Protein Expression and Purification in the Fast Lane

该项目的目标是为蛋白质表达和纯化创建一个统一的技术基础设施，适用于大规模的结构生物学计划。我们的方法的核心是使用多个基因工程亲和标签。我们目前正试图确定什么样的亲和标签组合是最有效的，以及如何以最大的效率使用它们。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，蛋白质以不溶的非活性形式积累的趋势。由于蛋白质的重折叠可能是一项艰巨而耗时的工作，因此避免形成包涵体的某种方法将是有利的。有时，这可以通过将易于聚集的多肽融合到高度可溶的配偶体来实现。我们已经证明，大肠杆菌麦芽糖结合蛋白（MBP）是一个非常有效的溶解度增强剂，在许多情况下，MBP可以促进其融合伙伴以及正确的折叠。这种类似伴侣的性质将MBP与其他亲和标签区分开来，并大大提高了其作为融合伴侣的价值。因此，MBP融合蛋白已成为我们蛋白表达策略的基石。在MBP融合蛋白的框架内使用额外的标签以促进靶蛋白的纯化。 如果删除它们不是那么困难的话，亲和标签可能会被更经常地使用。这通常是通过在设计的位点处的融合蛋白的内蛋白水解来实现的。这种方法的主要困难源于通常用于切割融合蛋白的蛋白酶的内在混杂性。这个问题由于购买足够的这些试剂中的任何一种以适合于结构研究的规模切割融合蛋白是极其昂贵的事实而变得复杂。为了克服这些问题，我们生产了我们自己供应的TEV蛋白酶，即烟草蚀纹病毒核包涵体蛋白酶的催化结构域。TEV蛋白酶以高特异性切割Q和G或Q和S之间的氨基酸序列ENLYPROTEIN G/S。与Xa因子、肠肽酶和凝血酶相反，从未有过TEV蛋白酶在融合蛋白的非典型位点切割的任何报道。过去，在大肠杆菌中生产TEV蛋白酶由于产率低和溶解性差而受到阻碍，但是我们已经能够通过进行同义密码子替换并以MBP融合蛋白的形式生产蛋白酶来解决这两个问题。TEV蛋白酶的一个更麻烦的缺点是它容易在特定位点切割自身，产生活性大大降低的截短蛋白酶。我们已经能够纠正这个问题，以及通过引入氨基酸取代，防止自动失活，而不妨碍蛋白酶的能力，切割典型的靶序列。酶的P1 '特异性的系统分析表明，除了G和S，许多不同的氨基酸可以容纳在这个位置，对加工效率的影响相对较小。与肽底物复合的无催化活性的TEV蛋白酶的晶体结构阐明了其严格的底物特异性的结构基础。烟草脉斑病毒（TVMV）的同源蛋白酶，TEV蛋白酶的近亲具有独特的序列特异性，目前正在开发作为替代试剂。

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.