Novel fusion tags to facilitate protein production and structure determination

新型融合标签促进蛋白质生产和结构测定

• 批准号: 1803618
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1803618
• 关键词: Novel; fusion; tags; facilitate; protein

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."

蛋白质生产对于许多生物技术和制药应用和工艺至关重要。此外，可溶性纯蛋白质的重组生产对于蛋白质表征是必不可少的，包括通过X射线晶体学的成功结晶和结构测定。在这个项目中，我们将利用结构数据库中的信息来创建新的融合标签，用于工业上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的困难通常通过使用基因融合而成功地克服。然而，到目前为止，促进蛋白质溶解度和结晶的标签的选择很少。最著名的例子是使用溶菌酶作为融合蛋白，该融合蛋白有助于确定G蛋白偶联受体晶体结构，这项工作导致了2012年诺贝尔奖的授予（1）。迄今为止，促进溶解度、结晶并且还可用于亲和纯化的唯一可用标签是工程化麦芽糖结合蛋白（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率，并且其使用导致蛋白质的结构测定，否则不适合结晶（2）。数据库，如蛋白质数据库，包括越来越多的蛋白质结构，并提供了丰富的资源，用于鉴定具有合适性质的蛋白质，这些蛋白质可以被工程化以用作亲和纯化和增强靶蛋白的溶解性和结晶性的新标签。我们将为此目的寻找候选蛋白质，考虑到分子量和表面特性等标准，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用途。我们将选择模型蛋白进行概念验证研究，随后使用这些新的标签来研究未知结构的靶蛋白。总之，这将为研究和工业应用提供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.等（2009）The structure and function of G-protein-coupled receptors，Nature 459，356-363. 2. Moon，A. F.、Mueller，G.一、Zhong，X.，中国科学院，和Pedersen，L. C.等人（2010）A synergistic approach to protein crystallization：combination of a fixed-arm carrier with surface entropy reduction，Protein Sci 19，901-913. "

项目成果

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel.

• DOI: 10.1074/jbc.ra118.003857
• 发表时间: 2018-11-09
• 期刊: The Journal of biological chemistry
• 作者: Ward SJ;Gratton HE;Indrayudha P;Michavila C;Mukhopadhyay R;Maurer SK;Caulton SG;Emsley J;Dreveny I
• 通讯作者: Dreveny I