Novel fusion tags to facilitate protein production and structure determination

蛋白质生产对许多生物技术和制药应用和过程至关重要。此外，重组生产的可溶性纯蛋白质对于蛋白质的特性包括成功的结晶和通过X射线结晶学确定结构是必不可少的。在这个项目中，我们将利用结构数据库中可用的信息来创建新的融合标签，用于生产、纯化和结晶工业感兴趣的选定蛋白质。蛋白质产量和溶解度方面的困难通常可以通过基因融合成功地克服。然而，到目前为止，促进蛋白质溶解和结晶的标签选择很少。最著名的例子是使用溶菌酶作为融合蛋白，这有助于确定G蛋白偶联受体的晶体结构，这项工作导致了2012年的诺贝尔奖(1)。到目前为止，唯一可以促进溶解性、结晶化并可用于亲和纯化的标签是工程麦芽糖结合蛋白(MBP)。用MBP标记蛋白质已被证明可以提高重组表达蛋白质的产量，它的使用导致了对否则不容易结晶的蛋白质的结构确定[2]。蛋白质数据库等数据库包括越来越多的蛋白质结构，并为鉴定具有合适性质的蛋白质提供了丰富的资源，这些蛋白质可以被设计为亲和纯化的新标签，并用于提高目标蛋白质的溶解性和结晶性。我们将在考虑分子质量和表面特征等标准的情况下为此目的寻找候选蛋白质，使用突变来增强所需的特性，然后测试这些候选标签在蛋白质生产和结晶中的使用。我们将选择模型蛋白质进行概念验证研究，并随后使用这些新的标签来研究未知结构的目标蛋白质。总而言之，这将产生用于研究和工业应用的蛋白质生产和结构确定的新工具。1.Rosenbaum，D.M.，Rasmussen，S.G.和Kobilka，B.K.(2009)《G蛋白偶联受体的结构和功能》，《自然》459,356-363。2.Moon，A.F.，Mueller，G.A.，Chung，X.和Pedersen，L.C.(2010)蛋白质结晶的协同方法：固定臂载体与表面熵降低的组合，蛋白质科学19,901-913。

Protein production is crucial for many biotechnological and pharmaceutical applications and processes. In addition, recombinant production of soluble pure protein is essential for protein characterisation including the successful crystallisation and structure determination by X-ray crystallography. In this project we will exploit the information available in structure databases to create novel fusion tags for the production, purification and crystallisation of selected proteins of interest to industry. Difficulties with protein yield and solubility are often successfully overcome by the use of gene fusions. However, very few options of tags that promote protein solubility and crystallisation are available to date. The most famous example is the use of lysozyme as fusion protein that has been instrumental in determining G-protein coupled receptor crystal structures, work which led to the award of the Nobel prize in 2012 (1). The only available tag to date that promotes solubility, crystallisation and can also be used for affinity purification is an engineered maltose binding protein (MBP). Tagging proteins with MBP has been shown to improve the yield of proteins expressed recombinantly and its use led to the structure determination of proteins otherwise not amenable to crystallisation (2). Data bases such as the Protein Data Bank include an ever increasing number of protein structures and provide a rich resource for the identification of proteins with suitable properties that can be engineered to be employed as novel tags for affinity purification and for enhancing the solubility and crystallisability of target proteins. We will search for candidate proteins for this purpose taking criteria such as molecular mass and surface characteristics into account, use mutagenesis to enhance desirable properties and then test these candidate tags for their use in protein production and crystallisation. We will select model proteins for proof-of-concept studies and subsequently use these novel tags to investigate target proteins of unknown structure. Together, this will result in novel tools for the production and structure determination of proteins for research and industrial applications. 1.Rosenbaum, D. M., Rasmussen, S. G., and Kobilka, B. K. (2009) The structure and function of G-protein-coupled receptors, Nature 459, 356-363. 2.Moon, A. F., Mueller, G. A., Zhong, X., and Pedersen, L. C. (2010) A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction, Protein Sci 19, 901-913."