Tailoring Fusion Proteins for Interfacial Nanoparticle Functionalization: Toward Fluorescence-Guided Imaging

Fusion proteins are specially designed molecules created by linking two or more peptides that were originally encoded by separate DNA sequences. To improve control of protein adsorption on poly-(3-hydroxybutyrate) (PHB) surfaces, we developed fusion proteins comprising a polyester substrate-binding domain (SBD) fused to superfolder green fluorescent protein (sfGFP). The subunits were connected via flexible (GGGGS)_n or rigid (EAAAK)_n linkers with n = 1, 2, or 4. The PHB immobilization efficiency provided by these fusion proteins was extensively evaluated on microparticles and nanoparticles by using various surface-sensitive techniques. Accordingly, constructs linked by rigid sequences with n = 2 and n = 4 exhibited higher PHB adsorption capability. This suggests that reduced interdomain conformational freedom may favor configurations compatible with SBD-mediated interaction with the PHB interface. The stable association of the constructs with PHB nanoparticles enabled fluorescence-based tracking during cellular uptake experiments. The spatial orientation and polyester-binding ability conferred by such constructs via a facile, mild, and persistent association process might represent a significant biotechnological advance in the development of advanced nanosystems sensitive to fluorescence imaging.