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Association of degradation and secretion of three chimeric polypeptides in Escherichia coli
Author(s) -
Reiner Gentz,
Y. M. Kuys,
Christian W Zwieb,
Douglas J. Taatjes,
H. Taatjes,
Willi Bannwarth,
Dietrich Stueber,
Ibrahim Ibrahimi
Publication year - 1988
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.170.5.2212-2220.1988
Subject(s) - biology , signal peptide , escherichia coli , fusion protein , secretion , biochemistry , peptide , chloramphenicol acetyltransferase , mutant , chimeric gene , peptide sequence , microbiology and biotechnology , recombinant dna , reporter gene , gene expression , gene
We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.

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