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Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.pdf

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Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

ORIGINAL RESEARCH PAPER Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli Amjad Abbas ? Stephan Plattner ? Kausar Hussain Shah ? Holger Bohlmann Received: 15 February 2013 / Accepted: 7 March 2013 / Published online: 21 March 2013 The Author(s) 2013. This article is published with open access at S 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. Keywords Fusion protein His-tag Maltose- binding protein Tobacco etch virus Thionin Thioredoxin Introduction Thionins are plant antimicrobial peptides (AMP) that are generally basic. They contain approximately 45 amino acids and 6 or 8 cysteine residues which form disulfide bridges (see Bohlmann 1994). Their general structure is that of two small a-helices and two small antiparallel b-sheets stabilized by three or four disul- fide bridges (Stec 2006). Their toxicity and antimi- crobial activity seem to depend on the destruction of membranes. Thionins have been isolated as 5 kDa peptides from various plant species but they are synthesized as much larger preproproteins (Ponz et al. 1986)