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

带电荷硫醇保护试剂的制备及其在蛋白质工程中的应用

基本信息

批准号：
06453128
负责人：
YAMADA Hidenori
金额：
$ 4.74万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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

为了提高使用大肠杆菌生产外源性含二硫键蛋白质作为包涵体的系统在蛋白质工程中的实用性，我们试图通过还原蛋白质中的SH基团的可逆修饰，而不使用变性剂，制备具有可以溶解变性蛋白质的电荷的巯基保护试剂。对于初步的实验，我们减少了四个蛋白质的二硫化物和烷基化的SH基团与带电的S-烷基化试剂，并检查所得变性蛋白质的溶解度和每个疏水残基的净电荷值之间的关系。结果，我们发现，当上述值大于+0.17或小于-0.32时，变性蛋白质在水中溶解良好（大于1 mg/ml）。由于S-烷基化是不可逆的，我们接下来制备了烷基甲硫基磺酸盐衍生物，其在烷基部分具有电荷，并且可以与SH基团反应，以混合物的形式将电荷引入还原的蛋白质中。 ...更多信息 d二硫化物（S-烷基亚磺酰基化）。这些试剂修饰的还原蛋白质表明，当碱性蛋白质用带正电荷的试剂三甲基铵丙基甲硫基磺酸盐（TAPS-磺酸盐）修饰时，变性蛋白质变得可溶，而酸性蛋白质用带负电荷的试剂修饰时，正如预期的那样。因此，在还原的蛋白质中引入的混合二硫键基团在蛋白质水解的条件下是足够稳定的，这是蛋白质工程中N-末端加工所需的，但可以通过还原容易地恢复为SH基团。此外，TAPS-磺酸盐可以成功地应用于溶解和提取重组人RNase 4和分泌形式的人成纤维细胞生长因子受体，这两者都是在大肠杆菌中作为包涵体产生的，并且由于它们的不溶性而不能通过先前的方法从细胞碎片中提取。纯化的变性核糖核酸酶4（TAPS-RNase 4）可通过谷胱甘肽氧化还原系统与SH SS交换反应折叠成具有活性的结构。所有这些结果表明，这里制备的TAPS-磺酸盐是非常有用的提取，N-末端加工和折叠的含二硫键的蛋白质在大肠杆菌中以包涵体形式产生。少