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Alterations in the carboxy‐termianl half of cloacin destabilize the protein and prevent its export by Escherichia coli
Author(s) -
Putten A. J.,
Stegehuis F.,
Bergen Henegouwen P. M. P.,
Graaf F. K.,
Oudega B.
Publication year - 1988
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1988.tb00063.x
Subject(s) - immunoelectron microscopy , cytoplasm , biology , mutant , escherichia coli , biochemistry , microbiology and biotechnology , gene , antibody , genetics
Summary Several overlapping carboxy‐terminal and internal deletions were constructed in the cloacin structural gene. The expression, the binding of the cloacin DF13 immunity protein and the release into the culture medium of the mutant cloacin polypeptides were studied by immunoblotting and ELISAs. Minor alterations at the carboxy‐terminal end of the cloacin did not affect protein expression, stability or release to a large extent, but larger carboxy‐terminal deletions strongly destabilized the protein and no release was observed. The removal of a particular region within the carboxy‐terminal portion of cloacin strongly destabilize the polypeptide and made it a target for proteolytic degradation. Binding of immunity protein did not affect stability and release of the mutant polypeptides. By using immunoelectron microscopy, the polypeptides that were not exported were located in the cytoplasm of producing cells. Large aggregates of these mutant polypeptides were not observed in the cytoplasm: the polypeptides were present in a soluble form.