In vivo assembly of active maltose binding protein from independently exported protein fragments.
The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is synthesized in the cytoplasm with an N-terminal signal peptide that is cleaved upon export. We examined whether active MBP could assemble into an active protein in bacteria, from N- and COOH-terminal complementary protein fragments encoded by distinct, engineered segments of its structural gene. We found export and functional periplasmic assembly of MBP fragments, despite the complex polypeptide chain topology of this protein, if two conditions were satisfied. First, each of the two fragments must carry a signal peptide. Second, the boundaries between the two fragments must correspond to a permissive site within the protein. Functional assembly of active MBP occurred in five cases where these conditions were met: sites after residues 133, 161, 206, 285 and 303; but not in three other cases where the break junction corresponded to a non-permissive site: after residues 31, 120 and 339. Thus, permissive sites which were initially characterized because they could accept extensive genetic insertion/deletion modifications without loss of most biological properties provide a means of defining complementing protein fragments. This observation opens a way to study genetically the relationships between protein export and folding into the periplasm.
Betton
J M
JM
Unité de Programmation Moléculaire et de Toxicologie Génétique (CNRS-UA1444), Département des Biotechnologies, Institut Pasteur, Paris, France.
Hofnung
M
M
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005816:Genetic Complementation Test; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D017433:Protein Structure, Secondary; D015183:Restriction Mapping
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli