Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria.
Heterologous protein secretion was studied in the gram-positive bacteria Bacillus subtilis and Staphylococcus carnosus by using the Escherichia coli outer membrane protein OmpA as a model protein. The OmpA protein was found to be translocated across the plasma membrane of both microorganisms. However, the majority of the translocated OmpA was similarly degraded in B. subtilis and S. carnosus despite the fact that the latter organism does not secrete soluble exoproteases into the culture medium. The finding that purified OmpA, which was added externally to the culture medium of growing S. carnosus cells, remained intact indicates that newly synthesized and exported OmpA is degraded by one or more cell-associated proteases rather than by a soluble exoprotease. Fusion of the mature part of OmpA to the pre-pro part of a lipase from Staphylococcus hyicus allowed the efficient release of the corresponding propeptide-OmpA hybrid protein into the supernatant and completely prevented the cell-associated proteolytic degradation of the mature OmpA, most likely reflecting an important function of the propeptide during secretion of its natural mature lipase moiety. The relevance of our findings for the biotechnological use of gram-positive bacteria as host organisms for the secretory production of heterologous proteins is discussed.
Meens
J
J
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany.
Herbort
M
M
Klein
M
M
Freudl
R
R
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008049:Lipase / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D011995:Recombination, Genetic; D013210:Staphylococcus / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D008049:Lipase; D010450:Endopeptidases