Molecular cloning of the genes for GDP-mannose 4, 6-dehydratase and GDP-l-fucose synthetase from Bacteroides thetaiotaomicron

Mi-Hee Jang, Won-Heong Lee, So-Yeon Shin, Nam Soo Han, Jin-Ho Seo, Myoung-Dong Kim

                                                                     

Abstract

Genes encoding GDP-mannose 4, 6-dehydratase (GMD) and GDP-l-fucose synthetase (GFS) were cloned from Bacteroides thetaiotaomicron and overexpressed in recombinant Escherichia coli by constructing isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression vectors. GMD and GFS genes from B. thetaiotaomicron were 60 and 45%, respectively, identical to those from E. coliK12 over their entire lengths. An optimum expression condition of 30oC and 0.1 mM IPTG was chosen for maximum soluble expression of B. thetaiotaomicron GMD and GFS in recombinant E. coli BL21(DE3). Functional expression of B. thetaiotaomicron GMD and GFS in recombinant E. colistrains was confirmed by measuring intracellular GDP-l-fucose content.

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Keywords :GDP-l-fucose ,GDP-mannose 4,6-dehydratase ,GDP-l-fucose synthetase ,Bacteroides thetaiotaomicron ,recombinant Escherichia col.