The normally periplasmic enzyme beta-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase.
Hybrid proteins were constructed in which C-terminal regions of the bacterial cell surface and extracellular protein pullulanase were replaced by the mature forms of the normally periplasmic Escherichia coli proteins beta-lactamase or alkaline phosphatase. In E. coli strains expressing all pullulanase secretion genes, pullulanase-beta-lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently and completely transported to the cell surface. This hybrid protein remained temporarily anchored to the cell surface, presumably via fatty acids attached to the N-terminal cysteine of the pullulanase segment, and was subsequently specifically released into the medium in a manner indistinguishable from that of pullulanase itself. These results suggest that the C-terminal extremity of pullulanase lacks signal(s) required for export to the cell surface. When beta-lactamase was replaced by alkaline phosphatase, the resulting hybrid also became exposed at the cell surface, but exposition was less efficient and specific release into the medium was not observed. We conclude that proteins that do not normally cross the outer membrane can be induced to do so when fused to a permissive site near the C-terminus of pullulanase.
Kornacker
M G
MG
Unité de Génétique Moléculaire (UA CNRS 1149), Institut Pasteur, Paris, France.
Pugsley
A P
AP
D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006026:Glycoside Hydrolases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D015183:Restriction Mapping; D001618:beta-Lactamases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase; D001618:beta-Lactamases