Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli.
The effects of 25-fold overproduction of Escherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from the Bacillus subtilis chromosome and the mature part of TEM-beta-lactamase, were studied in E. coli. One precursor (pre[A2d]-beta-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[A13i]-beta-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature beta-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-beta-lactamase and pre(A2)-beta-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export in E. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.
van Dijl
J M
JM
Department of Genetics, Centre of Biological Sciences, Haren, The Netherlands.
de Jong
A
A
Smith
H
H
Bron
S
S
Venema
G
G
D001412:Bacillus subtilis / Q000235:genetics; D015153:Blotting, Western; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008565:Membrane Proteins; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D015183:Restriction Mapping; D012697:Serine Endopeptidases; D013104:Spheroplasts / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D001618:beta-Lactamases