Production of cytotoxic proteins in Escherichia coli: a fermentation process for producing enzymatically active HIV-1 protease.
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 less than 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 degrees C; however, cultivation and induction at 28 degrees C resulted in active enzyme formation following induction at increased cell density (A550 = 10).
Department of Cellular and Molecular Biology, Merck, Sharp and Dohme Research Laboratories, West Point, PA 19486.
W K
WK
Herber
F J
FJ
Bailey
C E
CE
Carty
J C
JC
Heimbach
R Z
RZ
Maigetter
D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005798:Genes, Bacterial; D005814:Genes, Viral; D016333:HIV Protease / Q000096:biosynthesis*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014364:Tryptophan / Q000494:pharmacology; D015678:Viral Structural Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D014364:Tryptophan; D016333:HIV Protease