Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstr. 55, 38106, Braunschweig, Germany.
J Nanobiotechnology. 2020 Nov 6;18(1):159. doi: 10.1186/s12951-020-00715-1.
Purification of recombinant proteins is often a challenging matter because high purity and high recovery are desired. If the expressed recombinant protein is also in a complex matrix, such as from the silkworm expression system, purification becomes more challenging. Even if purification from the silkworm expression system is troublesome, it benefits from a high capacity for the production of recombinant proteins. In this study, magnetic nanoparticles (MNPs) were investigated as a suitable tool for the purification of proteins from the complex matrix of the silkworm fat body. The MNPs were modified with nickel so that they have an affinity for His-tagged proteins, as the MNP purification protocol itself does not need special equipment except for a magnet. Among the three different kinds of investigated MNPs, MNPs with sizes of 100 nm to 200 nm and approximately 20 nm-thick nickel shells were the most suitable for our purpose. With them, the total protein amount was reduced by up to at least approximately 77.7%, with a protein recovery of around 50.8% from the silkworm fat body. The minimum binding capacity was estimated to be 83.3 µg protein/mg MNP. Therefore, these MNPs are a promising tool as a purification pretreatment of complex sample matrices.
重组蛋白的纯化通常是一个具有挑战性的问题，因为需要高纯度和高回收率。如果表达的重组蛋白也处于复杂的基质中，例如来自家蚕表达系统，那么纯化就更加具有挑战性。即使从家蚕表达系统中进行纯化很麻烦，但它受益于生产重组蛋白的高容量。在这项研究中，磁性纳米颗粒 (MNP) 被研究作为从家蚕脂肪体的复杂基质中纯化蛋白质的合适工具。MNP 用镍进行修饰，使其对 His 标记的蛋白质具有亲和力，因为 MNP 纯化方案本身除了磁铁之外不需要特殊设备。在所研究的三种不同类型的 MNP 中，粒径为 100-200nm 且镍壳厚度约为 20nm 的 MNP 最适合我们的目的。使用它们，从家蚕脂肪体中可以将总蛋白量减少至少约 77.7%，蛋白回收率约为 50.8%。估计最小结合容量为 83.3µg 蛋白/mg MNP。因此，这些 MNP 作为复杂样品基质的纯化预处理工具具有广阔的应用前景。
