Proteolytic response to the expression of an abnormal beta-galactosidase in Escherichia coli.
Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant beta-galactosidase was investigated. A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome. Induction of expression of the mutant beta-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity. Growth at temperatures above 40 degrees C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37 degrees C, and the ATP-dependent activity increased 2.5-fold from 30 to 42 degrees C. Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal beta-galactosidase at 37 degrees C, but no such response was evident when mutant beta-galactosidase expression was induced at 30 degrees C. In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid. Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity. These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E. coli. These responses were reduced by induction at lower temperatures.
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125.
M J
MJ
Kosinski
U
U
Rinas
J E
JE
Bailey
D000255:Adenosine Triphosphate / Q000378:metabolism; D001709:Biotechnology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase; D010447:Peptide Hydrolases