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.