3. Inhibition of Protein Aggregation

3.1. Inhibition of amyloid protein aggregation

    A nascent protein (peptide) needs to fold to its specific tertiary structure to function in a living system, so how a protein folds is an essential issue of life science studies. If a protein fails to fold correctly or protein misfolding occurs in vivo, the misfolded protein will accumulate and aggregate in the living system, resulting in serious diseases. Currently, over 50 diseases have been identified to be related to in vivo protein misfolding and aggregation, including neurodegenerative diseases (e.g., Alzheimer's disease, AD), metabolic diseases and cancers. Hence, inhibition of in vivo protein aggregation is a major concern in medical science. We have made efforts on the discovery and design of new inhibitors against amyloid β-protein (Aβ) aggregation (fibrillogenesis) as well as metal (Zn2+ and Cu2+)-mediated Aβ aggregation, which have been recognized as the main hallmark of AD.
    By virtual screening with molecular dynamics simulations, we discovered a natural compound (brazilin) that inhibits Aβ fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity. Then, we proposed to modify serum albumin to explore protein-based Aβ aggregation inhibitors. Results revealed that acidulated human serum albumin (HSA) showed significantly higher inhibitory effects than native albumin, and a hydrophobic binding-electrostatic repulsion (HyBER) hypothesis was proposed to explain the phenomenon. Moreover, we succeeded in functionalizing HSA with iminodiacetic acid to create a multifunctional agent for inhibiting high-concentration metal-induced Aβ aggregation and remodeling mature metal-induced Aβ species. Recently, we have designed new peptide inhibitors and developed different peptide-nanoparticle conjugates and self-assembled nano-conjugates by incorporating peptide or small molecular inhibitors into nanoparticles or natural polymers. It was interesting to note that the nano-inhibitors displayed remarkably higher inhibitory efficiency than their free counterparts. The research has shed new light on the design and fabrication of multifunctional nano-inhibitors.
    Currently, we are researching into the development of new peptide-based inhibitors/chelators and their nanoconjugates, self-assembled nanoparticles of inhibitor-polymer conjugates, dendrimer-based inhibitors and protein-based inhibitors by surface functionalizations.

3.2. Protein refolding

    Heterologous proteins produced by recombinant microbial organisms (e.g., E. coli.) often form inactive aggregates known as inclusion bodies. In order for the recovery of biologically active protein, the aggregates must be separated from cell debris and solubilized by exposing them to a strong denaturant. Decreasing the denaturant concentration by dialysis or direct dilution can initiate the unfolded polypeptide chains to refold to their native state. However, only a fraction of the native-state protein can be recovered in this process since the unfolded protein tends to re-aggregate by intermolecular interactions. This is because of the fact that protein renaturation is a kinetically competitive process between the folding and aggregation. As a unimolecular process, protein folding is a first-order reaction, while the aggregate formation is higher order because it is caused by intermolecular interactions. Due to the kinetic nature of protein refolding process, suppression of the aggregating reaction is the key point to enhance protein renaturation yield. Moreover, efficient separation and purification of refolded proteins is another important part limiting the large-scale production of recombinant proteins. Our research has focused on the efficient refolding of denatured-reduced proteins at high concentrations by various methods, including the application of molecular chaperones, artificial chaperones, chromatography, and fed-batch dilution. Mathematical models for the refolding processes have been established and the kinetic behaviors of the processes are evaluated.
    In 2010, we found that charged particles such as ion exchange resins can greatly suppress the aggregation of like-charged folding protein intermediates, leading to the significant increase of protein refolding yield (native protein recovery). The working mechanism of the like-charged surface effect on folding proteins is considered due to the charge repulsion near like-charged surfaces. Namely, the charge repulsion at a solid (or polymer) surface can induce oriented alignment of folding protein molecules, which increases electrostatic repulsion between neighboring folding proteins and leads to the inhibition of protein aggregation. Besides solid particles, the like-charge effect was also confirmed with polyelectrolytes.
    Detailed research revealed that protein refolding yield increased with increasing ionic capacity and resin concentration. So higher ionic–capacity resin was favorable to offer higher refolding yield at lower added concentrations. Based on the findings, we developed extremely high charge density nanoparticles (4524 μmol/mL) by sequentially modifying silica nanoparticles (SNPs) with poly(ethylenimine) and 2-diethylaminoethyl chloride. Use of the highly charged nanoparticles efficiently facilitated the refolding of like-charged protein at extremely low utilization (e.g., 75% lysozyme refolding at 1 mg/mL with 3.3 μL/mL of the SNPs). Then, we proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. High efficiency refolding and purification of green fluorescent protein were realized with iminodiacetic acid-grafted SNPs.