Leong L E
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, USA.
Mol Biotechnol. 1999 Oct;12(3):269-74. doi: 10.1385/MB:12:3:269.
In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.
在重组融合蛋白的亲和纯化中，限速步骤通常是从纯化的重组蛋白中有效进行蛋白酶解切割并去除亲和标签和蛋白酶。我们开发了一种快速、便捷且高效的亲和纯化方法，可克服这一限制。在该方法的一个实例中，来自微小核糖核酸病毒的蛋白酶3C（3Cpro），其可切割包含至少6 - 7个氨基酸的特定序列，已被表达为与谷胱甘肽S - 转移酶的融合蛋白。所得的重组“融合蛋白酶”可切割带有（从氨基端起）与融合蛋白酶相同亲和标签、一个3Cpro切割识别位点以及目标重组蛋白的融合蛋白。重组蛋白通过单一色谱步骤进行纯化，该步骤可同时去除亲和标签和融合蛋白酶。相较于现有方法，其优势包括蛋白酶解切割的特异性显著提高、一步完全去除蛋白酶和亲和标签，以及可选择添加任意所需量的融合蛋白酶以确保有效切割。通过使用各种亲和标签和替代融合蛋白酶展示了该方法潜在的灵活性。
