Saez Natalie J, Cristofori-Armstrong Ben, Anangi Raveendra, King Glenn F
Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St. Lucia, QLD, 4067, Australia.
Methods Mol Biol. 2017;1586:155-180. doi: 10.1007/978-1-4939-6887-9_10.
Recombinant expression of disulfide-reticulated peptides and proteins is often challenging. We describe a method that exploits the periplasmic disulfide-bond forming machinery of Escherichia coli and combines this with a cleavable, solubility-enhancing fusion tag to obtain higher yields of correctly folded target protein than is achievable via cytoplasmic expression. The protocols provided herein cover all aspects of this approach, from vector construction and transformation to purification of the cleaved target protein and subsequent quality control.
二硫键交联肽和蛋白质的重组表达通常具有挑战性。我们描述了一种利用大肠杆菌周质中二硫键形成机制的方法，并将其与可裂解的、增强溶解性的融合标签相结合，以获得比通过细胞质表达更高产量的正确折叠的目标蛋白。本文提供的方案涵盖了该方法的所有方面，从载体构建、转化到裂解目标蛋白的纯化及后续质量控制。
