In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence |
| |
Authors: | Martin R Rohrbach Sabine Paul and Wolfgang K?ster |
| |
Institution: | (1) Mikrobiologie/Membranphysiologie, Universit?t Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany;(2) Present address: Abt. Infektionsbiologie, Max Planck Institut für Biologie, Spemannstr. 34, 72076 Tübingen, Germany |
| |
Abstract: | Transport of iron(III) hydroxamates across the inner membrane ofEscherichia coli depends on a binding protein-dependent transport system composed of the FhuB,C and D proteins. The FhuD protein, which is
synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the
system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying
an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified
from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro
by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport
in afhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD
contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein.
Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides
which are normally exported across the inner membrane and can cause toxicity problems when overproduced. |
| |
Keywords: | Solute-binding protein Periplasmic binding protein ABC transporter Iron(III) hydroxamate transport E coli |
本文献已被 SpringerLink 等数据库收录! |
|