Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Methods Mol Biol. 2023;2671:403-418. doi: 10.1007/978-1-0716-3222-2_23.
Protein refolding is a crucial procedure in bacterial recombinant expression. Aggregation and misfolding are the two challenges that can affect the overall yield and specific activity of the folded proteins. We demonstrated the in vitro use of nanoscale "thermostable exoshells" (tES) to encapsulate, fold and release diverse protein substrates. With tES, the soluble yield, functional yield, and specific activity increased from 2-fold to >100-fold when compared to folding in its absence. On average, the soluble yield was determined to be 6.5 mg/100 mg of tES for a set of 12 diverse substrates evaluated. The electrostatic charge complementation between the tES interior and the protein substrate was considered as the primary determinant for functional folding. We thus describe a useful and simple method for in vitro folding that has been evaluated and implemented in our laboratory.
蛋白质复性是细菌重组表达中的一个关键步骤。聚集和错误折叠是两个可能影响折叠蛋白整体产率和比活性的挑战。我们证明了纳米级“热稳定外壳”（tES）可在体外用于包裹、折叠和释放不同的蛋白质底物。与没有 tES 相比，使用 tES 可使可溶性产率、功能产率和比活性提高 2 倍以上。平均而言，对于 12 种不同的评估底物，每 100mg tES 的可溶性产率为 6.5mg。tES 内部与蛋白质底物之间的静电电荷互补被认为是功能折叠的主要决定因素。因此，我们描述了一种有用且简单的体外折叠方法，该方法已在我们的实验室中进行了评估和实施。
