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-mu-M. Thus, it was possible to engineer the quaternary structure of an exported, monomeric protein by using a structural motif taken from a natural protein.