细胞标签蛋白纯化中的难题及解决方法课件.ppt

My presentation today is as outlined as shown here. I will start by talking about affinity tags in general. I will also touch upon some features of various expression systems, which one may have to consider when planning both expression and purification of a protein. However, this presentation will put emphasis on the sections dealing with some of the most cited problems in tagged protein purification which are protein degradation, low yield, purity ,tag removal and protein stability. I will go through some of the reasons behind the problems and show you examples as well as give you some hints and tips what you can do in order to solve or at least minimize the problem. By the introduction of affinity tagging of proteins, protein purification has dramatically been simplified. Generic protocols are used which promotes much more efficient and easier protein purifications. Rather than before when complicated multistep purification schemes had to be set up. For some time now it has been recognized that certain affinity tags have the ability to promote the solubility of their fusion partners. Solubility-enhancing affinity tags tend to be proteins rather than petides. Some of the most frequently used tags to increase solubility are the maltose binding protein (MBP), glutathione-S transferase (GST) and Thioredoxin. We have also seen thatDual tagging of proteins can prevent proteolysis of the target protein and if different tags are positioned on each end of the protein, purification protocols can be set up in order to be sure that a full length protein is purified. For some applications, for example crystallography, it is important that the tag can be removed. This is possible when a specific cleavage site for a cleavage enzyme is incorporated adjacent to the sequence for the affinity tag in the cloning vector. When large affinity tags like GST are used, the tag can influence the conformation of the target protein leading to for example loss of biological activit