Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers
Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers
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DOI：
10.1016/j.bmc.2007.11.047
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发表时间：
2008-03-01
影响因子：
3.5
通讯作者：
Lees, Watson J.
中科院分区：
文献类型：
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作者：
Gurbhele-Tupkar, Minakshi C.;Perez, Lissette R.;Lees, Watson J.
几乎所有治疗性蛋白质和大多数细胞外蛋白质都含有二硫键。由于在折叠过程中需要形成正确匹配的二硫键，因此在细菌或体外生产这些蛋白质具有挑战性。有效体外折叠的一个重要参数是氧化还原缓冲液的组成，它是小分子硫醇和小分子二硫化物的混合物。然而，不同氧化还原缓冲液对蛋白质折叠的影响受到的关注有限。在含有不同浓度的五种不同芳香族硫醇或传统脂肪族硫醇谷胱甘肽 (GSH) 的氧化还原缓冲液存在下，跟踪变性还原型溶菌酶的氧化折叠。芳香族硫醇消除了低二硫键浓度下的滞后期，将折叠速率常数提高至 11 倍，并相对于 GSH 提高了活性蛋白的产量。五种芳香硫醇中的四种、pH 7 下的谷胱甘肽以及 pH 8.2 下的谷胱甘肽的活性蛋白产量相似。在 pH 6 时，带正电荷的芳香族硫醇比带负电荷的硫醇提供更高的产率。 (C) 2007 Elsevier Ltd. 保留所有权利。
Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important parameter for efficient in vitro folding is the composition of the redox buffer, a mixture of a small molecule thiol and small molecule disulfide. The effects of different redox buffers on protein folding, however, have received limited attention. The oxidative folding of denatured reduced lysozyme was followed in the presence of redox buffers containing varying concentrations of five different aromatic thiols or the traditional aliphatic thiol glutathione (GSH). Aromatic thiols eliminated the lag phase at low disulfide concentrations, increased the folding rate constant up to 11-fold, and improved the yield of active protein relative to GSH. The yield of active protein was similar for four of the five aromatic thiols and for glutathione at pH 7 as well as for glutathione at pH 8.2. At pH 6 the positively charged aromatic thiol provided a higher yield than the negatively charged thiols. (C) 2007 Elsevier Ltd. All rights reserved.