Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli

Abstract

We have constructed a set of expression vectors which contain synthetic DNA sequences comprising a computer-generated model ribosomal binding site located downstream from the tightly regulated phage λ p L promoter. These vectors have been used in several laboratories to produce significant amounts of eukaryotic and prokaryotic gene products in Escherichia coli, either as fusion proteins (with two to nine extra N-terminal amino acids) or as proteins containing the naturally occurring amino terminus. For inserting DNA sequences downstream of an initiation codon, we used synthetic oligonucleotides to introduce multiple-use restriction sites recognized by EcoRI, Bam HI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., Eco RI ∗, MboI, TaqI, and HpaII), in addition to their own. A set of three of these vectors was made to accommodate all three translational reading frames. In combination, the features of these vectors afford useful advantages over expression vectors previously described, especially for the application of shot-gun cloning of genomic DNA to generate expression libraries.