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

Abstract

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.

RGD peptide in cancer targeting: Benefits, challenges, solutions, and possible integrin–RGD interactions

“…[119][120][121][122] Producing RGD peptides using recombinant DNA technology in bacterial (e.g., E.coli), yeast (e.g., Pichia pastoris), insect cells (e.g., baculovirus system), or mammalian cell cultures can provide higher yields compared to traditional chemical synthesis methods at lower costs per batch. [123][124][125][126][127] Efficient bioconjugation strategies such as click chemistry allow for site-specific attachment of functional moieties like PEG chains without requiring additional protection/deprotection steps during peptide synthesis. 38,[128][129][130] By adopting these strategies individually or in combination, it is possible to overcome the complexity and cost challenges associated with the synthesis of RGD peptides without compromising their therapeutic potential.…”

“…For longer or more complex RGD‐containing peptides or proteins, chemical ligation techniques like native chemical ligation (NCL) enable the convergent assembly of multiple smaller peptide fragments with greater synthetic accessibility 119–122 . Producing RGD peptides using recombinant DNA technology in bacterial (e.g., E.coli), yeast (e.g., Pichia pastoris), insect cells (e.g., baculovirus system), or mammalian cell cultures can provide higher yields compared to traditional chemical synthesis methods at lower costs per batch 123–127 . Efficient bioconjugation strategies such as click chemistry allow for site‐specific attachment of functional moieties like PEG chains without requiring additional protection/deprotection steps during peptide synthesis 38,128–130 …”

“… 119 , 120 , 121 , 122 Producing RGD peptides using recombinant DNA technology in bacterial (e.g., E.coli), yeast (e.g., Pichia pastoris), insect cells (e.g., baculovirus system), or mammalian cell cultures can provide higher yields compared to traditional chemical synthesis methods at lower costs per batch. 123 , 124 , 125 , 126 , 127 Efficient bioconjugation strategies such as click chemistry allow for site‐specific attachment of functional moieties like PEG chains without requiring additional protection/deprotection steps during peptide synthesis. 38 , 128 , 129 , 130 …”