Fibronectin Recombinant

What is recombinant fibronectin and how does it differ from native fibronectin?

Recombinant fibronectin refers to artificially produced fibronectin protein or fragments manufactured using molecular biology techniques in expression systems such as HEK 293 cells. Unlike native fibronectin isolated from plasma or tissue sources, recombinant versions offer greater consistency, purity (typically ≥95%), and the ability to produce specific domains or fragments with precisely defined amino acid sequences. Recombinant fibronectin can be engineered with specific tags (such as 6-His tags) to facilitate purification and detection in experimental settings, providing researchers with greater control over the protein's characteristics . These engineered proteins can be produced as full-length molecules or as functional fragments containing specific domains of interest, such as the integrin-binding region (FNIII8-10) or heparin-binding domains (FNIII1H) . Compared to native fibronectin, recombinant versions minimize batch-to-batch variation and contamination risks, providing more reliable experimental outcomes for sensitive cellular and molecular assays.

What are the key structural domains in recombinant fibronectin and their functions?

Recombinant fibronectin maintains the modular structure of native fibronectin, consisting of three types of homologous structural motifs: FN type I, type II, and type III repeats. The integrin-binding domain containing the RGD (Arg-Gly-Asp) sequence is located in the type III-10 module and plays a crucial role in cell adhesion through interaction with multiple integrin receptors, including α5β1 and αvβ3 integrins . The first type III repeat (FNIII1) contains a heparin-binding fragment (FNIII1H) that remains cryptic in soluble fibronectin but becomes exposed during matrix assembly, providing regulatory signals to cells . Type III domains #9 and #10 form a critical functional unit where the tilt angle between them determines integrin binding affinity, influencing the different functions between fibrillar and soluble fibronectin . Additionally, the recombinant fibronectin fragment 2 (FN1.2) contains the first seven FN type III domains (III1-7), with III1 binding weakly to heparin and contributing to self-association and fibril formation . The structural organization enables fibronectin to interact with numerous molecules including collagen, fibrin, heparin, DNA, and actin, supporting its diverse biological functions.

How do recombinant fibronectin fragments support specific research applications?

Recombinant fibronectin fragments provide researchers with precise tools to investigate specific aspects of fibronectin biology that would be challenging to study using the full-length protein. The fragment containing amino acids 2177 to 2386 enables researchers to study specific binding interactions without interference from other domains, providing cleaner experimental readouts in binding assays and cellular response studies . The fibronectin matrix mimetics, which couple the heparin-binding FNIII1H fragment directly to the integrin-binding domains (FNIII8-10), allow researchers to investigate matricryptic sites that become exposed during matrix assembly or as a result of cellular tension on fibronectin fibrils . These engineered fragments support cell spreading, growth, migration, and contractility to a similar or greater extent than full-length fibronectin, making them valuable tools for tissue engineering applications . Additionally, researchers can use specialized fragments like the FN1.2 fragment (containing domains III1-7) to study self-association mechanisms without the complexity introduced by the RGD domain, which is absent in this particular construct . These tailored fragments allow for more controlled experimental designs when investigating specific cellular responses to fibronectin.

What are the optimal conditions for reconstitution and storage of recombinant fibronectin?

The reconstitution and storage conditions for recombinant fibronectin significantly impact its stability and biological activity in experimental applications. For carrier-free recombinant human fibronectin, reconstitution at 100 μg/mL in phosphate-buffered saline (PBS) is typically recommended to maintain protein stability and functionality . The lyophilized protein is commonly formulated in a 0.2 μm filtered solution containing HEPES, NaCl, and Tween® to protect the protein during the lyophilization process and subsequent storage . For long-term storage, it is critical to use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of biological activity . When working with recombinant fibronectin fragments, researchers should be aware that different fragments may have specific storage requirements based on their structural properties. For instance, fragments containing the heparin-binding domain may require different buffer conditions than those containing primarily the integrin-binding region. Prior to experimental use, researchers should verify protein activity through functional assays, as improper storage can lead to loss of biological function without visible precipitation or other physical indicators of degradation.

