Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli.
A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E. coli cell. The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P. Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography.
Blondel
A
A
Unité de Biochimie Cellulaire (Unité de Recherche associée D1129 du Centre National de la Recherche Scientifique), Institut Pasteur, Paris, France.
Bedouelle
H
H
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002501:Centrifugation, Zonal; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli