新型蛋白质氧化折叠助剂的分子设计和复性方法研究
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摘要
蛋白质复性是应用基因重组技术在菌体中生产贵重蛋白质的瓶颈。本文针对蛋白质复性过程的两个关键因素,二硫键形成和蛋白质聚集抑制,开展蛋白质氧化折叠助剂的分子设计和复性方法研究。
针对二硫键形成问题,开展蛋白质氧化折叠动力学研究,揭示二硫键形成是折叠过程中色氨酸包埋的前提条件,发现混合二硫键的形成是控制第一折叠相折叠速率的关键因素。
在此基础上开展氧化折叠助剂的分子设计。首先考察蛋白质二硫键异构酶(PDI)和DsbA的结构特性,发现其活性位点周围具有疏水性表面。因此,设计具有疏水尾的小分子折叠酶模拟物酰基胱胺作为新型氧化折叠助剂分子。研究表明,酰基胱胺能够在强还原环境下有效地促进蛋白质氧化复性,这是其它任何氧化剂所不能做到的。并且,酰基胱胺能够极大地提高蛋白质氧化复性速率,在较低浓度下依然有效,仅需胱胺浓度的一半即可获得相同的复性效果。其作用机理研究发现,酰基胱胺通过疏水烷基链介导与变性蛋白质之间形成疏水相互作用,获得其促进复性的显著效果。
其次,研究发现PDI的模拟分子氧化型寡肽CGC作为氧化折叠助剂可显著提高蛋白质氧化复性的速率和收率。以此为基础设计新型氧化折叠助剂分子RKCGC。研究表明,RKCGC能够与二硫苏糖醇很好地耦合,促进二硫键的形成和异构,具有较宽的适用pH范围。且与CGC相比,RKCGC能够更好地提高氧化复性的速率和收率。其作用机理研究发现,RKCGC具有更低的pKa值和更高的还原势,因而获得较好的辅助复性效果。
针对蛋白质聚集问题,开展抑制聚集方法研究,发现与蛋白质带有同种电荷的离子交换介质能够很好地抑制折叠中间体聚集从而促进蛋白质复性。通过四种带不同电荷的离子交换介质对三种带不同电荷的蛋白质辅助复性研究,证实同电荷离子交换介质能够有效促进蛋白质复性,且在高蛋白质浓度(4 mg/mL溶菌酶;2 mg/mL牛血清白蛋白)条件其促进复性效果依然非常显著,而且不会降低复性速率,优于其它化学添加剂。其作用机理研究发现,同电荷离子交换介质通过静电斥力诱导蛋白质在其带电表面形成定向排列,从而抑制聚集。进而将该方法应用到重组增强型绿色荧光蛋白(EGFP)的复性中,发现EGFP包含体的复性收率提高近一倍,并且在辅助复性的同时还具有纯化的效果。
在同电荷离子交换介质辅助复性的基础上拓展研究工作,提出同电荷聚电解质辅助蛋白质复性方法。通过比较聚电解质与离子交换介质的作用机制,揭示溶液中的存在形态是影响同电荷物质促进蛋白质复性效果的关键因素。
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.
针对二硫键形成问题,开展蛋白质氧化折叠动力学研究,揭示二硫键形成是折叠过程中色氨酸包埋的前提条件,发现混合二硫键的形成是控制第一折叠相折叠速率的关键因素。
在此基础上开展氧化折叠助剂的分子设计。首先考察蛋白质二硫键异构酶(PDI)和DsbA的结构特性,发现其活性位点周围具有疏水性表面。因此,设计具有疏水尾的小分子折叠酶模拟物酰基胱胺作为新型氧化折叠助剂分子。研究表明,酰基胱胺能够在强还原环境下有效地促进蛋白质氧化复性,这是其它任何氧化剂所不能做到的。并且,酰基胱胺能够极大地提高蛋白质氧化复性速率,在较低浓度下依然有效,仅需胱胺浓度的一半即可获得相同的复性效果。其作用机理研究发现,酰基胱胺通过疏水烷基链介导与变性蛋白质之间形成疏水相互作用,获得其促进复性的显著效果。
其次,研究发现PDI的模拟分子氧化型寡肽CGC作为氧化折叠助剂可显著提高蛋白质氧化复性的速率和收率。以此为基础设计新型氧化折叠助剂分子RKCGC。研究表明,RKCGC能够与二硫苏糖醇很好地耦合,促进二硫键的形成和异构,具有较宽的适用pH范围。且与CGC相比,RKCGC能够更好地提高氧化复性的速率和收率。其作用机理研究发现,RKCGC具有更低的pKa值和更高的还原势,因而获得较好的辅助复性效果。
针对蛋白质聚集问题,开展抑制聚集方法研究,发现与蛋白质带有同种电荷的离子交换介质能够很好地抑制折叠中间体聚集从而促进蛋白质复性。通过四种带不同电荷的离子交换介质对三种带不同电荷的蛋白质辅助复性研究,证实同电荷离子交换介质能够有效促进蛋白质复性,且在高蛋白质浓度(4 mg/mL溶菌酶;2 mg/mL牛血清白蛋白)条件其促进复性效果依然非常显著,而且不会降低复性速率,优于其它化学添加剂。其作用机理研究发现,同电荷离子交换介质通过静电斥力诱导蛋白质在其带电表面形成定向排列,从而抑制聚集。进而将该方法应用到重组增强型绿色荧光蛋白(EGFP)的复性中,发现EGFP包含体的复性收率提高近一倍,并且在辅助复性的同时还具有纯化的效果。
在同电荷离子交换介质辅助复性的基础上拓展研究工作,提出同电荷聚电解质辅助蛋白质复性方法。通过比较聚电解质与离子交换介质的作用机制,揭示溶液中的存在形态是影响同电荷物质促进蛋白质复性效果的关键因素。
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.
引文
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