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 initial recovery and purification of biomolecules. 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 and Pichia pastoris. In addition, mammalian cells such as HeLa, CHO, MDCK and HEK 293, insect cell such Sf 9, and High five for transient expression of recombinant proteins were propagated. The various products were needed for a number of collaborative research projects: Expression of P-glycoprotein (membrane protein transporter) from pichia pastoris (NCI), expression of human NPP1 in HEK 293 cells (Yale university medical school), peptidoglycan from Bacillus anthraces (NIH clinical center)and recombinant tetanus toxin (FDA). The lab continues its work on improving 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 improve the production we constructed a suspension T-REx-293 cell line that stably express an engineered neurotensin receptor 1 (NTS1) mutant, and the expression level and the protein quality was compared with the transient baculovirus-insect cell system. What was 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. The Lab also developed a novel method for recovery of potential HIV vaccine candidate based on HIV-gag that was produced in insect cells as virus like particle (VLP). The developed method is based on implementing two tangential flow filtration process steps using 0.45 micron membrane and 500kDa membrane at a medium cross flow rate under low trans-membrane pressure and low shear force.
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