Overexpression, purification, and refolding of link module from human TSG-6 in Escherichia coli: Effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons
Overexpression, purification, and refolding of link module from human TSG-6 in Escherichia coli: Effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons
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DOI：
10.1006/prep.1996.0068
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发表时间：
1996-08-01
影响因子：
1.6
通讯作者：
Willis, AC
中科院分区：
文献类型：
--
作者：
Day, AJ;Aplin, RT;Willis, AC
在大肠杆菌中过量表达Link模块，即在人肿瘤坏死因子刺激基因6的透明质酸结合蛋白中发现的98个氨基酸的结构域。电喷雾电离质谱显示，只有50%的表达蛋白具有预期的野生型分子量，剩余的材料具有1至4个精氨酸至赖氨酸的取代，这是由于在阿加密码子处的错误掺入而产生的，通过将4个阿加密码子突变为CGT，错误掺入的水平几乎完全消除。这种对高使用率精氨酸密码子的突变也增加了异源表达的水平。在纯化过程中发生的连接模块的重折叠产生了两种具有不同二硫键结构的物质，其可以通过高效液相色谱法分离，其中一个具有与其他Link模块中发现的一致的二硫键排列，并且通过核磁共振光谱显示是折叠的。(C)出版社：Academic Press，Inc.
The Link module, a 98-amino-acid domain found in hyaluronan binding proteins of human tumor necrosis factor stimulated gene 6 was overexpressed in Escherichia coli, Electrospray ionization mass spectrometry revealed that only 50% of the expressed protein had the expected wild-type molecular weight, with the remaining material having between 1 and 4 arginine to lysine substitutions, arising due to misincorporation at AGA codons, The level of misincorporation was almost completely abolished by mutation of the 4 AGA codons to CGT, This mutation to high-usage arginine codons also increased the level of heterologous expression, Refolding of the Link module, which occurred during the purification procedure, gave two species with different disulfide bond organizations that could be separated by high-performance liquid chromatography, One of these had a disulfide bond arrangement consistent with that found in other Link modules and, by nuclear magnetic resonance spectroscopy, was shown to be folded. (C) 1996 Academic Press, Inc.