Expression of an Aggregation-Prone Protein in the RTS 500 System

Abstract

The most widely used host for high-level production of recombinant proteins is Escherichia coli. However, many of these proteins are produced in biologically inactive and aggregated forms or inclusion bodies. The conventional molecular mechanism of this process suggests that nascent polypeptide chains have two alternative and competitive pathways between folding and aggregation (King et al. 1996). Although solubilization/refolding procedures exist to obtain proteins in soluble form, they are lengthy processes, often inefficient and not generalizable (Lilie et al. 1998). Roche Molecular Biochemicals’ recent development of an in vitro protein biosynthesis system, the Rapid Translation System or RTS 500, is a very attractive alternative for recombinant protein production. In this paper, the RTS 500 system was evaluated for the expression of the maltose-binding protein (MalE), an exported protein of E. coli, which serves as periplasmic receptor for the high-affinity transport of maltodextrins and for a defective MalE folding mutant, MalE31, that forms inclusion bodies when expressed in bacteria (Betton and Hofnung 1996).