3] Host strain selection for bacterial expression of toxic proteins

Abstract

This chapter focuses on the Escherichia coli T7 RNA polymerase expression system and the problems associated with the selection of a suitable bacterial host strain. Protocols are described in the chapter to determine if a host strain is unreceptive to transformation and expression of a cytotoxic gene product and to prepare an amenable host strain for transformation and expression of the desired gene product. The amenable host strain is prepared by curing a plasmid, encoding a toxic protein, from a bacterial strain that is capable of high expression of the toxic protein. An example of host strain determination using a plasmid encoding HIV-1 protease under transcriptional control of the T7 promoter and expression in the bacterial host strain BL21(DE3)pLysS is presented in the chapter. E. coli expression systems, the most popular system for large scale protein production, have been widely used for the expression of recombinant retroviral proteases. The major advantages are the ease of subcloning into expression vectors using standard molecular biological techniques and the cost effectiveness of growth, selection, and large scale production.