Engineering the quaternary structure of an exported protein with a leucine zipper.
The leucine zipper of the yeast transcriptional factor GCN4 was grafted to the C-terminal amino acid of the maltose binding protein (MalE) by fusing the malE gene of Escherichia coli to a synthetic gene coding for the leucine zipper. The hybrid protein, MalE-Lzp, was synthesized in large amounts from multicopy plasmids and efficiently exported into the periplasmic space of E. coli, up to 200,000 molecules per cell. Unlike hybrids between MalE and other proteins, MalE-Lzp was quite stable exhibiting only minimal degradation. The hybrid was purified from a periplasmic extract in one step by affinity chromatography on cross-linked amylose. Sedimentation velocity and gel filtration experiments showed that MalE-Lzp existed as a dimer in conditions where MalE was a monomer, at all concentrations tested down to 0.1 microM. Thus, it was possible to engineer the quaternary structure of an exported, monomeric protein by using a structural motif taken from a natural protein.
Blondel
A
A
Unité de Biochimie Cellulaire (CNRS URA D1129), Institut Pasteur, Paris, France.
Bedouelle
H
H
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005813:Genes, Synthetic; D016350:Leucine Zippers / Q000235:genetics*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D015202:Protein Engineering; D011494:Protein Kinases; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C461599:maltose transport system, E coli; D011494:Protein Kinases