Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system.
A fusion gene (ces-hlyAs) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyAs) of Escherichia coli hemolysin (HlyA) to the ces gene for a cholesterol esterase/lipase (CE) from a Pseudomonas species. Part (about 30%) of the expressed fusion protein CE-HlyAs was secreted in E. coli carrying hlyB and hlyD genes. Following the insertion between the reporter gene and hlyAs of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-HlyAs and CE-HlyAs) were shown to be cleaved by OmpT between the two parts during HlyB/HlyD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increased ompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.
Hanke
C
C
Institut für Genetik und Mikrobiologie, University of Würzburg, FRG.
Hess
J
J
Schumacher
G
G
Goebel
W
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D002787:Sterol Esterase / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002787:Sterol Esterase; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease