Decoration of silk fibroin by click chemistry for biomedical application

Silk-based stabilization of biomacromolecules

“…The addition of the diazonium group allows for a wide variety of functionalization strategies, including the addition of sulfonic acid groups, which inhibit β-sheet self-assembly in silk, and also results in a more anionic silk material. Recently, copper(1) catalyzed azide-alkyne cycloaddition (CuAAC), more commonly referred to as click chemistry, has been employed to overcome the inefficiencies of carbodiimide coupling [176]. The reaction involves conjugation of diazonium coupled silk to an alkyne-molecule complex via cycloaddition.…”

“…This prevents potentially immunogenic responses resulting from the aggregates. FGF-2 has been coupled to silk using click chemistry [176]. …”

Nanomechanics on FGF-2 and Heparin Reveal Slip Bond Characteristics with pH Dependency

“…[30][31] Briefly, protein expression of the fusionprotein pHisTrx-FGF-2 was induced with 0.2 mM IPTG at OD600 = 0.6 and expression was performed at 30° C at 200 rpm. After 5h cells were harvested by centrifugation and were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM PMSF, pH 7.5) and solubilized by sonification at 4° C. After centrifugation at 100,000 g for 1h at 4° C (L8-60M Ultracentrifuge, Beckman-Coulter, Brea, CA), the supernatant containing pHisTrx tagged FGF-2 was purified by heparin-sepharose affinity chromatography using an FPLC system (Aekta Purifier, fusionprotein pHisTrx-FGF-2 was cleaved by thrombin (GE, Freiburg, Germany) with a final concentration of 1U thrombin/mg fusionprotein at 4°C overnight.…”

“…1 mg Enoxaparin was added to the activation buffer (0.1 M MES, 0.5 M NaCl, pH 6.0) containing a tenfold molar excess of EDC and 30 mM Sulfo-NHS as described. 30 The mixture was vortexed and incubated for 15 min at room temperature. After activation, the solution was transferred into concentrated phosphate buffer, containing 0.2 mg PEG-NH2 beads, to raise the pH above neutral.…”

Bio-orthogonal Immobilization of Fibroblast Growth Factor 2 for Spatial Controlled Cell Proliferation

“…These and other concerns challenge the use of unspecific immobilization chemistries, including EDC/NHS coupling. 9,10 Site-specific chemistries remove these challenges through homogeneous decoration by strictly confining the covalent modification to, for example, an unnatural functional group or groups that are introduced into the biologic's backbone at the predefined site during recombinant protein expression. 11,12 This approach takes advantage of the translation machinery of archaebacteria, which incorporate the 22nd amino acid Lpyrrolysine (Pyl) in protein biosynthesis beyond the typically used 20 natural amino acid building blocks.…”