High Level Expression ofRicinus communisCasbene Synthase inEscherichia coliand Characterization of the Recombinant Enzyme

Abstract

Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communisL.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA inEscherichia colias insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in anE. colihost harboring the pSM102 vector that encodes thednaYgene fortArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lacpromoter.