Highly secretory expression of recombinant cowpea chlorotic mottle virus capsid proteins in Pichia pastoris and in-vitro encapsulation of ruthenium nanoparticles for catalysis

The applications of viral protein cages have expanded rapidly into the fields of bionanotechnology and materials science. However, the low-cost production of viral capsid proteins (CPs) on a large scale is always a challenge. Herein, we develop a highly efficient expression system by constructing recombinant Pichia pastoris cells as a “factory” for the secretion of soluble cowpea chlorotic mottle virus (CCMV) CPs. Under optimal induction conditions (0.9 mg/mL of methanol concentration at 30 °C for 96 h), a high yield of approximately 95 mg/L of CCMV CPs was harvested from the fermentation supernatant with CPs purity >90%, which has significantly simplified the rest of the purification process. The resultant CPs are employed to encapsulate Ruthenium (Ru) nanoparticles (NPs) via in-vitro self-assembly to prepare hybrid nanocatalyst , i.e. [email protected] particles (VLPs). The catalytic activity over [email protected] was evaluated by reducing 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). The results indicate that, with the protection of protein cages, Ru NPs were highly stabilized during the catalytic reaction. This results in enhanced catalytic activity (reaction rate constant k  = 0.14 min −1 ) in comparison with unsupported citrate-stabilized Ru NPs (Ru-CA) ( k  = 0.08 min −1 ). Additionally, comparatively lower activation energy over [email protected] (approximately 32 kJ/mol) than that over Ru-CA (approximately 39 kJ/mol) could be attributed to the synergistic effect between Ru NPs and some functional groups such as amino groups (–NH 2 ) on CPs that weakened the activation barrier of 4-NP reduction. Therefore, enhanced activity and decreased activation energy over [email protected] demonstrated the superiority of [email protected] to unsupported Ru-CA.