The calcium-binding property is essential for the protein's structure and function. Gloydius halys Coagulation factor IX-binding protein binds two calcium ions per molecule with dissociation constant (Kd) values of 16±0.7 and 103±10 μM . These calcium ions play crucial roles:

• Structural stabilization through coordination with specific amino acid residues

• Facilitation of proper protein folding

• Mediation of binding to coagulation factors IX and X

The protein contains conserved calcium-binding site residues, typically including Ser-41 and Glu-47. The calcium-dependent binding is a hallmark feature of C-type lectin-like proteins from snake venoms .

Interestingly, magnesium ions can affect the calcium requirement. Upon addition of magnesium ions, the required calcium concentration for optimal binding to factor IX and factor X is prominently reduced, and magnesium also increases the affinity of factor X for the venom protein .

What is known about the disulfide bond pattern in this protein?

The disulfide bond pattern is critical for the structural stability and proper folding of Gloydius halys Coagulation factor IX-binding protein subunit A. Based on studies of similar proteins, the typical disulfide bond pattern involves:

• Conserved disulfide bridges that stabilize the protein core structure

• An interchain disulfide bond that connects subunit A with subunit B to form the functional heterodimer

Specifically, patterns observed in related proteins include bridges at Cys2-Cys13, Cys30-Cys127, and Cys102-Cys119, coupled with an interchain bond involving Cys-79 .

The disulfide bond pattern follows a typical arrangement found in the C-type lectin-like domain, with a characteristic pattern: C0–C0′ (interchain), C1–C4, and C2–C3 (intrachain). This arrangement contributes to the formation of the "bow tie-like structure" that provides a central concave area serving as the ligand binding site .

What expression systems are most effective for producing this recombinant protein?

Several expression systems can be used for producing recombinant Gloydius halys Coagulation factor IX-binding protein subunit A, each with distinct advantages:

The commercially available recombinant protein is successfully produced in E. coli with >85% purity, suggesting this is a viable approach for most research applications . For E. coli expression, several factors must be optimized:

• Codon optimization for bacterial expression

• Selection of appropriate promoter and expression vector

• Optimization of induction conditions (temperature, inducer concentration)

• Selection of appropriate E. coli strain (BL21(DE3), Rosetta, SHuffle)

What are the critical challenges in expressing this protein and how can they be addressed?

Expressing recombinant Gloydius halys Coagulation factor IX-binding protein subunit A presents several key challenges:

• Disulfide bond formation: The protein contains multiple cysteine residues that form specific disulfide bonds critical for maintaining correct three-dimensional structure. In E. coli cytoplasm, the reducing environment can impede proper disulfide bond formation.

  • Solution: Use specialized E. coli strains engineered for enhanced disulfide bond formation (Origami, SHuffle) or direct the protein to the periplasmic space.

• Protein folding: C-type lectin-like proteins have a specific fold that may be challenging to achieve in heterologous systems.

  • Solution: Optimize expression conditions (temperature, pH) and consider co-expression with chaperone proteins that assist in proper folding.

• Calcium binding: The protein requires calcium ions for proper structure and function.

  • Solution: Include calcium in expression and purification buffers to facilitate proper folding and maintain stability.

• Heterodimeric structure: The native protein exists as a heterodimer with subunit B.

  • Solution: For full functionality, consider co-expression strategies for both subunits or reconstitute the heterodimer in vitro after separate purification.

• Solubility issues: Recombinant proteins may form inclusion bodies or aggregate when overexpressed.

  • Solution: Lower induction temperature (16-25°C), reduce inducer concentration, or add solubility-enhancing fusion tags like MBP or SUMO .

What purification strategies yield the highest purity and functional activity?

Optimal purification of recombinant Gloydius halys Coagulation factor IX-binding protein subunit A typically involves a multi-step approach:

• Initial capture: Affinity chromatography using an appropriate tag (His, GST, MBP) allows selective binding of the tagged protein to a specific resin.

• Intermediate purification: Ion exchange chromatography separates proteins based on their charge properties. Since the protein has a defined isoelectric point, this method can effectively remove contaminants with different charges.

• Polishing: Size exclusion chromatography (gel filtration) separates proteins based on size, removing aggregates and providing the final purity.

• Specialized approaches:

  • Calcium-dependent purification: Incorporating calcium-dependent elution steps can select for properly folded, functional protein.

  • Factor IX affinity chromatography: Using immobilized factor IX as an affinity ligand to select specifically for functional protein.

