Highly secretory expression of recombinant cowpea chlorotic mottle virus capsid proteins in Pichia pastoris and in-vitro encapsulation of ruthenium nanoparticles for catalysis.
Protein expression and purification(2020)
摘要
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. Ru@virus-like particles (VLPs). The catalytic activity over Ru@VLPs 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 Ru@VLPs (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 (-NH2) on CPs that weakened the activation barrier of 4-NP reduction. Therefore, enhanced activity and decreased activation energy over Ru@VLPs demonstrated the superiority of Ru@VLPs to unsupported Ru-CA.
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