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  • Title: Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus.
    Author: Bechlars S, Wüstenhagen DA, Drägert K, Dieckmann R, Strauch E, Kubick S.
    Journal: Toxicon; 2013 Dec 15; 76():132-42. PubMed ID: 24060377.
    Abstract:
    Vibrio parahaemolyticus is a recognized enteropathogen causing diarrhea in humans and is one of the major causes of seafoodborne gastroenteritis. An important virulence factor is thermostable direct hemolysin (TDH), a pore-forming toxin, which is able to lyse eukaryotic cells. The active toxin is a tetramer of four identical protein subunits, which is secreted by the pathogen after cleavage of a signal peptide. To establish diagnostic detection systems for TDH we expressed the hemolysin with and without the signal peptide in a prokaryotic cell-free system to obtain pure toxin. In order to purify and to facilitate the isolation from cell lysates we synthesized TDH variants with different tags. Important regulatory sequences for cell-free protein synthesis as well as sequences for N-terminal Strep-tag and C-terminal 6xHis-tag were added by a two-step PCR. For the expression in the cell-free system these linear tdh templates were subjected directly to prokaryotic cell extracts. Protein yields were in the range of 500-600 μg/ml for the preproteins and approx. 300-400 μg/ml for the mature proteins. The identities of expressed proteins were further confirmed by SDS-PAGE, immunological and MALDI-TOF mass spectrometric analyses. The functionality of newly synthesized toxin variants was tested by performing qualitative and semiquantitative hemolysis assays. Cell-free produced mature TDH and its variants were active while the preprotein and its derivatives lacked hemolytic activity. A C-terminal 6xHis-tag showed less influence on functionality compared to the N-terminal Strep-tag.
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