Recombinant Human Protein NOV homolog protein (NOV) (Active)

What is the molecular structure of Recombinant Human NOV protein?

Recombinant Human NOV (Nephroblastoma overexpressed) is a 36.2 kDa protein containing 331 amino acid residues (expression region 28-357aa). The protein comprises four distinct structural domains: the IGF binding protein (IGFBP) domain, von Willebrand Factor C (VWFC) domain, Thrombospondin type-I (TSP type-1) domain, and a C-terminal cysteine knot-like domain (CTCK). This multimodular structure enables NOV to interact with various cellular components and participate in diverse signaling pathways . The full amino acid sequence starts with M+QVAATQRCP and continues through a cysteine-rich sequence that facilitates its structural integrity and functional properties .

How does NOV relate to other CCN family proteins?

NOV (also known as CCN3) belongs to the CCN family of secreted, cysteine-rich regulatory proteins. These proteins share a common multimodular structure but exhibit distinct and sometimes opposing biological activities. While other family members like CCN1 and CCN2 may promote cell proliferation in certain contexts, NOV often shows differential effects depending on the cellular environment. Understanding these relationships is crucial when designing experiments that may involve multiple CCN family members, as cross-reactivity or compensatory mechanisms may influence experimental outcomes .

What are the recommended methods for reconstituting lyophilized NOV protein?

For optimal reconstitution of lyophilized NOV protein, first centrifuge the vial briefly to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being commonly recommended) and aliquot before storing at -20°C/-80°C. This approach minimizes protein degradation from repeated freeze-thaw cycles. The reconstituted protein maintains activity when properly stored, with a typical shelf life of 6 months in liquid form at -20°C/-80°C or 12 months in lyophilized form .

How can researchers verify the biological activity of NOV protein?

The biological activity of recombinant NOV protein can be assessed using a cell proliferation assay with murine Balb/c 3T3 cells. Active NOV protein typically exhibits an ED50 of less than 1.0 μg/ml, corresponding to a specific activity greater than 1000 IU/mg. This standardized assay provides a reliable measure of protein functionality. Additionally, binding assays to known interacting partners such as specific integrins, heparin sulfate proteoglycans, or fibulin 1C can provide alternative verification methods for activity assessment .

What are the optimal conditions for maintaining NOV protein stability?

To maintain optimal stability of NOV protein, researchers should avoid repeated freeze-thaw cycles, which can significantly compromise protein integrity and activity. For working solutions, store aliquots at 4°C for up to one week. For longer-term storage, maintain lyophilized protein at -20°C/-80°C, where it can remain stable for up to 12 months. When storing reconstituted protein, the addition of glycerol (typically to 50% final concentration) provides cryoprotection. Monitor protein stability periodically using activity assays rather than relying solely on storage duration .

How can researchers address stability issues when working with NOV protein in experimental systems?

When stability issues arise in experimental systems, consider adding protein stabilizers such as BSA (0.1-1%) to working solutions. For cell culture experiments, prepare fresh dilutions from stock solutions immediately before use. If activity decreases over time, verify protein integrity by SDS-PAGE before concluding experimental failure. The addition of protease inhibitors may be beneficial in systems where proteolytic degradation is suspected. Additionally, optimizing buffer conditions (pH, ionic strength) can significantly improve stability in specific experimental contexts .

How does NOV protein mediate cell adhesion and migration?

NOV protein mediates cell adhesion and migration through multiple mechanisms. It signals through specific cell-surface integrins, particularly αvβ3, α5β1, and α6β1, activating focal adhesion kinase (FAK) and related signaling cascades. Simultaneously, NOV binds to heparin sulfate proteoglycans on cell surfaces, providing additional adhesion support. Its interaction with fibulin 1C, an extracellular matrix protein, further modulates cellular adhesion properties. These combined interactions trigger cytoskeletal reorganization and activate migration-related signaling pathways, making NOV a powerful regulator of cellular movement and tissue remodeling .

How can NOV protein be used in angiogenesis research models?

For angiogenesis research, NOV protein can be employed in several model systems. In vitro, researchers can use NOV in endothelial tube formation assays, where it promotes endothelial cell reorganization into vessel-like structures. The protein can be applied at concentrations of 50-500 ng/mL to stimulate this process. In ex vivo models, NOV (100-200 ng/mL) enhances sprouting in aortic ring assays. For in vivo applications, NOV-loaded matrices (400-800 ng/matrix) can be implanted in corneal pocket or Matrigel plug assays to evaluate vessel formation. These approaches allow for comprehensive assessment of NOV's proangiogenic activities across different experimental contexts .

