Cellular and process engineering to improve mammalian membrane protein expression
Embargo until
2019-08-01
Date
2015-05-11
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Johns Hopkins University
Abstract
Improving the expression level of recombinant mammalian proteins has been pursued for production of commercial biotherapeutics in industry, as well as for biomedical studies in academia, as an adequate supply of correctly folded proteins is a prerequisite for all structure and function studies. Presented in this dissertation are different strategies to improve protein functional expression level, especially for membrane proteins. The model protein is neurotensin receptor 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR). GPCRs are integral membrane proteins playing a central role in cell signaling and are targets for most of the medicines sold worldwide. Obtaining adequate functional GPCRs has been a bottleneck in their structure studies because the expression of these proteins from mammalian cells is very low.
The first strategy is the adoption of mammalian inducible expression system. A stable and inducible T-REx-293 cell line overexpressing an engineered rat NTSR1 was constructed. 2.5 million Functional copies of NTSR1 per cell were detected on plasma membrane, which is 167 fold improvement comparing to NTSR1 constitutive expression.
The second strategy is production process development including suspension culture adaptation and induction parameter optimization. A further 3.5 fold improvement was achieved and approximately 1 milligram of purified functional NTSR1 per liter suspension culture was obtained. This was comparable yield to the transient baculovirus-insect cell system.
The third strategy is high throughput miRNA screening. MiRNAs are a novel class of small, non-coding RNAs that can simultaneously silence multiple genes. The NTSR1-expressing cell line was subjected to human miRNA mimic library screening and nine miRNA mimics were identified to improve functional expression of NTSR1 by as much as 48%. Interestingly, five out of nine identified miRNA mimics were effective in improving the functional expression of other proteins, including luciferase (cytosolic protein), serotonin transporter (membrane protein) and glypican-3 hFc protein (secreted protein). These indicated that the identified miRNAs could have a wide role in enhancing production of proteins with biomedical interest.
As genome-wide siRNA screens has emerged to be a powerful methodology for deducing gene functions in various diseases, we applied this technology on HEK293 cells constitutively expressing luciferase reporter to generate a genome-wide profile for recombinant protein expression process. Top 10 genes leading to greatest improvement of luciferase production were validated and tested with secreted and membrane proteins.
Investigation of these genes/pathways may provide profound information to understanding protein biosynthesis process in mammalian cells.
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Keywords
mammalian membrane protein expression, cellular engineering, process engineering, g-protein-coupled receptors, neurotensin receptor type 1, high-throughput RNAi screening