Enhanced Protein Adsorption and Facilitated Refolding of Like-Charged Protein with Highly Charged Silica Nanoparticles Fabricated by Sequential Double Modifications

Abstract

Silica nanoparticles (SNPs) were sequentially modified with poly(ethylenimine) (PEI) and 2-diethylaminoethyl chloride (DEAE) to prepare a series of positively charged SNPs-PEI and SNPs-PEI-DEAE. The sequential double-modification strategy produced a charge density as high as 1740 μmol/g (4524 μmol/mL), which offered a very high adsorption capacity for bovine serum albumin (314 mg/g). Most importantly, the highly charged SNPs-PEI and SNPs-PEI-DEAE could efficiently facilitate the refolding of like-charged protein at extremely low utilization. For instance, in the refolding of 1 mg/mL lysozyme, the refolding yield reached 75% with only 3.3 μL/mL SNPs-PEI-DEAE. The bead consumption was reduced by nearly 96% as compared to that of the charged microspheres used previously to reach a similar yield. The results proved that the polyelectrolyte-modified SNPs were promising for applications in facilitating like-charged protein refolding, and the research opened up a new way for biotechnology applications of highly charged nanoparticles.

Reversible Immobilization of Lipases on Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption

“…Other possibility is the modification of the support, e.g., adding anion groups [60], or even mixed cation/anion groups [61]. Some authors suggest that enzyme refolding of immobilized enzymes is easier if the support has the same ion nature of the enzyme, this may help in improving enzyme activity recovery during activity determination under mild conditions [62,63,64,65,66].…”

Elucidating the Role of Thermal Flexibility of Hydrogels in Protein Refolding

“…A number of experimental and theoretical models indicate that the presence of water in confined geometries plays an important role in the biological activities of macromolecules. 3,17,26,27 To understand the role of P(B5AMA)-10confined water in the protein refolding behavior of hydrogels, P(B5AMA)-10 were collapsed at 37 °C and the water collected from hydrogels was freeze-dried and analyzed by 1 H NMR and FTIR for the presence of any impurities that may have participated in the restoration of the enzymatic activities of proteins. However, only a trace amount of free B5AMA (less than 0.03% of the original feed ratio) was detected by 1 H NMR.…”

“…Recently, much interest has been focused on the development of synthetic chaperones to understand the mechanism of protein refolding and to aid the refolding of thermally or chemically denatured proteins into biologically active forms. − These synthetic chaperones are typically designed to mimic GroEL/GroES mechanism of protein refolding and are composed of polymers, ,,,,− , ionic liquids, nanoparticles, ,,,,− and hydrogels, ,, which assist in protein refolding, without them being incorporated into the final folded state of proteins. These macromolecules bind with denatured proteins via hydrophobic or ionic interactions, prevent their aggregation in solution, facilitate the refolding of denatured proteins, and release their biologically active form, as a function of external stimuli such as temperature, ,,, pH, , or stripper (detergent, cycloamylase, β-cyclodextrin). − ,, Poly­( N -isopropylmethacrylamide) (PNIPAM)-based mixed shell polymeric micelles, for example, closely simulate GroEL/GroES complex and confine the denatured proteins at higher temperatures, followed by the release of their refolded forms at lower temperatures .…”

“…These macromolecules bind with denatured proteins via hydrophobic or ionic interactions, prevent their aggregation in solution, facilitate the refolding of denatured proteins, and release their biologically active form, as a function of external stimuli such as temperature, ,,, pH, , or stripper (detergent, cycloamylase, β-cyclodextrin). − ,, Poly­( N -isopropylmethacrylamide) (PNIPAM)-based mixed shell polymeric micelles, for example, closely simulate GroEL/GroES complex and confine the denatured proteins at higher temperatures, followed by the release of their refolded forms at lower temperatures . In comparison to nanoparticles, thermally responsive hydrogels interact with denatured proteins via weak noncovalent interactions and solely depend on their thermal flexibility to refold the denatured proteins . Kisley et al have also demonstrated that weak noncovalent interactions between proteins and poly­(acrylamide) hydrogels are sufficient to impart thermal stability to the former and confinement of proteins in the porous architecture of hydrogels has minimum effect on protein stability .…”

“…16 nanoparticles, thermally responsive hydrogels interact with denatured proteins via weak noncovalent interactions and solely depend on their thermal flexibility to refold the denatured proteins. 3 Kisley et al have also demonstrated that weak noncovalent interactions between proteins and poly-(acrylamide) hydrogels are sufficient to impart thermal stability to the former and confinement of proteins in the porous architecture of hydrogels has minimum effect on protein stability. 23 However, a handful of studies on protein refolding efficacies of thermally responsive hydrogels simply describe that heating of unfolded proteins in the presence of hydrogel facilitates the refolding of proteins as a function of lower critical solution temperature (LCST) behavior of hydrogels, 5 and the mechanism by which hydrogels facilitate this process is currently unknown.…”