Studies On The Molecular Design Of Novel Oxidative Folding Aids And New Refolding Methods

Disulfide fromation and protein aggregation are the two key factors influencing protein refolding. Herein, a series of agents and methods that can facilitate disulfide formation and inhibit protein aggregation were developed to facilitate protein refolding.To investigate the disulfide bond formation, protein oxidative folding kinetics were examined. It was shown that disulfide bond formation was a prerequisite for tryptophan burial. Besides, the results elucidated that the rate of the first folding phase is determined by the formation of mixed disulfide bond.Based on these results, novel oxidative folding aids were designed. Firstly, structural characteristic of protein disulfide isomerase (PDI) and DsbA were investigated. Hydrophobic regions were observed around their active sites. Based on this finding, hydrophobic alkyl tails were linked to cystamine to design a novel small molecular foldase mimics, acyl cystamine. Acyl cystamine was proven very effective to facilitate oxidative protein refolding at strong reducing environments, which can not be achieved by any other oxidants. Besides, Acyl cystamine can greatly increase protein folding rate and effectively facilitate protein folding at much lower concentration. To achieve the same refolding effect, the concentration of n-hexanoyl cystamine was only about half of cystamine. Further investigation was performed to explore the mechanism. It was shown that the specific hydrophobic interaction between the alkyl tail and unfolded peptide or folding intermediates make acyl cystamine strikingly better oxidative folding aids.Moreover, the disulfide form of a small peptide mimic of PDI, CGC, was explored to facilitate oxidative protein refolding. It was found that CGC increased both the folding rate and refolding yield. Based on this finding, a new pentapeptide, RKCGC was designed. It was confirmed that RKCGC can effectively facilitate protein folding with DTT, and assist both disulfide formation and isomerization. It was effective in a wide pH range. Meanwhile, RKCGC has better effect to increase the folding rate and yield as compared with CGC. Further investigation was perfomed to explore the mechanism. It was shown that the better effect of RKCGC than CGC was contributed by its lower pKa and higher reduction potential.The inhibition of protein aggregation during refolding was investigated. Herein we found that electrostatic repulsion between like-charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding was extensively demonstrated with three different proteins and four kinds of ion-exchange resins when the protein and ion-exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect was significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics was not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. This was not realistic by any other approaches. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The method was further applied to the renaturation of enhanced green fluorescent protein (EGFP) inclusion bodies. Like-charged exchanger increased the refolding yield by almost 100%. Moreover, it has purification effect at the same time.Based on the research in protein refolding assisted by like-charged ion-exchange resin, a protein refolding method with like-charged polyelectrolyte as additive was developed. The working mechanisms of like-charged polyelectrolytes and ion exchangers revealed that the structure was the key factor influencing the effectiveness of like-charged agents in facilitating protein refolding.