Expression and purification of the extracellular domain of wild-type humanRET and the dimeric oncogenic mutant C634R

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Liu , Y , Ribeiro , O D C , Robinson , J & Goldman , A 2020 , ' Expression and purification of the extracellular domain of wild-type humanRET and the dimeric oncogenic mutant C634R ' , International Journal of Biological Macromolecules , vol. 164 , pp. 1621-1630 . https://doi.org/10.1016/j.ijbiomac.2020.07.290

Title: Expression and purification of the extracellular domain of wild-type humanRET and the dimeric oncogenic mutant C634R
Author: Liu, Yixin; Ribeiro, Orquidea De Castro; Robinson, James; Goldman, Adrian
Contributor organization: Molecular and Integrative Biosciences Research Programme
Biochemistry and Biotechnology
Date: 2020-12-01
Language: eng
Number of pages: 10
Belongs to series: International Journal of Biological Macromolecules
ISSN: 0141-8130
DOI: https://doi.org/10.1016/j.ijbiomac.2020.07.290
URI: http://hdl.handle.net/10138/332918
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. (C) 2020 Published by Elsevier B.V.
Subject: Receptor tyrosine kinase
Recombinant protein expression
Cysteine-rich domain
EPIDERMAL-GROWTH-FACTOR
PROTEIN N-GLYCOSYLATION
CYSTEINE-RICH DOMAIN
RET RECEPTOR
KINASE INHIBITORS
LIGAND
GDNF
MUTATIONS
BINDING
GDF15
1182 Biochemistry, cell and molecular biology
Peer reviewed: Yes
Rights: cc_by_nc_nd
Usage restriction: openAccess
Self-archived version: acceptedVersion


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