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579801 
Journal Article 
Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli 
Picaud, S; Olsson, ME; Brodelius, PE 
2007 
1 
Protein Expression and Purification
ISSN: 1046-5928 
51 
1 
71-79 
Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner(TM), BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner(TM) using different inducing agents (IPTG, The Inducer(TM)). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer(TM). For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner(TM), induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer(TM). Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively. 
Amorpha-4; 11-diene synthase; Artemisia annua; Germacrene D synthase; Protein expression; Protein solubility; Recombinant protein; Sesquiterpene synthase; Zingiber officinale