Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli.
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 lambda pL. 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, BamHI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., EcoRI, 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.
D002455:Cell Division; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D007158:Immunologic Techniques; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D007376:Interleukin-2; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes