Impact of epidermal growth factor tethering strategy on cellular response.

In an effort to evaluate the impact of various epidermal growth factor (EGF) grafting strategies upon cell surface receptor activation and cell adhesion, we generated low-fouling surfaces by homogeneously grafting carboxymethylated dextran (CMD) on amino-coated glass substrate. By preventing nonspecific cell adhesion while providing reactive groups facilitating subsequent protein grafting, CMD allowed achieving specific cell/tethered EGF interactions and therefore deriving unambiguous conclusions about various EGF grafting strategies. We demonstrate here that A-431 cell response to immobilized EGF is highly dependent on the bioactivity of the tagged protein being tethered, its proper orientation, and its surface density.

New ELISA approach based on coiled-coil interactions.

The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF).

Human corneal epithelial cell response to epidermal growth factor tethered via coiled-coil interactions.

The development of new strategies for protein immobilization to control cell adhesion, growth and differentiation is of prime interest in the field of tissue engineering. Here we propose a versatile approach based on the interaction between two de novo designed peptides, Ecoil and Kcoil, for oriented immobilization of epidermal growth factor (EGF) on polyethylene terephthalate (PET) films. After amination of PET surfaces by ammonia plasma treatment, Kcoil peptides were covalently grafted in an oriented fashion using succinimidyl 6-[30-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) linker, and the Kcoil-functionalized films were characterized by X-ray photoelectron spectroscopy (XPS).

Protein detection by Western blot via coiled-coil interactions.

We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing.

Epidermal growth factor tethered through coiled-coil interactions induces cell surface receptor phosphorylation.

We have elaborated and validated a novel approach for the oriented tethering of proteins such as the epidermal growth factor (EGF) on aminated surfaces. The grafting reactions were optimized to generate a dense and homogeneous EGF layer. Impact of EGF orientation on A-431 cellular response was investigated.

Tyrosinase-catalyzed synthesis of a universal coil-chitosan bioconjugate for protein immobilization.

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 bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags.

For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure.