High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme.
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 communis L.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA in Escherichia coli as 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 an E. coli host harboring the pSM102 vector that encodes the dnaY gene for tArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter.
Hill
A M
AM
Department of Chemistry, Brown University, Providence, Rhode Island, 02912, USA. DavidvCane@brown.edu
Cane
D E
DE
Mau
C J
CJ
West
C A
CA
D002367:Ricinus communis / Q000201:enzymology*; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry; D002479:Inclusion Bodies / Q000201:enzymology; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D011994:Recombinant Proteins; D008190:Lyases; C044955:casbene synthetase; D019761:Phosphorus-Oxygen Lyases