title: Active expression of the ubiA gene from E. coli in tobacco: influence of plant ER-specific signal peptides on the expression of a membrane-bound prenyltransferase in plant cells.
abstract: The ubiA gene from E. coli codes for 4-hydroxybenzoate: polyprenyldiphosphate 3-polyprenyltransferase, an integral membrane protein involved in ubiquinone biosynthesis. This prokaryotic membrane protein was stably expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Transgenic lines containing a direct fusion of the ubiA structural gene to a 35S-derived promoter gave very low enzyme activity levels (average 0.16 pkat/mg). Inclusion of an N-terminal ER-specific signal peptide from a lectin gene from Phaseolus vulgaris resulted in an average activity of 1.08 pkat/mg in the transgenic tobacco lines. The additional inclusion of a C-terminal HDEL tetrapeptide, responsible for the retention of proteins in the endoplasmic reticulum of eukaryotic cells, increased the activity to 18.6 pkat/mg. When the promotor of this construct was changed from the 35S derivative to the recently described very strong plant promoter (ocs)3mas, the activity increased further to 128.6 pkat/mg. The most active tobacco line showed activities of the introduced enzyme which exceeded those of wild-type E. coli (the source of ubiA) by a factor of 1100. These results demonstrate the efficacy of plant ER-specific signal peptides for the active expression of a prokaryotic membrane protein in plants.
authors.affiliation: Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Boehm
authors.forename: S
authors.initials: S
authors.lastname: Sommer
authors.forename: K
authors.initials: K
authors.lastname: Severin
authors.forename: S M
authors.initials: SM
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Heide
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D015152:Blotting, Northern; D015139:Blotting, Southern; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D014451:Ubiquinone / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C021386:4-hydroxybenzoate polyprenyltransferase