scholarly journals Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

2021 ◽  
Vol 22 (14) ◽  
pp. 7362
Author(s):  
Amina Ben Abla ◽  
Guilhem Boeuf ◽  
Ahmed Elmarjou ◽  
Cyrine Dridi ◽  
Florence Poirier ◽  
...  
Keyword(s):  
Specific Binding ◽  
Cellular Adhesion ◽  
High Yield ◽  
Adhesion Assay ◽  
Cell Binding ◽  
Affinity Tag ◽  
Binding Domains ◽  
Rational Development ◽  
Fibronectin Type Iii ◽  
Arginine Glycine Aspartic Acid

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

scholarly journals Fibronectin–collagen binding and requirement during cellular adhesion

1980 ◽  
Vol 186 (2) ◽  
pp. 551-559 ◽  
Author(s):  
Leslie I. Gold ◽  
Edward Pearlstein
Keyword(s):  
Chick Embryo ◽  
Human Plasma ◽  
Hydrophobic Interactions ◽  
Human Fibroblasts ◽  
Sodium Dodecyl ◽  
Cellular Adhesion ◽  
Adhesion Assay ◽  
Human Umbilical Cord ◽  
Ionic Detergents ◽  
Kinetics Of

Fibronectin isolated from human plasma and from the extracellular matrices of cell monolayers mediates the attachment in vitro and spreading of trypsin-treated cells on a collagen substratum. Fibronectin-dependent kinetics of cellular attachment to collagen were studied for several adherent cell types. It was shown that trypsin-treated human umbilical-cord cells, mouse sarcoma CMT81 cells, endothelial cells, and human fibroblasts from a patient with Glanzmann's disease were completely dependent on fibronectin for their attachment to collagen, whereas guinea-pig and monkey smooth-muscle cells and chick-embryo secondary fibroblasts displayed varying degrees of dependence on fibronectin for their attachment. Radiolabelled human plasma fibronectin possessed similar affinity for collagen types I, II and III from a variety of sources. The fibronectin bound equally well to the collagens with or without prior urea treatment. However, in the fibronectin-mediated adhesion assay using PyBHK fibroblasts, a greater number of cells adhered and more spreading was observed on urea-treated collagen. Fibronectin extracted from the extracellular matrix of chick-embryo fibroblasts and that purified from human plasma demonstrated very similar kinetics of complexing to collagencoated tissue-culture dishes. Fibronectin from both sources bound to collagen in the presence of 0.05–4.0m-NaCl and over the pH range 2.6–10.6. The binding was inhibited when fibronectin was incubated with 40–80% ethylene glycol, the ionic detergents sodium dodecyl sulphate and deoxycholate, and the non-ionic detergents Nonidet P-40, Tween 80 and Triton X-100, all at a concentration of 0.1%. From these results we proposed that fibronectin–collagen complexing is mainly attributable to hydrophobic interactions.

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

2020 ◽  
Author(s):  
Cecy Xi ◽  
Arianna Arianna Di Fazio ◽  
Naveed Nadvi ◽  
Karishma Patel ◽  
Michelle Xiang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Specific Activity ◽  
Specific Binding ◽  
Exchange Chromatography ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure ◽  
Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

scholarly journals The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains

2009 ◽  
Vol 390 (5/6) ◽  
Author(s):  
Nina M. Link ◽  
Cornelia Hunke ◽  
Jonathan W. Mueller ◽  
Jutta Eichler ◽  
Peter Bayer
Keyword(s):  
Solution Structure ◽  
Specific Binding ◽  
Transmembrane Protein ◽  
Structural Data ◽  
Adaptor Proteins ◽  
Axonal Guidance ◽  
High Affinity ◽  
Binding Domains ◽  
Wide Range ◽  
Pancreatic Peptide

Abstract Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the amino-terminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains.

scholarly journals Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

2016 ◽  
Vol 213 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Holly A. Ping ◽  
Lauren M. Kraft ◽  
WeiTing Chen ◽  
Amy E. Nilles ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Coiled Coil ◽  
Lipid Binding ◽  
General Mechanism ◽  
Cell Cortex ◽  
Pleckstrin Homology Domain ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Mitochondrial Distribution

