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 50000 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. Copyright © 1990, Wiley Blackwell. All rights reserved