Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus

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.