Tyrosinase-Catalyzed Synthesis of a Universal Coil-Chitosan Bioconjugate for Protein Immobilization

Abstract

Chitosan has been reported as a promising material for gene and drug delivery as well as for tissue engineering and regenerative medicine. We report here the conjugation of a de novo designed coil peptide (Kcoil) to chitosan ( M(n) = 200 kDa) to achieve a universal Kcoil-chitosan scaffold for subsequent immobilization of proteins tagged with the Kcoil partner, i.e., the Ecoil peptide. Kcoil-chitosan conjugate was synthesized using a tyrosinase-catalyzed protocol. Extensive UV/vis and IR characterization demonstrated that Kcoil peptide was covalently grafted to amines of chitosan. The ability of Kcoil-chitosan conjugate to recruit Ecoil tagged epidermal growth factor (EGF) was assessed by surface plasmon resonance measurements (SPR). Despite nonspecific interactions between chitosan and EGF, the specific formation of an E/K coiled coil complex was observed at slightly acidic pH and high salt concentration conditions, demonstrating that grafting to chitosan did not negatively impact binding characteristics of Kcoil peptide. Finally, the benefits of such bioconjugates for biomedical applications are discussed.

Immobilization of cellulase onto a recyclable thermo-responsive polymer as bioconjugate

“…To reduce cost associated with enzymes, various techniques have been developed to enable their repeated use in multiple rounds of processing. To render them reusable, enzymes have been immobilized on solid matrices [2][3][4] such as sepharose [5], polyester [6], chitosan [7], magnetic affinity sorbent [8] and carbon nanotubes [9], either via covalent attachment or adsorption. However, biocatalysts in solid-liquid interfaces are often accompanied by a reduced activity as the accessibility of the enzymes to the substrate is potentially limited.…”

Biomimetic Materials for Medical Application Through Enzymatic Modification

“…Recently, a tyrosinase-catalyzed oxidation-grafting reaction has been used by [29] for the conjugation of de novo designed coil peptides, called Kcoil and Ecoil, that heterodimerize in a highly specific and stable fashion to adopt a coiled-coil structure, characterized by the repetition (five times in each peptide) of K-V-S-A-L-K-E and E-V-S-A-L-E-K heptads, respectively. Demolliens et al [29] studied the conjugation of the Ecoil/Kcoil system with chitosan to obtain a scaffold or particles for subsequent immobilization of proteins tagged with the Ecoil peptide.…”

“…Demolliens et al [29] studied the conjugation of the Ecoil/Kcoil system with chitosan to obtain a scaffold or particles for subsequent immobilization of proteins tagged with the Ecoil peptide. Precisely, the system Kcoil/chitosan has been found to be able to recruit the Ecoiltagged epidermal growth factor (EGF) via coiled-coil interactions.…”

“…To achieve this purpose, the same enzymatic protocol described above was used, in which tyrosinase-catalyzed oxidation of a tyrosine-containing Kcoil peptide was followed by its condensation onto chitosan in a non-enzymatic step. A limitation of the approach was the limited stability of the coiled-coil complex [29].…”

The Signal‐Enhanced Label‐Free Immunosensor Based on Assembly of Prussian Blue‐SiO2 Nanocomposite for Amperometric Measurement of Neuron‐Specific Enolase

“…Chitosan, a cationic polysaccharide with abundant amines has been largely studied as its attractive properties including excellent film-forming ability, good biocompatibility and adhesion. However, it is reported that chitosan film may block the electron transfer between electrode and target molecules because of its electroinactivity [19,20]. In this paper, gold-chitosan nanocomposites with the adsorption of chitosan molecules on particle surfaces was prepared which obtained the better electron transfer ability than pure chitosan and better stability than pure gold nanoparticles as chitosan is very effective stabilizing agent for gold nanoparticles.…”