Protein Expression and Purification in the Fast Lane

该项目的目标是为蛋白质的表达和纯化创建一个统一的技术基础设施，这将适合大规模的结构生物学倡议。我们方法的核心是使用多个基因工程的亲和标签。我们目前正在努力确定什么组合的亲和标签是最有效的，以及如何最大限度地使用它们。与此同时，由于大多数亲和标签都有可能干扰结构研究，我们也在努力开发更可靠的方法来去除它们。 我们面临的最大技术障碍之一是“包涵体问题”--即蛋白质以不溶的、不活跃的形式积累的趋势。由于蛋白质的复性可能是一项艰巨而耗时的工作，因此一些绕过包涵体形成的方法将是有利的。有时，这可以通过将易于聚集的多肽与高度溶解的伙伴融合来实现。我们已经证明，大肠杆菌麦芽糖结合蛋白(MBP)是一种非常有效的溶解性增强剂，在许多情况下，MBP也可以促进其融合伙伴的正确折叠。这种类似伴侣的性质使MBP有别于其他亲和标签，并极大地增强了其作为融合伙伴的价值。因此，MBP融合蛋白已成为我们蛋白质表达策略的基石。在MBP融合蛋白的框架内利用额外的标签来促进目标蛋白的纯化。 如果去掉亲和力标签不是那么困难的话，它们可能会被更频繁地使用。这通常是通过在指定的位置对融合蛋白进行内切蛋白分解来实现的。这种方法的主要困难来自于通常用于裂解融合蛋白的蛋白酶的内在混杂。使这一问题更加复杂的事实是，购买足够多的这些试剂来切割符合结构研究规模的融合蛋白，价格高得令人望而却步。为了克服这些问题，我们生产了我们自己供应的TEV蛋白酶，这是烟草蚀刻病毒核包涵体蛋白酶的催化结构域。TeV蛋白水解酶切割Q和G之间或Q和S之间的氨基酸序列ENLYFQG/S具有高度的特异性。与凝血因子Xa、肠肽酶和凝血酶相比，TEV蛋白酶在融合蛋白的非正则位点处被切割的报道尚未见报道。过去，TEV蛋白酶在大肠杆菌中的生产一直受到产量低和溶解性差的阻碍，但我们已经能够通过进行同义密码子替换和以MBP融合蛋白的形式生产该酶来解决这两个问题。TEV蛋白酶的一个更麻烦的缺点是，它很容易在特定的位置裂解自己，产生一种活性大大降低的截短的蛋白酶。我们也能够通过引入氨基酸取代来纠正这个问题，这种取代可以防止自动失活，而不会阻碍蛋白酶切割典型靶序列的能力。对该酶的P1‘专一性的系统分析表明，除了G和S外，许多不同的氨基酸都可以在这个位置上被容纳，而对加工效率的影响相对较小。催化失活的TEV蛋白水解酶的晶体结构说明了其严格的底物专一性的结构基础。烟草静脉斑驳病毒(TVMV)的同源蛋白水解酶是TEV的近亲，具有独特的序列特异性，目前正被开发为替代试剂。

The goal of this project is to create a unified technological infrastructure for protein expression and purification that will be suitable for large-scale structural biology initiatives. Central to our approach is the use of multiple genetically engineered affinity tags. We are currently trying to determine what combination of affinity tags is most effective and how to use them with maximal efficiency. At the same time, because most affinity tags have the potential to interfere with structural studies, we are also striving to develop more reliable methods for removing them. One of the greatest technical obstacles that we face is "the inclusion body problem"-i.e., the tendency of proteins to accumulate in an insoluble, inactive form. Because refolding of proteins can be an arduous and time consuming undertaking, some way to circumvent the formation of inclusion bodies would be advantageous. Sometimes this can be accomplished by fusing an aggregation-prone polypeptide to a highly soluble partner. We have demonstrated that Escherichia coli maltose-binding protein (MBP) is a remarkably effective solubility enhancer, and that in many cases MBP can promote the proper folding of its fusion partners as well. This chaperone-like quality distinguishes MBP from other affinity tags and greatly enhances its value as a fusion partner. Accordingly, MBP fusion proteins have become the cornerstone of our strategy for protein expression. Additional tags are utilized within the framework of an MBP fusion protein to facilitate purification of the target protein. Affinity tags would probably be used more often if it were not so difficult to remove them. This is usually accomplished by endoproteolysis of a fusion protein at a designed site. The main difficulty with this approach stems from the intrinsic promiscuity of the proteases that are commonly used to cleave fusion proteins. This problem is compounded by the fact that it is prohibitively expensive to purchase enough of any of these reagents to cleave fusion proteins on a scale amenable for structural studies. To overcome these problems, we produce our own supply of TEV protease, the catalytic domain of the nuclear inclusion protease from tobacco etch virus. TEV protease cleaves the amino acid sequence ENLYFQG/S between Q and G or Q and S with high specificity. In contrast to factor Xa, enteropeptidase and thrombin, there have never been any reports of cleavage at noncanonical sites in fusion proteins by TEV protease. The production of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility, but we have been able to solve both problems by making synonymous codon replacements and producing the protease in the form of an MBP fusion protein. A more troublesome shortcoming of TEV protease is that it readily cleaves itself at a specific site, generating a truncated protease with greatly diminished activity. We have been able to rectify this problem as well by introducing amino acid substitutions that prevent autoinactivation without impeding the ability of the protease to cleave canonical target sequences. A systematic analysis of the enzyme's P1' specificity revealed that, in addition to G and S, many different amino acids can be accommodated in this position with relatively little impact on the efficiency of processing. The crystal structure of catalytically inactive TEV protease in complex with a peptide substrate illuminated the structural basis of its stringent substrate specificity. A homologous protease from tobacco vein mottling virus (TVMV), a close relative of TEV protease with a distinct sequence specificity, is currently being developed as an alternative reagent.