Liu, Y, Ribeiro, ODC, Robinson, J orcid.org/0000-0001-5425-7520 et al. (1 more author) (2020) Expression and purification of the extracellular domain of wild-type humanRET and the dimeric oncogenic mutant C634R. International Journal of Biological Macromolecules, 164. pp. 1621-1630. ISSN 0141-8130
Abstract
The receptor tyrosine kinase RET is essential in a variety of cellular processes. RET gain-of-function is strongly associated with several cancers, notably multiple endocrine neoplasia type 2A (MEN 2A), while RET loss-of-function causes Hirschsprung's disease and Parkinson's disease. To investigate the activation mechanism of RET as well as to enable drug development, over-expressed recombinant protein is needed for in vitro functional and structural studies. By comparing insect and mammalian cells expression of the RET extracellular domain (RETECD), we showed that the expression yields of RETECD using both systems were comparable, but mammalian cells produced monomeric functional RETECD, whereas RETECD expressed in insect cells was non-functional and multimeric. This was most likely due to incorrect disulfide formation. By fusing an Fc tag to the C-terminus of RETECD, we were able to produce, in HEK293T cells, dimeric oncogenic RETECD (C634R) for the first time. The protein remained dimeric even after cleavage of the tag via the cysteine disulfide, as in full-length RET in the context of MEN 2A and related pathologies. Our work thus provides valuable tools for functional and structural studies of the RET signaling system and its oncogenic activation mechanisms.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2020 Published by Elsevier. This is an author produced version of a paper published in International Journal of Biological Macromolecules. Uploaded in accordance with the publisher's self-archiving policy. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/. |
Keywords: | Receptor tyrosine kinase; Recombinant protein expression; Cysteine-rich domain |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 12 Aug 2020 13:35 |
Last Modified: | 02 Jun 2023 17:28 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.ijbiomac.2020.07.290 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:164269 |
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