Open AccessAlteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli.
Author(s) -
Ping Li,
Jon Beckwith,
Hiroshi Inouye
Publication year - 1988
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.85.20.7685
Subject(s) - periplasmic space , signal peptide , escherichia coli , biology , amino acid , biochemistry , peptide sequence , alkaline phosphatase , phosphatase , mutagenesis , mutant , nuclear export signal , cytoplasm , gene , enzyme , rna
Escherichia coli alkaline phosphatase, coded for by the phoA gene, is normally translocated across the cytoplasmic membrane into the periplasm with high efficiency. We have constructed a series of derivatives of the phoA gene that code for a wild-type signal sequence but result in altered amino acid sequences at the amino terminus of the mature alkaline phosphatase. Our results suggest that the presence of two positively charged amino acids very early in the mature sequence interferes significantly with protein export. In one case, phoA2AB, the presence of the sequence Arg-Ile-Arg at the amino terminus of alkaline phosphatase results in a 50-times reduction in the export of the protein. By using oligonucleotide-directed mutagenesis, we have constructed mutant derivatives of phoA2AB that are greatly enhanced for export. In all cases, these derivatives reduce the net positive charge in the region. Our results may explain the failure of E. coli to export a number of proteins coded for by artificial constructs and suggest a way to improve export in these cases.