Multipurpose Expression Cloning Vehicles in Escherichia coli

Publisher Summary This chapter reviews the construction of several high-level expression vectors using the Escherichia coli lipoprotein promoter and part of the protein. These vectors are designed to produce a large amount of an inserted gene product and to localize it to a specific compartment of the E. coli cell—cytoplasm, cytoplasmic membrane, periplasm, or outer membrane. These vectors have three restriction enzyme sites—EcoRI, HindIII, and BamHI—in each of the three reading frames at various positions along the lipoprotein gene. The pIN-I vectors constitutively produce a large amount of the inserted gene product. The pIN-II and pIN-III vectors produce the foreign gene product only in the presence of an inducer. Once a gene has been inserted properly into one of these vectors it can easily be moved to the other vectors, like a cassette, with the use of several possible unique restriction-enzyme sites on either side of the gene. The chapter also describes two promoter cloning vectors that produce a protein—tetracycline resistance or β-galactosidase—when a promoter is inserted.