Expression and Purification of Recombinant Proteins Using the pET System

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in Escherichia coli . Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell (1 –3 ). T7 RNA polymerase is so selective and active that almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein after a few hours of induction. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability caused by the production of proteins potentially toxic to the host cell. Once established in a nonexpression host, plasmids are then transferred into expression hosts containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, and expression is induced by the addition of IPTG. Two types of T7 promoter and several hosts that differ in their stringency of suppressing basal expression levels are available, providing great flexibility and optimizing the expression of a wide variety of target genes. This chapter describes the vectors, hosts, and basic protocols for cloning, expression, and purification of target proteins in the pET System.