[11] Use of Secretion Cloning Vectors for Guiding the Localization of Proteins in Escherichia Coli

This chapter discusses three cloning systems, which direct the cloned gene product out of the cytoplasm. These vectors provide several advantages not afforded by standard vector systems for cloning foreign genes into Escherichia coli. First, the transport of cloned proteins out of the cytoplasm allows cloning of genes whose products are lethal to the host cell. Second, gene products sensitive to the high proteolytic activity present in the bacterial cytoplasm can be translocated to the periplasmic space. Third, production of proteins in the cytoplasm requires an initiator methionine at the polypeptides amino-terminus. In cases in which the terminal amino acid is not methionine, the processing that occurs during translocation provides a method of resurrecting a native gene product from a transcriptionally active precursor. Transport out of the cytoplasm affords a several fold purification of the cloned gene product, based on the relative protein concentration in cytoplasmic fractions versus membrane or periplasmic fractions. For all of these reasons, secretion cloning vectors provide powerful tools for the production of biologically interesting proteins.