title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.
abstract: Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.
authors.affiliation: Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, UFT Tulln, Tulln, Austria. amjad.abbas@boku.ac.at
authors.forename: Amjad
authors.initials: A
authors.lastname: Abbas
authors.forename: Stephan
authors.initials: S
authors.lastname: Plattner
authors.forename: Kausar Hussain
authors.initials: KH
authors.lastname: Shah
authors.forename: Holger
authors.initials: H
authors.lastname: Bohlmann
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D019897:Periplasm / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D054809:Thionins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D054809:Thionins