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 |
| |
Authors: | Boehm Robert Sommer Susanne Severin Klaus Li Shu-Ming Heide Lutz |
| |
Affiliation: | (1) Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany;(2) Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany |
| |
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 0.16pkat/mg). Inclusion of an N-terminal ER-specific signal peptide from a lectin gene from Phaseolus vulgaris resulted in an average activity of 1.08pkat/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 18.6pkat/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 128.6pkat/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. |
| |
Keywords: | intracellular targeting Nicotiana tabacum prenyl transferase signal peptide ubiA ubiquinone |
本文献已被 PubMed SpringerLink 等数据库收录! |
|