Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, 2000, Argentina.
Protein Sci. 2019 Apr;28(4):800-807. doi: 10.1002/pro.3574. Epub 2019 Feb 20.
The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.
近年来，细菌中重组蛋白的生产有了显著的增长，成为研究和工业生产蛋白质的常用工具。这种方法的一个要求是获得没有污染物的所需蛋白质。然而，这个目标并不总是能够轻易实现。已经开发了多种策略来提高所需蛋白质产品的质量。然而，与分子伴侣的污染仍然是影响获得蛋白质质量的一个顽固问题。伴侣蛋白结合未折叠蛋白质或暴露多肽链的区域的能力使得在纯化过程中难以去除污染。这项工作旨在开发一种从纯化的重组蛋白中去除污染的 DnaK（大肠杆菌中同源 Hsp70 分子伴侣之一）的策略。为此，我们开发了一种方法，通过与 GST-cleanser 蛋白共孵育来捕获污染蛋白中的 DnaK，该蛋白具有与伴侣蛋白自由结合的功能结合位点。然后可以很容易地将清洁蛋白与捕获的 DnaK 一起去除。在这里，我们通过批量处理去除了 His 标记的重组蛋白来证明我们系统的实用性。在 ATP-Mg 的存在下添加 GST-cleanser 蛋白可以大大去除 DnaK 污染。因此，我们的去污策略是多功能且简单的，可以应用于不同表达和纯化系统获得的蛋白质以及小样本或大体积制剂。