Throughout the purification process, critical parameters must be maintained:

• Presence of calcium ions in buffers (typically 1-5 mM)

• Appropriate pH (usually pH 7-8)

• Suitable ionic strength to maintain protein stability

• Inclusion of reducing agents (at low concentrations) to prevent unwanted disulfide bond formation

Using this approach, commercial preparations achieve >85% purity as assessed by SDS-PAGE . Functional activity should be verified through binding assays with factors IX and X and calcium-binding assessments.

How can this recombinant protein be used to study coagulation mechanisms?

Recombinant Gloydius halys Coagulation factor IX-binding protein subunit A offers valuable tools for studying coagulation mechanisms:

• Investigation of factor IX/X binding mechanisms:

  • The protein binds differentially to factors IX and X (Kd values of 3 nM and 0.4 nM, respectively), allowing for investigation of structural requirements for this differential binding .

  • Structure-function studies can identify key residues involved in the interaction between the protein and coagulation factors.

• Dissection of coagulation pathways:

  • The protein can be used as a specific inhibitor to block the activities of factors IX and X selectively.

  • This selective inhibition allows researchers to determine the relative contributions of these factors in different experimental contexts.

• Evaluation of calcium and magnesium effects on coagulation:

  • The protein's binding to factors IX and X is calcium-dependent, and magnesium modulates this calcium requirement .

  • This property can be exploited to study the roles of these divalent cations in coagulation.

• Structure-based studies of the Gla domain:

  • The protein binds to the Gla domain (GD) of factor X, with the binding inhibited by GD peptide 1-44 but not by GD peptide 1-41 or Gla domainless factor X .

  • This specificity can be used to study structure-function relationships in the Gla domain.

• Comparative studies with other anticoagulant proteins:

  • The protein can be compared with other C-type lectin-like proteins from different snake species to understand evolutionary adaptations in anticoagulant mechanisms .

What experimental methods can verify the functional activity of the recombinant protein?

Verifying the functional activity of recombinant Gloydius halys Coagulation factor IX-binding protein subunit A requires multiple complementary approaches:

For a comprehensive assessment, it is advisable to use multiple methods that evaluate different aspects of protein function, as no single assay can confirm complete functional integrity.

How can structural studies inform understanding of target binding specificity?

Structural studies of recombinant Gloydius halys Coagulation factor IX-binding protein subunit A can provide critical insights into binding specificity through several approaches:

• Crystallographic analysis:

  • X-ray crystallography of the protein alone or in complex with factors IX or X can reveal:

    • Binding interface residues

    • Conformational changes upon ligand binding

    • Role of calcium ions in the binding site

  • These structures would complement the existing three-dimensional models constructed from known folds of similar proteins .

• Molecular determinants of binding specificity:

  • Analysis of the concave surface between the subunits, which is considered the coagulation factor-binding site .

  • Identification of amino acid residues that differ from other C-type lectin-like proteins and act as "discriminators" for ligand binding .

  • Investigation of the molecular basis for the differential binding to factors IX versus X.

• Calcium coordination geometry:

  • Determination of the exact coordination geometry of the two calcium ions bound by the protein.

  • Understanding how calcium binding influences the conformation of the binding site.

  • Elucidation of how magnesium modulates calcium binding and subsequent interactions with coagulation factors .

• Comparative structural biology:

  • Comparison with other C-type lectin-like proteins that target different molecules in the coagulation cascade.

  • Phylogenetic analysis has shown that C-type lectin-like proteins cluster based on both species of origin and target specificity .

  • Structural comparison can reveal convergent or divergent evolution of binding mechanisms.

• Gla domain interaction analysis:

  • Detailed analysis of how the protein interacts with the Gla domain of factor X.

  • Understanding why the C-terminal region of the Gla domain peptide (residues 41-44) is critical for binding .

  • Elucidation of the role of calcium in mediating this interaction.

What protein engineering approaches could enhance this protein's research utility?

Several protein engineering strategies could enhance the research utility of recombinant Gloydius halys Coagulation factor IX-binding protein subunit A:

How might this protein be utilized in developing new anticoagulant therapies?

Recombinant Gloydius halys Coagulation factor IX-binding protein subunit A offers several promising avenues for developing new anticoagulant therapies:

• Template for rational drug design:

  • The protein's specific binding to factors IX and X provides a structural template for designing small molecule inhibitors.

