Jenny Richard J, Mann Kenneth G, Lundblad Roger L
Haematologic Technologies, Inc., Essex Junction, VT, USA.
Protein Expr Purif. 2003 Sep;31(1):1-11. doi: 10.1016/s1046-5928(03)00168-2.
Expression and purification of proteins in recombinant DNA systems is a powerful and widely used technique. Frequently there is the need to express the protein of interest as a fusion protein or chimeric protein. Fusion protein technology is frequently used to attach a "signal" which can be used for subsequent localization of the protein or a "carrier" which can be used to deliver a "therapeutic" such as a radioactive molecule to a specific site. In addition to these applications, fusion protein technology can be employed for several other useful purposes. Of these, the most frequent reason is to provide a 'tag' or 'handle' which will aid in the purification of the protein. Another useful purpose is to improve the expression or folding of the protein of interest. In these latter two situations, it is often necessary to remove the fusion partner before the recombinant protein of interest can be used for further studies. This removal process involves the insertion of a unique amino acid sequence that is susceptible to cleavage by a highly specific protease. Thrombin and factor Xa are the most frequently used proteases for this application. The purpose of this review is to discuss the application of thrombin and factor Xa for the cleavage of fusion proteins. It is emphasized that while these enzymes are quite specific for cleavage at the inserted cleavage site, proteolysis can frequently occur at other site(s) in the protein of interest. It is necessary to characterize the protein of interest after cleavage from the affinity label to assure that there are no changes in the covalent structure of the protein of interest. Examples are presented which describe the proteolysis of the protein of interest by either factor Xa or thrombin.
在重组DNA系统中表达和纯化蛋白质是一项强大且广泛应用的技术。通常需要将感兴趣的蛋白质表达为融合蛋白或嵌合蛋白。融合蛋白技术经常用于连接一个可用于后续蛋白质定位的“信号”或一个可用于将“治疗剂”（如放射性分子）递送至特定部位的“载体”。除了这些应用外，融合蛋白技术还可用于其他几个有用的目的。其中，最常见的原因是提供一个“标签”或“把手”，以帮助蛋白质的纯化。另一个有用的目的是改善感兴趣蛋白质的表达或折叠。在这后两种情况下，通常需要在感兴趣的重组蛋白可用于进一步研究之前去除融合伙伴。这个去除过程涉及插入一个独特的氨基酸序列，该序列易被高度特异性的蛋白酶切割。凝血酶和因子Xa是该应用中最常用的蛋白酶。本综述的目的是讨论凝血酶和因子Xa在切割融合蛋白中的应用。需要强调的是，虽然这些酶在插入的切割位点的切割具有很高的特异性，但蛋白水解经常会在感兴趣蛋白质的其他位点发生。从亲和标签切割后，有必要对感兴趣的蛋白质进行表征，以确保感兴趣蛋白质的共价结构没有变化。文中给出了描述因子Xa或凝血酶对感兴趣蛋白质进行蛋白水解的实例。
