Production of biologically active Bacillus anthracis edema factor in Escherichia coli.
Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.
Cooksey
Bridget A
BA
Fermentation & Cell Culture Sciences, Human Genome Sciences, Inc., Rockville, Maryland 20850, USA.
Sampey
Gavin C
GC
Pierre
Jennifer L
JL
Zhang
Xiaozhen
X
Karwoski
Jeffrey D
JD
Choi
Gil H
GH
Laird
Michael W
MW
D000262:Adenylyl Cyclases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D014169:Transformation, Bacterial
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D000262:Adenylyl Cyclases