The Stadtman Way A Tale of Two Biochemists at NIH

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Div class usa-width-two-thirds Anaerobic Laboratory-An NIH First In 1967, the first anaerobic laboratory for biomedical research was built in Building 3 on the NIH's Bethesda campus. This quarter of a million dollar facility demonstrated the NIH's strong commitment to the research programs led by Thressa and Earl Stadtman. Thressa's research on methane biosynthesis and selenium biochemistry particularly benefited from this unique facility. Until the closing of Building 3 in 2002, the anaerobic laboratory served numerous researchers at NIH as well as visiting scientists from around the world.The anaerobic laboratory was furnished with the usual laboratory equipment, but it was filled with a mixture of nitrogen and hydrogen, instead of ordinary air. An "oxygen-free" atmosphere is crucial in dealing with bacteria that are killed in the presence of oxygen or with biological compounds that are inactivated when exposed to air. This unique facility allowed researchers to conduct multi-step experiments with various instruments, including manipulations that were extremely difficult in conventional anaerobic "glove boxes." Because of the danger of working in an anaerobic atmosphere, a researcher must wear a special mask fitted with an air-delivery tube while an observer outside monitors a two-way communication system. Div class floatright Image Added Floor plan of the anaerobic laboratory Video: Michael Poston on the anaerobic laboratory. Running time: 3:00 minutes  Small (56k modem) Large (ISDN/DSL) Image Removed Div class usa-width-one-third Floor plan of the anaerobic laboratory Inside the anaerobic laboratory Respirator mask used iside the anaerobic laboratory Fermenter Room

Fermenter Room

The fermenter room in Building 3 had facilities for growing and harvesting a large quantity of bacterial or yeast cells from which various enzymes could be extracted. Earl's research on metabolic regulation was particularly dependent upon the mass-production of glutamine synthetase in this room. The cell-growing procedure starts with a small culture of cells. This culture is scaled up to 10 or 20 liters in flasks, and then is introduced into the large fermenter which has a maximum volume of 500 liters. Typically, a volume of 350 liters is used for cells growing in an aerobic condition. The cells in the fermenter are then moved to a continuous flow centrifuge. As the centrifuge bowl spins at high speed, the cells are collected on the bowl's inside. The supernatant liquid overlying centrifuged cells flows out of the top and goes to a holding tank. This liquid is discarded after being chemically treated. The cells are scraped off, frozen in liquid nitrogen, and stored for enzyme purification. The frozen cells are suspended in a buffer solution and passed through a homogenizer, which uses high pressure to break the cells open and release the proteins. Finally, the enzyme is purified from cell-free extracts, according to various precipitation protocols. The 500-liter fermenter, installed around 1986, had an automatically-controlled valve system for sterilization, temperature control, agitation, and gas or air sparging . The fermenter prior to this model had been manually operated with about fifty different valves to open and close at various times. Earl and his assistants usually grew two fermenter-loads of E. coli a week. They then harvested 1200-1500 grams of cells, from which one gram of glutamine synthetase was purified. Now, the development of genetic engineering makes a large-scale fermenter almost obsolete. Only about 10 liters of the genetically modified organism that specifically overproduces glutamine synthetase are needed to harvest 30-50 grams of cells from which one gram of pure enzyme is obtained.

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