Novel fusion tags to facilitate protein production and structure determination
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Pharmacy
Abstract
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."
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."
Organisations
People |
ORCID iD |
Ingrid Dreveny (Primary Supervisor) |
Publications
Ward S
(2018)
The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel
in Journal of Biological Chemistry
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/M008770/1 | 01/10/2015 | 31/03/2024 | |||
1803618 | Studentship | BB/M008770/1 | 01/10/2016 | 31/03/2021 |
Description | Goal of the award was to develop new protein tags to facilitate structural characterisation of proteins of unknown structure. -2 selected tags increased solubility of model protein -2 selected tags increased crystallisability of model protein -1 selected tag allowed visualisation of model protein by Cryo-EM Studies of tags to determine novel protein structures completed -1 crystallisation tag in fusion to protein of interest led to solving of a novel protein structure |
Exploitation Route | -use new tags for increasing expression and solubility of proteins -use new tags for facilitating crystallisability of proteins of unknown structure -use new tags for facilitating Cryo-EM approaches for proteins of unknown structure -use new tags for allowing crystallisation of proteins in different crystal forms for binding studies -use of novel crystal structure solved in this project for further research into protein of interest |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Title | novel protein crystallisation tag |
Description | new fusion tag for protein crystallisation developed for loop insertion into target proteins |
Type Of Material | Biological samples |
Year Produced | 2020 |
Provided To Others? | No |
Impact | led to solving of novel structure of an important cancer target protein |