  • Crystallographic analysis of protein-factor complexes could reveal binding pockets suitable for small molecule targeting.

  • Computational approaches could screen for compounds that mimic the protein's interaction with coagulation factors.

• Peptide-based anticoagulants:

  • Identification of minimal binding regions that retain anticoagulant activity.

  • Development of peptide mimetics based on the active regions of the protein.

  • Optimization of these peptides for improved pharmacokinetics and reduced immunogenicity.

• Recombinant protein therapeutics:

  • Engineering the protein itself for:

    • Increased half-life in circulation

    • Reduced immunogenicity

    • Enhanced specificity for target coagulation factors

    • Improved stability under physiological conditions

  • Development of fusion proteins with albumin or Fc regions to extend circulation time.

• Targeted anticoagulation strategies:

  • Creation of fusion proteins that combine the factor-binding domain with targeting moieties.

  • These could direct anticoagulant activity to specific sites, such as forming thrombi.

  • This approach could reduce bleeding risk associated with current anticoagulants.

• Combination therapy approaches:

  • Development of strategies that combine the protein's specific inhibition of factors IX and X with other anticoagulant mechanisms.

  • This could allow for lower doses of individual agents and reduced side effects.

• Diagnostic applications supporting therapy:

  • Development of assays to monitor levels of factors IX and X during anticoagulant therapy.

  • Creation of point-of-care testing devices for rapid assessment of coagulation status.

  • These tools would enable more precise anticoagulant dosing.

• Novel delivery systems:

  • Exploration of nanoparticle-based delivery systems for the protein or derived peptides.

  • Investigation of controlled-release formulations for sustained anticoagulant activity.

What strategies can improve expression yields and solubility of this recombinant protein?

Optimizing expression yields and solubility of recombinant Gloydius halys Coagulation factor IX-binding protein subunit A involves systematic approaches:

• Expression vector optimization:

  • Codon optimization: Adapt the coding sequence to match the codon usage bias of the expression host.

  • Promoter selection: Compare different promoters (T7, tac, pBAD) for optimal expression levels.

  • Fusion tags: Incorporate solubility-enhancing tags such as:

    • Maltose-binding protein (MBP)

    • Small ubiquitin-like modifier (SUMO)

    • Thioredoxin (TrxA)

    • These tags can dramatically improve solubility and can be removed after purification.

• Expression condition optimization:

  • Temperature modulation: Lower temperatures (16-25°C) often improve solubility significantly.

  • Induction parameters: Test different inducer concentrations and induction timings.

  • Media composition: Evaluate defined media vs. rich media formulations.

  • Growth phase: Induce at different cell densities to find optimal balance.

• Host strain selection:

  • For disulfide bond formation: Use specialized strains like Origami or SHuffle.

  • For rare codons: Consider Rosetta or CodonPlus strains.

  • For toxic proteins: C41/C43 strains are designed for toxic protein expression.

  • For general high-level expression: BL21(DE3) and its derivatives.

• Co-expression strategies:

  • Chaperone co-expression: Include molecular chaperones like GroEL/ES or DnaK to assist folding.

  • Disulfide isomerases: Co-express DsbA or DsbC to facilitate correct disulfide bond formation.

  • Both subunits: For heterodimer formation, co-express both subunits A and B.

• Buffer optimization during cell lysis and purification:

  • pH optimization: Test different pH values to find optimal solubility conditions.

  • Salt concentration: Evaluate various ionic strengths.

  • Additives: Include stabilizing agents such as:

    • Glycerol (5-10%)

    • Low concentrations of detergents (0.05-0.1% Triton X-100)

    • Arginine (50-500 mM)

    • Calcium ions (1-5 mM)

• Refolding strategies (if inclusion bodies form):

  • Controlled denaturation and refolding: Use urea or guanidine hydrochloride for denaturation followed by stepwise dialysis.

  • Oxidative refolding: Create conditions for proper disulfide bond formation during refolding.

  • Pulse renaturation: Add denatured protein gradually to refolding buffer.

What are the common pitfalls in working with this protein and how can they be avoided?

Working with recombinant Gloydius halys Coagulation factor IX-binding protein subunit A presents several challenges that researchers should anticipate:

• Incorrect disulfide bond formation:

  • Pitfall: Random disulfide bond formation leading to misfolded, inactive protein.

  • Solution: Use oxidizing environments for expression (specialized E. coli strains or periplasmic expression) or controlled oxidative refolding protocols with appropriate redox buffers (typically containing reduced and oxidized glutathione).

