Chong S, Mersha F B, Comb D G, Scott M E, Landry D, Vence L M, Perler F B, Benner J, Kucera R B, Hirvonen C A, Pelletier J J, Paulus H, Xu M Q
New England Biolabs, Beverly, MA 01915, USA.
Gene. 1997 Jun 19;192(2):271-81. doi: 10.1016/s0378-1119(97)00105-4.
A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.
已开发出一种新型蛋白质纯化系统，该系统能够在单个色谱步骤中纯化游离的重组蛋白。该系统利用来自酿酒酵母的修饰蛋白剪接元件（内含肽）（Sce VMA内含肽）与来自环状芽孢杆菌的几丁质结合结构域（CBD）作为亲和标签。这一概念基于以下观察结果：修饰后的Sce VMA内含肽在低温和较宽的pH范围内，可被1,4-二硫苏糖醇（DTT）、β-巯基乙醇（β-ME）或半胱氨酸诱导在其N端肽键处发生自切割反应。将目标蛋白与内含肽-CBD融合体的N端进行框内克隆，稳定的融合蛋白通过吸附到几丁质柱上进行纯化。然后在温和条件下诱导固定化的融合蛋白进行自切割，从而释放目标蛋白，而内含肽-CBD融合体仍与柱结合。无需外源蛋白水解切割。此外，使用该方法，纯化的游离目标蛋白可在其C端进行特异性标记。
