Beyond bottlenecks in membrane protein production
PhD ceremony: Mr. R.K.R. Marreddy, 13.15 uur, Academiegebouw, Broerstraat 5, Groningen
Thesis: Beyond bottlenecks in membrane protein production
Promotor(s): prof. B. Poolman
Faculty: Mathematics and Natural Sciences
The structural and functional analysis of complex multi-domain (membrane) proteins is hampered in large part due to problems associated with their overproduction in a functional state. The bacterium Lactococcus lactis is a suitable host for overexpression of membrane proteins. Although many pro- and eukaryotic proteins are expressed well in L. lactis, other proteins are difficult to (over)produce. In many cases L. lactis and other expression hosts are grown in complex media as proteins are expressed best under those conditions. However, for the incorporation into proteins of specific amino acid analogues, which is advantageous for various biophysical studies, one requires a chemically defined medium. We have used a comparative proteomeics approach to determine why proteins are produced at higher levels in complex than in synthetic media. We could show that in the synthetic media the intracellular levels of branched-chain amino acids become limiting for biosynthesis, and, importantly, we could overcome this limitation either by overexpressing the corresponding amino acid transport protein or providing to cell with an alternative path for amino acid accumulation (e.g. via uptake of peptides). Subsequentely, we determined why certain membrane proteins are not well expressed while others do. Here, the physiological response of the cell was studied by a combined proteomics and transcriptomics approach. The overproduction of membrane proteins in L. lactis invoked a general stress response (upregulation of various chaperones), a severe metabolic burden and a specific cell envelope stress response. With this basic knowledge of the physiological response of the cells, it should be possible engineer to engineert the expression hosts for improved membrane protein production. Initial successes in improving the biosynthesis of medically-important membrane proteins have been obtained.
Last modified: | 13 March 2020 01.16 a.m. |
More news
-
08 November 2024
Two 6.7 million grants for FSE researchers from Dutch Research Agenda
Researchers of the Faculty of Science and Engineering have been awarded two large NWO grants for global biodiversity restoration and research into the origin of life.
-
05 November 2024
Five million ERC Synergy Grant for synthetic cell research
Professor Bert Poolman, together with Prof. Petra Schwille, receives an ERC Synergy grant of five million euros for synthetic cell research.
-
28 October 2024
CogniGron: A revolution in future-proof computing
In this first article of the two-part CogniGron series, Beatriz Noheda, Niels Taatgen, and Erika Covi tell us about drawing inspiration from the human brain to make smart devices even smarter.