Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation
Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation
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DOI：
10.1021/bp970123w
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发表时间：
1998-01-01
影响因子：
2.9
通讯作者：
Maachupalli-Reddy, J
中科院分区：
文献类型：
--
作者：
Clark, ED;Hevehan, D;Maachupalli-Reddy, J
自从重组 DNA 技术诞生以来，在大肠杆菌中作为不溶性包涵体产生的蛋白质的分离、复性和天然二硫键形成方面已经开发了不同的策略。优化复性过程的主要挑战之一是防止路径外非活性和聚集物种的形成。基于描述折叠和聚集之间竞争的简化动力学模型，可以分析变性剂和硫醇/二硫化物浓度对这种竞争的影响。虽然较高的氯化胍 (GdmCl) 浓度会导致较高的复性产率，但折叠率会受到负面影响，这表明 GdmCl 的最佳范围可实现最佳复性率和产率。类似地，较高的总谷胱甘肽浓度导致较高的产量但降低了速率，也表明最佳复性速率和产量的最佳总谷胱甘肽浓度（6-16 mM），还原谷胱甘肽与氧化型谷胱甘肽的最佳比例在1和3之间。为了表征聚集体的性质，在不同的氧化/还原条件下进行了聚集实验。结果表明，部分折叠的多肽链之间的疏水相互作用是聚集的主要原因。聚集速度很快，并且聚集体浓度在复性的第一分钟之后不会显着增加。在促进二硫键键合的条件下，由于二硫键的形成，聚集体尺寸可能会增加，但浓度不会增加，从而形成共价键合的聚集体。
Since the inception of recombinant DNA technology, different strategies have been developed in the isolation, renaturation, and native disulfide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges in optimizing renaturation processes is to prevent the formation of off-pathway inactive and aggregated species. On the basis of a simplified kinetic model describing the competition between folding and aggregation, it was possible to analyze the effects of denaturant and thiol/disulfide concentrations on this competition. Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rate was negatively affected, indicating an optimum range of GdmCl for optimum renaturation rates and yields. Similarly, higher total glutathione concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum renaturation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize the nature of aggregates, aggregation experiments were performed under different oxidizing/reducing conditions. It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of aggregation. Aggregation is fast and aggregate concentration does not significantly increase beyond the first minute of renaturation. Under conditions which promote disulfide bonding, aggregate size, but not concentration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.