title: High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis.
abstract: The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene 97403.
authors.forename: J A
authors.initials: JA
authors.lastname: Koke
authors.forename: M
authors.initials: M
authors.lastname: Yang
authors.forename: D J
authors.initials: DJ
authors.lastname: Henner
authors.forename: J J
authors.initials: JJ
authors.lastname: Volwerk
authors.forename: O H
authors.initials: OH
authors.lastname: Griffith
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids
chemical_list: D004269:DNA, Bacterial; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase