An important portion of the Lab activities is the production of high-quality and unique biologicals that are not available from commercial sources. On average, the Unit performs around 100 processes per year, which include fermentation of bacteria, yeast, insect cells, and mammalian cells (in volumes ranging from 2 to 100 liters), and recovery and purification of biomolecules, such as proteins and polysaccharides, from different sources. The following are examples of processes performed: growth of bacteria such as Escherichia coli, (recombinant and native) various recombinant yeast strains such as Saccharomyces cerevisiae cerivisea and Pichia pastoris. In addition, mammalian cells such as HeLa, CHO, MDCK and HEK 293, and insect cell such Sf 9, and High five for transient expression of recombinant proteins were propagated. Several proteins and molecules were produced and purified in gram quantities. The various products were needed for a number of collaborative research projects: human adiponectin was made for biological studies, bacterial toxins for vaccine development and several proteins were produced for structural studies. The lab put a specific effort to improve the production of membrane proteins needed for crystallography especially G protein coupled receptor (GPCR). Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. To gain better understanding of the preferred way to produce these proteins we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. What we found is that the two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist 3H neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. The work demonstrated two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provided useful input to users in choosing and optimizing an appropriate expression host for other GPCRs.

Project Start
Project End
Budget Start
Budget End
Support Year
52
Fiscal Year
2013
Total Cost
$571,866
Indirect Cost
City
State
Country
Zip Code
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