A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures

Abstract

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high‐molecular‐weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.

Characterizing the Dynamic Disassembly/Reassembly Mechanisms of Encapsulin Protein Nanocages

“…Indeed, ITF spectroscopy has previously been used to monitor the refolding of T = 1 encapsulin from Rhodococcus erythropolis N771 when desorbed from a zeolite substrate. 26 ITF spectroscopy is also an appealing technique to monitor the process of encapsulin assembly/disassembly owing to its relative simplicity and its ability to report on a dynamic ensemble of structures in solution due to its nondestructive nature, which enables measurements to be performed in real time and allows the same sample to be subject to additional techniques. ITF spectroscopy also provides a method to monitor encapsulins in more complex physiological solutions, such as blood, which would be beneficial in investigating their biomedical potential.…”

“…Therefore, these intrinsic Trp residues are likely to be suitable reporters for both assembly/disassembly of the encapsulin macrostructure (i.e., tertiary/quaternary structure) and folding of the individual subunit polypeptide chains. Indeed, ITF spectroscopy has previously been used to monitor the refolding of T = 1 encapsulin from Rhodococcus erythropolis N771 when desorbed from a zeolite substrate . ITF spectroscopy is also an appealing technique to monitor the process of encapsulin assembly/disassembly owing to its relative simplicity and its ability to report on a dynamic ensemble of structures in solution due to its nondestructive nature, which enables measurements to be performed in real time and allows the same sample to be subject to additional techniques.…”

Nanotechnological Applications Based on Bacterial Encapsulins

“…Biocompatibility of encapsulin nanocompartments can be increased by PEGylation, the coating of the nanoparticle surface with polyethylene glycol [104,105]. This is a safe procedure that increases bloodstream retention time and evades immune system surveillance [106].…”

Exploring the Self-Assembly of Encapsulin Protein Nanocages from Different Structural Classes

“…Indeed, intrinsic Trp fluorescence (ITF) has previously been used to monitor the re-folding of T = 1 encapsulin from Rhodococcus erythropolis N771 when desorbed from a zeolite substrate. 31 ITF is also an appealing technique to monitor the process of encapsulin assembly/disassembly owing to its relative simplicity, ability to report on a dynamic ensemble of structures in solution and non-destructive nature, which enables measurements to be performed in real-time. Additionally, unlike fluorescence resonance energy transfer (FRET)-based techniques, ITF does not require modification of the protein with any extrinsic labels that may alter the assembly/disassembly dynamics.…”