Journal article
Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-39182 Kalmar, Sweden.1
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, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer).
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.
For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, 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.
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.
Language: | English |
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Year: | 2007 |
Pages: | 71-79 |
ISSN: | 10960279 and 10465928 |
Types: | Journal article |
DOI: | 10.1016/j.pep.2006.06.025 |
11-diene synthase Alkyl and Aryl Transferases Amorpha-4 Artemisia annua Betaine Enzyme Induction Escherichia coli Germacrene D synthase Isopropyl Thiogalactoside Molecular Biology Osmotic Pressure Protein Folding Protein expression Protein solubility Recombinant Fusion Proteins Recombinant protein Sesquiterpene synthase Sorbitol Zingiber officinale amorpha-4,11-diene synthase germacrene D synthase