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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

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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.

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Authors and Affiliations

  1. Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, D-72076, Tübingen, Germany

    Robert Boehm, Susanne Sommer, Klaus Severin & Shu-Ming Li

  2. Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, D-72076, Tübingen, Germany

    Lutz Heide

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  1. Robert Boehm
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  2. Susanne Sommer
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  3. Klaus Severin
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  4. Shu-Ming Li
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  5. Lutz Heide
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Boehm, R., Sommer, S., Severin, K. et al. 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. Transgenic Res 9, 477–486 (2000). https://doi.org/10.1023/A:1026507803067

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