Wu Wan-Yi, Miller Keith D, Coolbaugh Michael, Wood David W
Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA.
Protein Expr Purif. 2011 Apr;76(2):221-8. doi: 10.1016/j.pep.2010.12.004. Epub 2010 Dec 16.
In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.
在这项工作中，我们将自切割亲和标签技术应用于几种分泌到大肠杆菌周质中的靶蛋白，其中包括两种含有二硫键的蛋白。将靶蛋白与自切割几丁质结合结构域-内含肽标签进行基因融合，以便通过几丁质-琼脂糖亲和树脂进行纯化。通过将内含肽标记的融合基因连接到PelB分泌前导序列，标记的靶蛋白被分泌到周质空间，并且可以通过简单的渗透压休克以活性形式回收。经过几丁质亲和纯化后，靶蛋白通过内含肽自切割从几丁质结合结构域标签上释放出来。这是通过在室温下将pH值从8.5轻微改变至6.5来诱导的，从而允许从几丁质亲和树脂上直接洗脱切割后的靶蛋白。靶蛋白包括大肠杆菌麦芽糖结合蛋白和β-内酰胺酶，以及两种含有二硫键的人抗体片段。在所有情况下，靶蛋白均以良好的活性和产量进行了纯化，无需重折叠。总体而言，这项工作证明了ΔI-CM内含肽与大肠杆菌中的PelB分泌系统具有兼容性，极大地扩展了其对更复杂蛋白质的应用潜力。
