Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease.
Lon protease from Escherichia coli is an ATP-dependent protease which plays important roles in regulating the levels of specific proteins and in eliminating abnormal proteins. A major problem of working with Lon protease, the inability to substantially overproduce the enzyme, has been overcome by placing the lon gene under the control of an inducible trp promoter within a copy-number-controllable plasmid. Induction resulted in higher levels of production of the protease (approximately 100 micrograms/ml of cell culture) than were previously possible. The enzyme has been purified to apparent homogeneity and shown to possess the characteristic ATP-dependent proteolytic activity. Sequence verification during DNA manipulations revealed differences from two previously published sequences for the lon gene.
Thomas
C D
CD
Department of Biochemistry, University of Leicester, UK.
Modha
J
J
Razzaq
T M
TM
Cullis
P M
PM
Rivett
A J
AJ
D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids; D049070:Protease La; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La