What methodologies are recommended for studying NOV interactions with integrins?

To study NOV-integrin interactions, researchers should consider multiple complementary approaches. Solid-phase binding assays using purified integrins (particularly αvβ3, α5β1, and α6β1) and immobilized NOV can quantify direct binding parameters. Cell adhesion assays using cells expressing specific integrins, with and without function-blocking antibodies, can confirm biological relevance. Co-immunoprecipitation experiments in cell lysates can verify complex formation in cellular contexts. For advanced studies, surface plasmon resonance (SPR) provides kinetic binding parameters, while FRET-based approaches can visualize these interactions in living cells. These methods collectively provide robust evidence for specific NOV-integrin interactions .

How can researchers address issues with protein aggregation when working with NOV?

When encountering NOV protein aggregation, implement a systematic troubleshooting approach. First, verify reconstitution protocol adherence—ensure proper buffer composition and pH (optimally PBS at pH 7.0). Filter solutions through 0.22 μm filters to remove existing aggregates. Adding low concentrations (0.01-0.05%) of non-ionic detergents like Tween-20 can help maintain solubility without compromising activity. If aggregation persists, try reconstituting at lower protein concentrations (0.1-0.5 mg/mL) or adjusting salt concentration. For experimental applications, centrifuge solutions at 10,000g for 10 minutes immediately before use to remove any insoluble material .

What strategies can be employed when NOV protein exhibits unexpectedly low activity in functional assays?

When NOV protein shows unexpectedly low activity, first verify protein integrity by SDS-PAGE and western blotting. Ensure proper storage conditions were maintained and freeze-thaw cycles minimized. Check experimental conditions, particularly the presence of divalent cations (Ca²⁺, Mg²⁺) which are often required for proper protein folding and activity. Evaluate for potential inhibitors in the experimental system, especially serum components that may contain endogenous inhibitors. Consider pre-incubating the protein at room temperature for 15-30 minutes before the assay to allow proper folding. If using cell-based assays, verify cell responsiveness with a positive control stimulus. Titrating protein concentration over a wider range than originally planned may reveal a shifted dose-response relationship .

How should researchers design experiments to distinguish between direct NOV effects and indirect effects mediated through IGF pathways?

To differentiate between direct NOV effects and those mediated through IGF pathways, implement a comprehensive experimental design. Begin with parallel treatments using NOV alone, IGF alone, and their combination to identify synergistic or antagonistic effects. Incorporate IGF receptor inhibitors (such as NVP-AEW541 for IGF-1R) to block potential IGF-mediated effects while preserving direct NOV signaling. Use NOV mutants with disrupted IGFBP domains to eliminate IGF binding while maintaining other functional domains. Additionally, evaluate downstream signaling pathways unique to each stimulus—NOV typically activates integrin-associated pathways while IGF predominantly signals through IRS and PI3K/AKT. Time-course experiments can also help distinguish between immediate direct effects and delayed indirect effects .

How can NOV protein be utilized in tissue engineering applications?

For tissue engineering applications, NOV protein can be incorporated into biomaterials through several approaches. Direct adsorption onto scaffold surfaces at concentrations of 5-20 μg/cm² provides a simple method for promoting cell attachment and migration. For sustained release, NOV can be encapsulated in polymer microspheres or hydrogels at 50-200 μg/mL, providing gradual protein delivery over days to weeks. Alternatively, NOV can be chemically conjugated to scaffolds using carbodiimide chemistry to create surfaces with stable bioactivity. These NOV-functionalized materials promote angiogenesis, cell migration, and tissue integration, making them particularly valuable for vascularized tissue constructs and wound healing applications .

What are the considerations for using NOV protein in developing models of fibrotic disease?

When developing fibrotic disease models using NOV protein, several methodological considerations are critical. First, establish baseline NOV expression in the target tissue, as both increased and decreased expression have been linked to fibrosis depending on the tissue context. For in vitro models, treat fibroblasts with NOV (200-500 ng/mL) alongside established profibrotic factors (TGF-β, PDGF) to assess synergistic or antagonistic effects on myofibroblast differentiation and extracellular matrix production. For in vivo applications, consider both systemic administration (1-5 μg/g body weight) and local delivery approaches. Monitor multiple fibrosis markers (α-SMA, collagen types, fibronectin, tissue stiffness) rather than relying on single readouts. Time-course studies are essential as NOV may have different effects during initiation versus progression or resolution phases of fibrosis .