The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli.
The kinetics of Photinus pyralis and Luciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible lambda PR promoter in Escherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay. E. coli cells were cultivated at temperature schemes 28-42-21 degrees C or 28-21 degrees C, or at alkali pH shift. In the cases of thermoinduction and pH shift, the luciferase expressions have similar features. The 3-h thermoinduction (42 degrees C) followed by the incubation at 21 degrees C, for 10 h resulted in the maximal amount of the luciferase protein of 4-5% of the total cell proteins. The yield did not change further. The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21 degrees C and reached the maximum after 50-60 h when the synthesized luciferase protein adopted the native-like conformation. At the same time, only 50% of the latter appeared to be catalytically active. An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content. Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.
Leont'eva
O
O
Chemistry Faculty, Lomonosov Moscow State University, Russia.
Kutuzova
G
G
Skripkin
E
E
Ugarova
N
N
D000818:Animals; D001517:Coleoptera / Q000201:enzymology*; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D010957:Plasmids / Q000378:metabolism
D008156:Luciferases