Enhancing the stability and solubility of TEV protease using in silico design
Enhancing the stability and solubility of TEV protease using in silico design
复制标题
DOI：
10.1110/ps.072822507
复制
发表时间：
2007-11-01
期刊：
影响因子：
8
通讯作者：
Bottomley, Stephen P.
中科院分区：
文献类型：
--
作者：
Cabrita, Lisa D.;Gilis, Dimitri;Bottomley, Stephen P.
合理提高重组蛋白的稳定性和溶解度的能力长期以来一直是生物技术的目标，并且对生物医学研究具有重要意义。例如，溶解性差的酶需要更大的反应体积、更长的孵育时间和更严格的反应条件，所有这些都会增加成本并对工艺的可行性产生负面影响。这里通过 PoPMuSiC 程序实现了合理设计，该程序在计算机中对单位点突变的稳定性变化进行预测。我们使用该程序来提高烟草蚀纹病毒 (TEV) 蛋白的稳定性。 TEV 是一种具有严格特异性的 27 kDa 核包涵蛋白酶，通常用于在蛋白质纯化方案中去除溶解度标签。然而，虽然重组 TEV 可以大量生产，但其限制是其溶解度相对较差（通常类似于 1 mg/mL），这意味着有效裂解需要大体积和较长的孵育时间。在对 TEV 进行 PoPMuSiC 分析后，选择了五个预计比野生型更稳定的变体，对其稳定性、溶解度和活性进行实验分析。其中，有两种被发现可以增强 TEV 的溶解度而不损害其功能活性。此外，发现完全活性的双突变体在浓度超过 40 mg/mL 时仍然可溶。因此，这种改良的 TEV 似乎是重组蛋白技术中使用的一个有趣的候选者。
The ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. Poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. Rational design is achieved here by means of the PoPMuSiC program, which performs in silico predictions of stability changes upon single-site mutations. We have used this program to increase the stability of the tobacco etch virus (TEV) protein. TEV is a 27-kDa nuclear inclusion protease with stringent specificity that is commonly used for the removal of solubility tags during protein purification protocols. However, while recombinant TEV can be produced in large quantities, a limitation is its relatively poor solubility (generally similar to 1 mg/mL), which means that large volumes and often long incubation times are required for efficient cleavage. Following PoPMuSiC analysis of TEV, five variants predicted to be more stable than the wild type were selected for experimental analysis of their stability, solubility, and activity. Of these, two were found to enhance the solubility of TEV without compromising its functional activity. In addition, a fully active double mutant was found to remain soluble at concentrations in excess of 40 mg/mL. This modified TEV appears thus as an interesting candidate to be used in recombinant protein technology.