The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

A fluorescent cellular adhesion assay using insect cell produced human VCAM1

1994 ◽  
Vol 175 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Janet K. Stoltenborg ◽  
Peter W. Tsao ◽  
Henry J. George ◽  
Peter J. Bouchard ◽  
Eric J. Wexler ◽  
...  
Keyword(s):  
Insect Cell ◽  
Cellular Adhesion ◽  
Adhesion Assay

scholarly journals Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 117-128 ◽  
Author(s):  
JH Beer ◽  
KT Springer ◽  
BS Coller
Keyword(s):  
Protein Interactions ◽  
Platelet Inhibition ◽  
Cell Protein ◽  
Platelet Glycoprotein ◽  
Integrin Receptors ◽  
Recognition Sequence ◽  
Rgd Peptides ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Arginine Glycine Aspartic Acid

Abstract The interactions between ligands containing the recognition sequence arginine-glycine-aspartic acid (RGD) and integrin receptors are important in many cell-cell and cell-protein interactions. The platelet contains five integrin receptors and they contribute significantly to platelet adhesion and aggregation. To investigate the RGD binding domains on platelet integrins, we immobilized a series of RGD peptides containing variable numbers of glycine residues [(G)n-RGDF] on polyacrylonitrile beads and evaluated the ability of the beads to interact with platelets. With native platelets, virtually no interaction occurred with G1-RGDF beads, but the interactions increased as the number of glycine residues increased, plateauing with the G9- RGDF and G11-RGDF beads. ADP pretreatment enhanced the interactions with all of the beads, whereas prostaglandin E1 pretreatment eliminated the interactions with the shortest peptide beads, but only partially inhibited interactions with the longer peptide beads. Monoclonal antibodies to glycoprotein (GP) IIb/IIIa were most effective in inhibiting the interactions, but antibodies to GPIIb/IIIa with similar inhibitory effects on fibrinogen binding varied dramatically in their ability to inhibit the interaction between platelets and immobilized RGD peptides. Our data indicate that the majority of RGD binding sites on GPIIb/IIIa can be reached by peptides that extend out approximately 11 to 32 A from the surface of the bead, and these results are in accord with the dimensions of integrin receptors deduced from electron microscopy. Activation of GPIIb/IIIa facilitates the interactions, but platelet inhibition fails to eliminate the interactions with the longer peptide beads, suggesting that access to the RGD binding site on at least a fraction of the GPIIb/IIIa receptors is always possible for preferred ligands. Finally, we found that the G3-RGDF peptide beads were uniquely sensitive to the activation state of the GPIIb/IIIa receptor.

scholarly journals Modulating wall shear stress gradient via equilateral triangular channel for in situ cellular adhesion assay

2016 ◽  
Vol 10 (5) ◽  
pp. 054119 ◽  
Author(s):  
Hyung Woo Kim ◽  
Seonjin Han ◽  
Wonkyoung Kim ◽  
Jiwon Lim ◽  
Dong Sung Kim
Keyword(s):  
Shear Stress ◽  
Wall Shear Stress ◽  
Stress Gradient ◽  
Cellular Adhesion ◽  
Adhesion Assay ◽  
Triangular Channel ◽  
Wall Shear Stress Gradient ◽  
Shear Stress Gradient ◽  
Wall Shear

Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 718-724 ◽  
Author(s):  
Nicholas A. Watkins ◽  
Lily M. Du ◽  
J. Paul Scott ◽  
Willem H. Ouwehand ◽  
Cheryl A. Hillery
Keyword(s):  
Sickle Cell ◽  
Matrix Protein ◽  
Binding Domain ◽  
Extracellular Matrix Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adhesion Assay ◽  
Single Chain ◽  
Cell Binding

AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

scholarly journals Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons.

1996 ◽  
Vol 132 (4) ◽  
pp. 681-699 ◽  
Author(s):  
B Götz ◽  
A Scholze ◽  
A Clement ◽  
A Joester ◽  
K Schütte ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neural Cells ◽  
Cell Binding ◽  
Tenascin C ◽  
Functional Studies ◽  
Distinct Cell ◽  
Fibronectin Type Iii ◽  
Alternatively Spliced ◽  
Fibronectin Type

The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Götz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell-binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins.

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