How should cell adhesion assays be optimized when using recombinant fibronectin as a substrate?

Cell adhesion assays using recombinant fibronectin require careful optimization to ensure reproducible and physiologically relevant results. For plates coated with fibronectin matrix mimetics like GST/III1H,8-10 or GST/III1H,8,10, cells may need to be centrifuged into contact with the adhesive substrate (70×g force at 4°C for approximately 4 minutes) to facilitate initial attachment, followed by washing with PBS and fixation with 1% paraformaldehyde . When using GST/III1H,8 RGD-coated plates, a 30-minute adhesion period at 4°C may be sufficient before washing and counting adherent cells . Researchers should consider preincubating cells with integrin function-blocking antibodies (25 μg/mL for 45 minutes) as a control to confirm the specificity of integrin-mediated adhesion to the recombinant fibronectin . The coating concentration of recombinant fibronectin is another critical parameter, with optimal concentrations ranging from 0.5-50 μg/mL for supporting cell spreading, requiring individual optimization for different cell types and experimental objectives . Additionally, researchers should implement appropriate controls, including non-specific binding surfaces and comparison with full-length fibronectin, to accurately interpret cellular responses to recombinant fibronectin fragments.

What techniques are recommended for visualizing fibronectin fibrillogenesis induced by recombinant fibronectin?

Visualizing fibronectin fibrillogenesis induced by recombinant fibronectin requires specialized techniques that can track the conversion of soluble fibronectin into insoluble fibrils. Immunofluorescence microscopy represents a powerful approach, where cells seeded onto tissue culture plates precoated with recombinant fibronectin proteins are allowed to adhere and spread (typically for 4 hours at 37°C) before fixation with 2% paraformaldehyde . For optimal visualization, researchers may supplement the cell culture medium with full-length fibronectin after the initial adhesion period to provide the substrate for fibrillogenesis . Confocal microscopy with z-stack imaging enables three-dimensional reconstruction of fibronectin matrices, allowing researchers to assess the spatial organization and density of the fibrils. Time-lapse imaging using fluorescently labeled fibronectin can provide insights into the kinetics of fibril formation and remodeling in response to different recombinant fibronectin fragments. For higher resolution analysis, techniques such as super-resolution microscopy or atomic force microscopy can reveal nanoscale features of fibronectin fibrils that might not be visible with conventional fluorescence microscopy. Additionally, biochemical approaches such as deoxycholate solubility assays can complement microscopy techniques by quantifying the conversion of soluble fibronectin into the insoluble matrix form.

How do fibronectin matrix mimetics direct extracellular matrix deposition and organization?

Fibronectin matrix mimetics, particularly those engineered to couple the heparin-binding FNIII1H fragment directly to the integrin-binding domains (FNIII8-10), play a sophisticated role in directing extracellular matrix deposition and organization. These mimetics expose the normally cryptic heparin-binding site in FNIII1, which becomes naturally exposed during matrix assembly or as a result of tension exerted by cells on fibronectin fibrils . When used as adhesive substrates, fibronectin matrix mimetics like GST/III1H,8-10, GST/III1H,8,10, and GST/III1H,8 RGD support cell spreading, growth, migration, and contractility to a similar or greater extent than full-length fibronectin . The ability of these mimetics to support cell-dependent extracellular matrix deposition stems from their presentation of specific binding sites that initiate the assembly process, which is typically an active, cell-dependent conversion of soluble fibronectin into insoluble fibrils . Through the strategic presentation of both integrin-binding and heparin-binding domains, these engineered proteins provide cells with regulatory signals similar to those from extracellular matrix fibronectin, effectively bypassing the need for the initial conformational change that exposes the cryptic sites . This enables researchers to create scaffolds that stimulate cells to produce a new, native extracellular matrix capable of promoting functions critical for tissue homeostasis.

What role does recombinant fibronectin play in tissue engineering applications?

