Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus.
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.
Federal Institute for Risk Assessment, Berlin, Germany.
Silke
S
Bechlars
Doreen A
DA
Wüstenhagen
Katja
K
Drägert
Ralf
R
Dieckmann
Eckhard
E
Strauch
Stefan
S
Kubick
D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002474:Cell-Free System / Q000378:metabolism; D003001:Cloning, Molecular; D048168:Escherichia coli K12 / Q000235:genetics; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006461:Hemolysis; D016133:Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014736:Vibrio parahaemolyticus / Q000235:genetics; D037521:Virulence Factors / Q000096:biosynthesis* / Q000302:isolation & purification
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D037521:Virulence Factors; C068489:thermostable direct hemolysin
Cell-free protein synthesis; In vitro translation; Thermostable direct hemolysin (TDH); Vibrio parahaemolyticus