Stabilization of a Virus-Like Particle and Its Application as a Nanoreactor at Physiological Conditions

Abstract

Virus-like particles are very interesting tools for application in bionanotechnology, due to their monodisperse features and biocompatibility. In particular, the cowpea chlorotic mottle virus (CCMV) capsid has been studied extensively as it can be assembled and disassembled reversibly, facilitating cargo encapsulation. CCMV is, however, only stable at physiological conditions when its endogenous nucleic acid cargo is present. To gain more flexibility in the type of cargo encapsulated and to broaden the window of operation, it is interesting to improve the stability of the empty virus-like particles. Here, a method is described to utilize the CCMV capsid at close to physiological conditions as a stable, enzyme-filled nanoreactor. As a proof-of-principle, the encapsulation of T4 lysozyme (T4L) was chosen; this enzyme is a promising antibiotic, but its clinical application is hampered by, for example, its cationic character. It was shown that four T4L molecules can successfully be encapsulated inside CCMV capsids, while remaining catalytically active, which could thus improve the enzyme’s application potential.

Enzyme encapsulation by protein cages

“…42 A second generation of ELP-CCMV was further developed using the sortase-based encapsulation method to load cargo proteins without the need for high salt conditions ($2 M NaCl). 43 This strategy used metal ions to stabilize the assembly process and bypass the high salt concentration effect. Here, a hexahistidine tag was added to ELP-CCMV at the N-terminus and the presence of divalent nickel ions resulted in local clustering 44 of the ELP-CCMV capsid proteins to subsequently trigger the assembly at pH 7.5 without the necessity for high salt concentrations.…”

“…A possible explanation is that the action of EDTA could be hampered by the crowding effect inside the cavity. 43…”

Structural nanotechnology: three-dimensional cryo-EM and its use in the development of nanoplatforms forin vitrocatalysis

“…9 In other work, T4 lysozyme was attached covalently to the CP N terminus using sortase A as a catalyst. 76 CP assembly resulted in encapsulation of the enzyme, since the N terminus now pointed inwards.…”

Protein‐Based Nanoparticles for the Delivery of Enzymes with Antibacterial Activity

“…Lysozyme (LYZ) is often used a model protein for encapsulation in nanoparticles . LYZ is a small antibacterial enzyme with a molecular weight of 14.3 kDa, exists in many human body fluids and tissues, and plays an important role in the innate immune system .…”