Preparation and application to protein engineering of thiol-protecting reagent having charges.

为了提高大肠杆菌产生外源含二硫化物蛋白作为包涵体的系统在蛋白质工程中的实用性，我们试图制备巯基保护试剂，这些试剂可以通过对还原蛋白中的SH基团进行可逆修饰来溶解变性蛋白质，而不使用变性剂。在初步实验中，我们还原了四种蛋白质的二硫化物，并用带电荷的s烷基化试剂将SH基团烷基化，并检测了所得变性蛋白质的溶解度与每个疏水残基的净电荷值之间的关系。因此，我们发现，当上述值大于+0.17或小于-0.32时，变性蛋白质在水中的可溶性较好（大于1mg/ml）。由于s -烷基化是不可逆的，我们下一步制备了烷基甲基乙硫代磺酸衍生物，它在烷基部分带有电荷，可以与SH基团反应，将电荷引入还原蛋白中作为混合物。用这些试剂修饰的还原蛋白表明，碱性蛋白用带正电的试剂修饰时，变性蛋白可以溶解，带负电的试剂修饰酸性蛋白时，变性蛋白可以溶解。在蛋白质工程中n端加工所需的蛋白质水解条件下，在还原蛋白中引入的混合二硫基团足够稳定，但可以很容易地通过还原还原回SH基团。此外，taps -磺酸盐可以成功地用于溶解和提取重组人RNase 4和人成纤维细胞生长因子受体的分泌形式，这两者都是在大肠杆菌中作为包体体产生的，由于它们的不溶性，用以前的方法无法从细胞碎片中提取。纯化的变性RNase 4 （TAPS-RNase 4）可与谷胱甘肽氧化还原体系通过SH-SS交换反应折叠成活性结构。这些结果表明，本文制备的taps -磺酸盐对大肠杆菌外源产生的含二硫化物蛋白的提取、n端加工和折叠具有重要意义。少

In order to enhance the utility in protein engineering of a system using Escherichia coli producing extraneous disulfide-containing proteins as inclusion bodies, we attempted to prepare thiol-protecting reagents having charges that can solubilize denatured proteins, by reversible modification of SH groups in reduced proteins, without using denaturants. For preliminary experiments, we reduced disulfides of four proteins and alkylated the SH groups with charged S-alkylating reagents, and examined the relationship between the solubilities and the values of net charge per hydrophobic residue of the resultant denatured proteins. As a result, we found that a denatured protein is well soluble in water (more than 1mg/ml) when the above value is more than +0.17 or less than -0.32. Since S-alkylation is not reversible, we next prepared alkyl methanethiosulfonate derivatives which possess charges in the alkyl moieties and can react with SH groups to intorduce charges into reduced proteins as mixe … More d disulfides (S-alkylsulfenylation). Reduced protein modified with these reagents indicated that the denatured protein became soluble when basic protein was modified with a positively charged reagent, trimethylammoniopropyl methanethiosulfonate (TAPS-sulfonate), and acidic one with negatively charged reagent, as expected. Mixed disulfide groups thus introduced in the reduced protein were enough stable under the conditions of proteolysis, which is required for N-terminal processing in protein engineering, but could be easily reverted back to SH groups by reduction. Furthermore, TAPS-sulfonate could be successfully applied to solubilize and extract recombinant human RNase 4 and the secreted form of human fibroblast growth factor receptor, both of which were produced in E.coli as inclusion bodies and had not been able to be extracted form cell debris by means of the previous methods because of their insolubility. Purified denatured RNase 4 (TAPS-RNase 4) could be folded into the active structure by SH-SS interchange reaction with a glutathione redox system. All of these results indicate that TAPS-sulfonate prepared here is very useful for extraction, N-terminal processing and folding of disulfide-containing proteins extraneously produced in E.coli as inclusion bodies. Less