Reeves, Philip J (2023) Expression systems for bovine rhodopsin: a review of the progress made in the Khorana laboratory. Biophysical Reviews, 15 (1). pp. 93-101. DOI https://doi.org/10.1007/s12551-022-01037-2
Reeves, Philip J (2023) Expression systems for bovine rhodopsin: a review of the progress made in the Khorana laboratory. Biophysical Reviews, 15 (1). pp. 93-101. DOI https://doi.org/10.1007/s12551-022-01037-2
Reeves, Philip J (2023) Expression systems for bovine rhodopsin: a review of the progress made in the Khorana laboratory. Biophysical Reviews, 15 (1). pp. 93-101. DOI https://doi.org/10.1007/s12551-022-01037-2
Abstract
Here I will review the development of gene expression systems for production of bovine rhodopsin in the Khorana laboratory with particular focus on stable mammalian cell lines made using human embryonic kidney cells (HEK293S). The synthesis of a gene encoding bovine rhodopsin was completed in 1986. This gene was expertly designed with the built-in capacity for DNA duplex cassette replacement mutagenesis which made site-directed mutagenesis relatively straightforward. Intense effort was expended over several years in order to identify a gene expression system capable of producing rhodopsin in milligram amounts as required for biophysical studies. Mammalian expression systems, both transient and stable, were found to be the most favourable based on several criteria including receptor expression levels, correct folding and post translational processing, and capacity for purification of fully functional receptor. Transient expression using COS-1 cells was preferred for routine small-scale production of rhodopsin mutants, while HEK293S stable cell lines were used when milligram amounts of rhodopsin mutants were needed; for example, when conducting NMR studies.
Item Type: | Article |
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Uncontrolled Keywords: | G protein-coupled receptors; Membrane proteins; Glycosylation; Tetracycline-inducible; Sodium butyrate |
Divisions: | Faculty of Science and Health Faculty of Science and Health > Life Sciences, School of |
SWORD Depositor: | Unnamed user with email elements@essex.ac.uk |
Depositing User: | Unnamed user with email elements@essex.ac.uk |
Date Deposited: | 25 Jan 2023 15:46 |
Last Modified: | 16 May 2024 21:41 |
URI: | http://repository.essex.ac.uk/id/eprint/34714 |
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Licence: Creative Commons: Attribution 4.0