Department of Pathology, Stanford School of Medicine, Stanford, California, United States of America.
PLoS One. 2009 Dec 2;4(12):e8119. doi: 10.1371/journal.pone.0008119.
We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6)), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6)-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6) to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.
我们介绍了一种新的方法，用于使用高度特异性、诱导性、自切割蛋白酶标签从细菌中纯化重组蛋白。该标签由霍乱弧菌 MARTX 毒素半胱氨酸蛋白酶结构域 (CPD) 组成，这是一种自动加工酶，仅在靶蛋白-CPD 连接处的亮氨酸残基后切割。重要的是，V. cholerae CPD 被肌醇六磷酸 (InsP(6)) 特异性激活，InsP(6) 是一种真核特异性小分子，不存在于细菌细胞质中。因此，当 His(6)-标记的 CPD 融合到靶蛋白的 C 末端并在大肠杆菌中表达时，全长融合蛋白可以使用金属离子亲和层析从细菌裂解物中纯化出来。随后向固定化融合蛋白中添加 InsP(6)，诱导 CPD 介导的靶蛋白-CPD 连接处切割，将未标记的靶蛋白释放到上清液中。该方法将亲和层析和融合标签切割缩合到一个步骤中，无需添加外源性蛋白酶来去除融合标签，从而提高了标签分离的效率。此外，除了省时、多功能和廉价之外，我们的结果还表明，CPD 纯化系统可以提高来自不同生物体的难以表达的蛋白质的表达、完整性和可溶性。
