Successful PEGylation of hollow encapsulin nanoparticles from Rhodococcus erythropolis N771 without affecting their disassembly and reassembly properties

Abstract

We developed a hollow PEGylated encapsulin nanoparticle from Rhodococcus erythropolis N771. The hollow engineered encapsulin nanoparticles with His-Tag and Lys residues on the surface were constructed by means of genetic recombination. The Lys residues on the particle surface were successfully PEGylated with a PEG derivative, methoxy-PEG-SCM. Consequently, we demonstrated that the hollow PEGylated engineered encapsulin nanoparticle could successfully disassemble or reassemble even after PEGylation in the presence or absence of a protein denaturing agent. The nanoparticle obtained in the present study has the potential to incorporate hydrophilic compounds in the internal cavity of the particle by reversibly controllable disassembly and reassembly. The hollow PEGylated encapsulin nanoparticle can be used as a drug carrier for the delivery of hydrophilic biopolymers in future medical applications.

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

“…Moreover, the EH7‐K1 encapsulin nanoparticles were perfectly purified by gel filtration chromatography, because the nanoparticles are larger than contaminant proteins. These results are consistent with our previous report …”

“…The difference demonstrates that the C ‐terminal Lys residue of the EH7‐K1 subunit functions as the target of PEGylation effectively. We confirmed that the encapsulin nanoparticles that derived from R. erythropolis N771 tended to aggregate as the amount of PEG bound to the Lys residues at C ‐termins increased . The EH7‐K1 encapsulin nanoparticles with high dispersibility are highly applicable in diverse research.…”

“…Although previous research reported that the hollow encapsulin nanoparticles derived from R. jostii are definitely disassembled under an acidic condition of pH 3, the PEGylated EH7‐K1 encapsulin nanoparticles were not dissociated under the condition. Therefore, we developed a method for the disassembly of nanoparticles with GdnHCl . GdnHCl, a protein denaturant, denatures proteins by inhibiting the formation of hydrogen bonds.…”

“…In the present study, we have examined zeolites to establish a method for successfully preparing refolded and reassembled PEGylated protein nanoparticle without the use of protein denaturants through the proteins' reassembly process. In previous research, we reported that the removal of protein denaturants, accomplished by means of a dialysis method, can contribute to the reassembly of PEGylated encapsulin into hollow nanoparticles . However, this method has a problem: encapsulated proteins' contact with protein denaturants may, through the encapsulation process, denature the proteins.…”

“…We have reported that PEGylated hollow engineered encapsulin nanoparticles can successfully disassemble and reassemble even after PEGylation . We employed a hollow encapsulin nanoparticle derived from Rhodococcus erythropolis N771 .…”