Novel fusion tags to facilitate protein production and structure determination

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."