Escherichia coli is one of the most powerful and versatile hosts for highlevel protein production. Its well-characterized genetics and biochemistry have led to the development of many different systems for heterologous protein expression (1,2). In this chapter, we focus on the use of the T7 expression system (3,4) as we have found this system to produce protein most consistently. Many different vectors and host strains have been developed for use with the T7 system. In the format we most commonly use, employing the vector pET24d (Fig. 1), transcription originates from a T7lac promoter (5) in response to induction of a chromosomally encoded T7 RNA polymerase gene. Inclusion of a lac operator in the T7 promoter and the presence of a vector encoded lactose repressor, lacI, helps to reduce basal transcription levels. Background expression can be further reduced by cotransformation with plasmids encoding T7 lysozyme such as plysS (6). Kanamycin selection due to intracellular expression of aminoglycoside 3′-phosphotransferase by pET24d allows more stringent selection for plasmid maintenance than if a secreted β-lactamase is used. Tight control of expression and the continued application of selective pressure are important for high-level protein production as leaky expression can select for lower expressing clones (7) and high-level protein expression produces a strong negative selection pressure, even when the expressed proteins are nontoxic (8).

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