Recombinant fibronectin serves as a crucial component in advanced tissue engineering approaches, particularly in designing biologically active scaffolds that support key aspects of tissue regeneration. In perfusion-decellularized whole organ systems designed to generate intact extracellular matrix scaffolds, recombinant fibronectin can be used as an adhesive coating material to stimulate cells to produce a new, native extracellular matrix that promotes functions critical for tissue homeostasis . When incorporated into biomaterials, recombinant fibronectin matrix mimetics like GST/III1H,8-10 have been shown to promote cell growth, migration, and contractility through FNIII1H-dependent mechanisms, facilitating the integration of cells with the engineered scaffold . Additionally, the ability of recombinant fibronectin to bind to multiple extracellular matrix components, including collagen, heparin, and fibrin, makes it an excellent bridging molecule for comprehensive matrix assembly in engineered tissues . Researchers have demonstrated that recombinant fibronectin can be added to culture media at concentrations of 0.5-50 μg/mL to support cell spreading, with optimal concentrations needing to be determined for specific applications and cell types . Through these mechanisms, recombinant fibronectin contributes to the development of complex, functional tissues that more accurately mimic the native cellular microenvironment.

How does recombinant fibronectin modulate cellular signaling pathways in research models?

Recombinant fibronectin exerts profound effects on cellular signaling pathways through its multiple functional domains and their interaction with cell surface receptors. The RGD sequence in the type III-10 module interacts with several different integrin receptors, including α5β1 and αvβ3 integrins, initiating integrin-mediated signaling cascades that regulate cell adhesion, migration, and survival . Additional amino acid sequences in neighboring modules contribute to the affinity and specificity of integrin receptor binding, allowing for fine-tuned cellular responses . Anastellin, a fibronectin fragment, activates p38 MAPK and inhibits lysophospholipid signaling, while the superfibronectin polymer formed through anastellin-induced fibril formation exhibits enhanced adhesive properties and inhibitory effects on tumor growth, angiogenesis, and metastasis . When secreted by contracting muscle, fibronectin induces liver autophagy (a degradative pathway for nutrient mobilization and damage removal) and systemic insulin sensitization via hepatic ITGA5:ITGB1 integrin receptor signaling . Furthermore, fibronectin acts as a ligand for the LILRB4 receptor, inhibiting FCGR1A/CD64-mediated monocyte activation, which has implications for immune regulation in experimental models . These diverse signaling effects make recombinant fibronectin a valuable tool for studying cellular response mechanisms in various physiological and pathological contexts.

How can researchers validate the functionality of recombinant fibronectin in their experimental systems?

Validating the functionality of recombinant fibronectin in experimental systems requires a multi-faceted approach to ensure that the protein retains its intended biological activities. Cell adhesion assays represent a fundamental validation method, where cells are seeded onto surfaces coated with recombinant fibronectin and their ability to attach and spread is quantified using crystal violet staining and absorbance measurement at 590 nm or by manual counting of adherent cells . Including integrin function-blocking antibodies as controls can confirm that the observed adhesion is specifically mediated through the expected integrin-fibronectin interactions . Immunofluorescence microscopy following cell seeding on recombinant fibronectin-coated surfaces can be used to visualize focal adhesion formation and cytoskeletal organization, providing evidence of proper integrin engagement and downstream signaling . For recombinant fibronectin intended to support matrix assembly, researchers should examine the protein's ability to induce fibrillogenesis, either alone or in combination with full-length fibronectin, using appropriate immunostaining techniques . Biochemical validation can include surface plasmon resonance or ELISA-based binding assays to confirm that the recombinant fibronectin interacts with known binding partners such as integrins, heparin, collagen, or other matrix components. Finally, functional assays specific to the research question, such as cell migration, proliferation, or differentiation studies, provide the most relevant validation of the recombinant fibronectin's utility in the particular experimental context.

How is recombinant fibronectin being utilized in regenerative medicine research?

