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工艺至关重要。此外，可溶性纯蛋白的重组生产对于蛋白质表征至关重要，包括成功的结晶和x射线晶体学的结构测定。在这个项目中，我们将利用结构数据库中的可用信息来创建新的融合标签，用于工业感兴趣的选定蛋白质的生产，纯化和结晶。蛋白质产量和溶解度方面的困难通常通过基因融合得以成功克服。然而，迄今为止，很少有标签可以促进蛋白质的溶解度和结晶。最著名的例子是利用溶菌酶作为融合蛋白，在确定g蛋白偶联受体晶体结构方面发挥了重要作用，这项工作获得了2012年诺贝尔奖(1)。迄今为止唯一可用的标签，促进溶解度，结晶，也可以用于亲和纯化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，并且它的使用可以确定蛋白质的结构，否则不适合结晶(2)。像蛋白质数据库这样的数据库包含了越来越多的蛋白质结构，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成用于亲和纯化和增强目标蛋白质的溶解度和结晶性的新标签。我们将为此目的寻找候选蛋白质，考虑到分子质量和表面特征等标准，使用诱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进行概念验证研究，随后使用这些新标签研究未知结构的靶蛋白。总之，这将产生用于研究和工业应用的蛋白质生产和结构测定的新工具。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，Nature 459, 356-363。2.Moon, a.f, Mueller, g.a, Zhong， 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减少的结合，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."