• Inadequate calcium binding:

  • Pitfall: Protein lacking proper calcium incorporation, resulting in unstable structure and reduced activity.

  • Solution: Include calcium ions (1-5 mM) in all buffers during purification and storage. Verify calcium binding through functional assays.

• Heterogeneity in protein preparations:

  • Pitfall: Multiple protein species with different folding states or disulfide arrangements.

  • Solution: Implement additional purification steps to isolate the correctly folded species, such as ion exchange chromatography under conditions that separate properly folded from misfolded forms.

• Protein aggregation during storage:

  • Pitfall: Loss of activity due to protein aggregation over time.

  • Solution: Optimize storage conditions (temperature, buffer composition), consider adding stabilizers like glycerol (10-20%), and aliquot protein to avoid freeze-thaw cycles.

• Inconsistent activity in functional assays:

  • Pitfall: Variable results in binding or anticoagulant assays.

  • Solution: Standardize assay conditions, particularly calcium concentration, pH, and temperature. Include positive controls (such as native snake venom fractions if available).

• Proteolytic degradation:

  • Pitfall: Loss of intact protein due to proteolytic cleavage.

  • Solution: Include protease inhibitors during purification, minimize processing time, and consider engineering protease-resistant variants.

• Tag interference with function:

  • Pitfall: Affinity tags affecting protein structure or function.

  • Solution: Compare activity before and after tag removal, or test different tag positions (N-terminal vs. C-terminal) to find the optimal configuration.

• Single subunit limitations:

  • Pitfall: Expressing only subunit A when the native protein functions as a heterodimer.

  • Solution: If complete biological activity is required, express and purify both subunits and reconstitute the heterodimer, or engineer a single-chain version.

By anticipating these challenges and implementing appropriate strategies, researchers can significantly improve their success in working with this complex protein.

How does this protein compare to other snake venom C-type lectin-like proteins affecting coagulation?

Gloydius halys Coagulation factor IX-binding protein subunit A can be compared to other snake venom C-type lectin-like proteins (CTLPs) across several dimensions:

Specific comparisons with other well-characterized CTLPs include:

The diversity of CTLPs across snake species represents a fascinating example of convergent evolution toward manipulating host coagulation systems through different molecular targets in the coagulation cascade.

What unique characteristics distinguish this protein for research applications?

Recombinant Gloydius halys Coagulation factor IX-binding protein subunit A possesses several distinctive characteristics that make it valuable for research applications:

• Dual-target specificity with differential affinity:

  • The protein binds both factor IX (Kd = 3 nM) and factor X (Kd = 0.4 nM) with different affinities .

  • This differential binding enables comparative studies of structural requirements for interaction with these closely related but distinct coagulation factors.

  • Few other proteins offer this specific combination of target selectivity.

• Well-characterized calcium dependence:

  • The protein binds two calcium ions with distinct affinities (Kd values of 16±0.7 and 103±10 μM) .

  • This property makes it an excellent model for studying calcium-dependent protein-protein interactions in the coagulation cascade.

  • The interplay between calcium and magnesium binding provides additional dimensions for research .

• Gla domain recognition specificity:

  • The protein specifically recognizes the C-terminal region of the Gla domain, with binding inhibited by Gla domain peptide 1-44 but not by peptide 1-41 .

  • This precise recognition specificity makes it a valuable tool for structure-function studies of Gla domains.

• Evolutionary significance:

  • The protein represents one group in a phylogenetically diverse family of C-type lectin-like proteins .

  • This evolutionary context provides opportunities for comparative studies on protein evolution and adaptation.

• Recombinant expression advantages:

  • The protein can be successfully expressed in E. coli with good yield and purity (>85%) .

  • This enables cost-effective production for research purposes without requiring venomous snake handling.

  • The availability of the recombinant form allows for protein engineering and modification studies.

• Structural predictability:

  • The protein adopts the well-characterized C-type lectin fold, making it amenable to homology modeling and structure prediction .

  • This structural understanding facilitates rational design of experiments and interpretation of results.

• Coagulation research applications:

  • The specific inhibition of factors IX and X allows for targeted disruption of specific pathways in the coagulation cascade.

  • This selectivity makes it valuable for dissecting the roles of these factors in different experimental contexts.

These unique characteristics make recombinant Gloydius halys Coagulation factor IX-binding protein subunit A a particularly valuable tool for coagulation research, protein-protein interaction studies, and anticoagulant drug development.

