Redesigning amino/carboxyl ends of DARPin G3 for high thermostability and production in tobacco transplastomic plants.

Key message: Redesigning the N- and C-capping repeats of the native DARPin G3 significantly improved its stability, and may facilitate its purification from the total soluble proteins of high-temperature dried leaf materials of transplastomic plants. Designed ankyrin repeat proteins (DARPins) constitute a promising class of binding molecules that can overcome the limitations of monoclonal antibodies and enable the development of novel therapeutic approaches. Despite their inherent stability, detailed studies have revealed that the original capping repeats derived from natural ankyrin repeat proteins impair the stability of the initial DARPin design. Consequently, the development of thermodynamically stabilized antibody mimetics may facilitate the development of innovative drugs in the future. In this study, we replaced the original N- and C-capping repeats with improved caps to enhance the thermostability of native DARPin G3. Computational analyses suggested that the redesigned thermostable DARPin G3 structure possessed optimal quality and stability. Molecular dynamics simulations verified the stability of the redesigned thermostable DARPin G3 at high temperatures. The redesigned thermostable DARPin G3 was expressed at high levels in tobacco transplastomic plants and subsequently purified from high-temperature dried leaf materials. Thermal denaturation results revealed that the redesigned thermostable DARPin G3 had a higher Tm value than the native DARPin G3, with a Tm of 35.51 °C greater than that of native DARPin G3. The results of the in vitro bioassays confirmed that the purified thermostable DARPin G3 from high-temperature dried leaf materials maintained its binding activity without any loss of affinity and specifically bound to the HER2 receptor on the cell surface. These findings demonstrate the successful improvement in the thermostability of DARPin G3 without compromising its biological activity.