Production of cytotoxic proteins in Escherichia coli: a fermentation process for producing enzymatically active HIV-1 protease |
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Authors: | Wayne K. Herber F. James Bailey Christine E. Carty Jill C. Heimbach Robert Z. Maigetter |
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Affiliation: | (1) Department of Cellular and Molecular Biology, Merck, Sharp and Dohme Research Laboratories, 19486 West Point, PA, USA |
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Abstract: | Summary Two fermentation processes for the tryptophan-regulated expression of active HIV protease (HIV-1 prt) in Escherichia coli are described. Since overexpression of HIV-1 prt results in cell death, stringent control of product expression was necessary to attain high enzyme levels. Such control was achieved by separation of growth and production phases in a two-step process or by implementation of nutrient feed in a one-step process. When the two-stage process was used, soluble product was detectable only when induction occurred at low culture density (A550 < 3.5). Short induction periods of 1–2 h and rapid harvesting were necessary to recover active product. Similar results were obtained when the single-stage process was operated at 37°C; however, cultivation and induction at 28°C resulted in active enzyme formation following induction at increased cell density (A550=10).Offprint requests to: W. K. Herber |
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