What are the emerging applications for this protein in advanced coagulation research?

Recombinant Gloydius halys Coagulation factor IX-binding protein subunit A is positioned at the forefront of several emerging research areas:

• Precision anticoagulation strategies:

  • Development of highly targeted anticoagulants that specifically inhibit factors IX and X.

  • Design of zone-specific anticoagulation therapies that target only the sites of pathological coagulation.

  • This approach could minimize bleeding risks associated with conventional anticoagulants.

• Biomarker development for coagulation disorders:

  • Creation of novel diagnostic tools using the protein's specific binding properties.

  • Development of assays to detect abnormal levels or functions of factors IX and X.

  • These tools could improve diagnosis and management of coagulation disorders.

• Structural biology of coagulation factors:

  • Use of the protein as a crystallization chaperone to facilitate structure determination of coagulation factors.

  • Investigation of conformational changes in factors IX and X upon binding.

  • These studies could reveal new druggable pockets in coagulation factors.

• Nanotechnology applications:

  • Integration of the protein into nanoscale diagnostic devices for point-of-care coagulation testing.

  • Development of nanoparticles functionalized with the protein for targeted delivery of anticoagulants.

  • These applications could revolutionize both diagnosis and treatment of thrombotic disorders.

• Synthetic biology approaches:

  • Creation of engineered cells expressing the protein for localized anticoagulation in artificial tissues or organs.

  • Development of cell-based delivery systems for sustained anticoagulant therapy.

  • These approaches could address challenges in long-term anticoagulation management.

• Mathematical modeling of coagulation dynamics:

  • Use of the protein as a specific inhibitor in systems biology approaches to understand the coagulation network.

  • Development of predictive models for coagulation behavior under different conditions.

  • These models could improve individualized anticoagulant dosing strategies.

• Biomaterial development:

  • Incorporation of the protein or derived peptides into blood-contacting materials to create non-thrombogenic surfaces.

  • Development of wound dressings with controlled anticoagulant properties for management of bleeding.

  • These materials could find applications in medical devices and tissue engineering.

What interdisciplinary research opportunities exist combining this protein with emerging technologies?

The unique properties of recombinant Gloydius halys Coagulation factor IX-binding protein subunit A create exciting opportunities for interdisciplinary research:

• Integration with microfluidic technologies:

  • Development of "organ-on-a-chip" models incorporating the protein to study coagulation dynamics under flow conditions.

  • Creation of microfluidic diagnostic devices for rapid assessment of coagulation status.

  • These systems could provide insights into thrombosis formation in various pathological states.

• Computational biology and artificial intelligence:

  • Application of machine learning to predict binding interactions with modified coagulation factors.

  • Development of in silico models to design optimized variants with enhanced specificity or activity.

  • These approaches could accelerate the development of new anticoagulant strategies.

• Biosensor development:

  • Creation of electrochemical or optical biosensors using the protein as a recognition element.

  • Development of continuous monitoring systems for coagulation status.

  • These sensors could transform management of patients requiring anticoagulation.

• CRISPR-Cas9 gene editing:

  • Use of the protein to validate therapeutic targets identified through gene editing of coagulation factors.

  • Development of cell-based models with engineered coagulation factors to study binding interactions.

  • This could provide new insights into structure-function relationships in coagulation factors.

• Proteomics and systems biology:

  • Application of proteomics approaches to identify additional binding partners beyond factors IX and X.

  • Integration of these findings into systems biology models of coagulation.

  • This could reveal previously unknown connections in the coagulation network.

• Materials science:

  • Development of smart biomaterials that release the protein in response to coagulation triggers.

  • Creation of surface-functionalized materials with controlled anticoagulant properties.

  • These materials could find applications in extracorporeal circulation devices and implantable medical devices.

• Imaging technology integration:

  • Development of molecular imaging probes based on the protein for visualization of active coagulation sites.

  • Creation of dual-function molecules that both detect and inhibit pathological coagulation.

  • These tools could transform diagnosis and treatment of thrombotic disorders.

• Immunoengineering:

  • Investigation of immune responses to the protein and development of strategies to minimize immunogenicity.

  • Creation of immunomodulatory fusion proteins combining coagulation factor binding with immune regulation.

  • This could lead to novel approaches for treating coagulation disorders with inflammatory components.

By pursuing these interdisciplinary opportunities, researchers can leverage the unique properties of this protein to address complex challenges in understanding and managing coagulation disorders.