Recombinant fibronectin is emerging as a crucial component in regenerative medicine strategies aimed at enhancing tissue repair and functional recovery. Current research is exploring how fibronectin matrix mimetics, which directly couple the heparin-binding fragment of the first type III repeat (FNIII1H) to the integrin-binding repeats (FNIII8–10), can be incorporated into biomaterial scaffolds to promote cell growth, migration, and contractility through FNIII1H-dependent mechanisms . These engineered proteins are particularly valuable in perfusion-decellularized whole organ systems, where they serve as adhesive coating materials that stimulate cells to produce new, native extracellular matrix capable of supporting tissue homeostasis . Researchers are investigating how recombinant fibronectin fragments can be used to functionalize synthetic biomaterials, improving their integration with host tissues by providing specific binding sites for cellular integrins and other receptors . Additionally, the ability of anastellin (a fibronectin fragment) and superfibronectin to inhibit tumor growth, angiogenesis, and metastasis has opened new avenues for exploration in cancer therapy and tissue regeneration applications . The modulatory effects of fibronectin on osteoblast compaction, mineralization, and type I collagen deposition are being leveraged in bone tissue engineering strategies, where recombinant fragments can provide cell-type specific cues to guide proper tissue formation . As research continues to unravel the complex roles of fibronectin in tissue development and repair, recombinant versions offer precisely engineered tools to harness these functions in regenerative medicine applications.

What are the current approaches for studying fibronectin mechanobiology using recombinant proteins?

Cutting-edge research in fibronectin mechanobiology leverages recombinant proteins to investigate how mechanical forces influence fibronectin structure and function in cellular microenvironments. Scientists are studying how the tilt angle between type III domains #9 and #10 (which contains the RGD motif) determines integrin binding affinity, providing insights into the structural differences between fibrillar and soluble fibronectin and their functional implications . Recombinant fibronectin fragments are being used on stretchable substrates to investigate how mechanical tension exposes the matricryptic heparin-binding site in FNIII1, which becomes accessible during matrix assembly or as a result of tension exerted by cells on fibronectin fibrils . This approach allows researchers to precisely control the mechanical environment while monitoring cellular responses to specific fibronectin domains. Single-molecule force spectroscopy techniques combined with recombinant fibronectin fragments are revealing how mechanical forces unfold individual fibronectin domains, exposing cryptic binding sites that trigger specific cellular responses . Additionally, engineered fibronectin matrix mimetics that directly couple the open heparin-binding FNIII1 fragment to the integrin-binding domain enable researchers to bypass the need for mechanical unfolding, providing cells with regulatory signals similar to those from extracellular matrix fibronectin under tension . These approaches are elucidating how fibronectin acts as a mechanosensitive switch in the extracellular matrix, converting mechanical stimuli into biochemical signals that regulate cell behavior.

How do recombinant fibronectin constructs contribute to understanding disease mechanisms?

Recombinant fibronectin constructs have become instrumental in deciphering the molecular mechanisms underlying various pathological conditions where extracellular matrix dysregulation plays a significant role. In cancer research, the ability of anastellin (a fibronectin fragment) and superfibronectin to inhibit tumor growth, angiogenesis, and metastasis provides valuable insights into how fibronectin fragments might influence tumor progression and offers potential therapeutic strategies . Researchers are using recombinant fibronectin to study fibrotic disorders, where excessive extracellular matrix deposition leads to tissue dysfunction, by investigating how specific fibronectin domains contribute to myofibroblast activation and collagen production. In the context of wound healing and tissue repair, recombinant fibronectin fragments help elucidate the sequential processes of matrix assembly and remodeling, identifying potential intervention points for chronic wounds or excessive scarring . Studies on autoimmune diseases are exploring how antibodies targeting specific fibronectin epitopes might disrupt normal matrix function, with recombinant fragments serving as precise tools to map pathogenic antibody binding sites. Additionally, the role of fibronectin in metabolic regulation—including its ability to induce liver autophagy and systemic insulin sensitization via hepatic ITGA5:ITGB1 integrin receptor signaling when secreted by contracting muscle—opens new avenues for understanding metabolic disorders using recombinant fibronectin constructs . Through these diverse applications, recombinant fibronectin is advancing our understanding of disease pathogenesis and identifying potential therapeutic targets.