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首页> 外文期刊>Journal of Biotechnology >Comparison of the oxidative folding of lysozyme at a high protein concentration using aromatic thiols versus glutathione
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Comparison of the oxidative folding of lysozyme at a high protein concentration using aromatic thiols versus glutathione

机译：使用芳族硫醇与谷胱甘肽比较高蛋白浓度下溶菌酶的氧化折叠

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摘要

The production of proteins using recombinant DNA technology often requires the use of in vitro protein folding. In order to facilitate in vitro protein folding, a redox buffer is added to the protein folding mixture. The redox buffer is composed of a small molecule disulfide and/or a small molecule thiol. Recently, redox buffers containing aromatic thiols have been shown to be an improvement over traditional redox buffers such as glutathione. For in vitro protein folding to be relevant to protein production on a larger scale, high protein concentrations are required to avoid large volumes of folding buffer. Therefore, we investigated the in vitro folding of lysozyme at 1mg/mL instead of the traditional 0.1mg/mL. Aromatic thiols and aromatic disulfides were compared directly with glutathione and glutathione disulfide, the most commonly used redox buffer. Folding experiments at pH 7 using aromatic thiols increased the yield by 20-40% and the folding rate constants by as much as 11 times relative to glutathione. At pH 8, improvements in yields of up to 25% and up to a 7-fold increase in folding rate constants were demonstrated. The effect of aromatic disulfide concentration was also investigated.
机译：使用重组DNA技术生产蛋白质通常需要使用体外蛋白质折叠。为了促进体外蛋白质折叠，将氧化还原缓冲液添加到蛋白质折叠混合物中。氧化还原缓冲液由小分子二硫化物和/或小分子硫醇组成。最近，已显示出含有芳族硫醇的氧化还原缓冲液是对传统氧化还原缓冲液如谷胱甘肽的一种改进。为了使体外蛋白质折叠与大规模生产蛋白质相关，需要高蛋白质浓度以避免大量折叠缓冲液。因此，我们研究了溶菌酶在体外以1mg / mL代替传统的0.1mg / mL折叠的情况。将芳香族硫醇和芳香族二硫化物直接与最常用的氧化还原缓冲液谷胱甘肽和谷胱甘肽二硫化物进行了比较。相对于谷胱甘肽，使用芳族硫醇在pH 7下进行折叠实验可使收率提高20-40％，折叠速率常数提高11倍。在pH 8时，产率提高了25％，折叠速率常数提高了7倍。还研究了芳族二硫化物浓度的影响。

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