Temperature‐Responsive Mixed‐Shell Polymeric Micelles for the Refolding of Thermally Denatured Proteins

Abstract

We have fabricated a mixed-shell polymeric micelle (MSPM) that closely mimics the natural molecular chaperone GroEL-GroES complex in terms of structure and functionality. This MSPM, which possesses a shared PLA core and a homogeneously mixed PEG and PNIAPM shell, is constructed through the co-assembly of block copolymers poly(lactide-b-poly(ethylene oxide) (PLA-b-PEG) and poly(lactide)-b-poly(N-isopropylacryamide) (PLA-b-PNIPAM). Above the lower critical solution temperature (LCST) of PNIPAM, the MSPM evolves into a core-shell-corona micelle (CSCM), as a functional state with hydrophobic PNIPAM domains on its surface. Light scattering (LS), TEM, and fluorescence and circular dichroism (CD) spectroscopy were performed to investigate the working mechanism of the chaperone-like behavior of this system. Unfolded protein intermediates are captured by the hydrophobic PNIPAM domains of the CSCM, which prevent harmful protein aggregation. During cooling, PNIPAM reverts into its hydrophilic state, thereby inducing the release of the bound unfolded proteins. The refolding process of the released proteins is spontaneously accomplished by the presence of PEG in the mixed shell. Carbonic anhydrase B (CAB) was chosen as a model to investigate the refolding efficiency of the released proteins. In the presence of MSPM, almost 93 % CAB activity was recovered during cooling after complete denaturation at 70 °C. Further results reveal that this MSPM also works with a wide spectrum of proteins with more-complicated structures, including some multimeric proteins. Given the convenience and generality in preventing the thermal aggregation of proteins, this MSPM-based chaperone might be useful for preventing the toxic aggregation of misfolded proteins in some diseases.

Refolding of Aggregation-Prone ScFv Antibody Fragments Assisted by Hydrophobically Modified Poly(sodium acrylate) Derivatives

“…The protection and/or association was attributed primarily to hydrophobic binding of the polymers with unfolded proteins or folding intermediates and, less frequently, to Coulomb interactions . The protection of partly folded proteins was ascribed to the increased solubility of the protein:polymer complexes, for instance in the cases of carbonic anhydrase, lysozyme alpha‐chymotrypsin, and alpha‐amylase), to the hydrophobically mediated interfacial immobilization of proteins on polymer nanoparticles or on polymer micelles, or to confinement in nanogels (of pullulan or in PNIPAM). In each case, it was concluded that sequestration of the protein resulted in their stabilization against aggregation by immobilizing aggregation‐prone species.…”

Thermosensitive mixed shell polymeric micelles decorated with gold nanoparticles at the outmost surface: tunable surface plasmon resonance and enhanced catalytic properties with excellent colloidal stability

“…46 The subscripts in the block copolymers designate the degrees of polymerization. The two block polymers, poly-(3-caprolactone) 88 -block-(ethylene glycol) 113 (PCL 88 -b-PEG 113 ) and poly(3-caprolactone) 83 -block-poly(N-isopropylacrylamide) 90 (PCL 78 -b-PNIPAM 90 ), were prepared as reported in our previous work.…”

“…46 A tailor-designed PCL coupled with a thiocarbonylthio group at one of the chain ends was used as the macro chain transfer agent for reversible addition fragmentation chain transfer (RAFT) polymerization of NIPAM, resulting in PCL-b-PNIPAM with the thiocarbonylthio group sitting at the end of the PNI-PAM block (insets of Scheme 1). 46 A tailor-designed PCL coupled with a thiocarbonylthio group at one of the chain ends was used as the macro chain transfer agent for reversible addition fragmentation chain transfer (RAFT) polymerization of NIPAM, resulting in PCL-b-PNIPAM with the thiocarbonylthio group sitting at the end of the PNI-PAM block (insets of Scheme 1).…”

Polymer antidotes for toxin sequestration

“…In the general protocol for affinity purification of proteins, isolated proteins on the solid affinity support are released by selective elution induced by either pH and/or salt gradients or by adding an excess of competing ligand. However, NIPAm polymers and many NIPAm-TBAm copolymers are thermoresponsive [23,24]. Above their lower critical solution temperature (LCST; ∼10 °C), NPs containing both negatively charged (AAc) and hydrophobic (TBAm) functional monomers have a collapsed structure.…”