Wu Wan-Yi, Mee Courtney, Califano Filomena, Banki Reza, Wood David W
Department of Chemical Engineering, A-217 Engineering Quadrangle, Princeton University, New Jersey 08544-5263, USA.
Nat Protoc. 2006;1(5):2257-62. doi: 10.1038/nprot.2006.314.
A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters.
本文介绍了一种用于非色谱法纯化在大肠杆菌中表达的重组蛋白的简单技术。该方法基于一种可逆沉淀、自我切割的纯化标签。该标签由两个部分组成：一个类弹性蛋白多肽（ELP），它在高于30摄氏度的高盐缓冲液中可逆地自我缔合；一个内含肽，它会使ELP标签在温和的pH值变化时自我切割。因此，一个三方的ELP-内含肽-目标蛋白前体可以通过加盐、加热和离心循环来纯化。一旦纯化，内含肽介导的自我切割，随后切割后的ELP标签沉淀，无需色谱分离就能轻松有效地分离出纯的、天然的目标蛋白。对于十几种蛋白质，每升摇瓶培养物可回收50-100毫克切割后的天然目标蛋白，通常根据具体工艺参数在8-24小时内完成。
