CRMs were diluted twofold according to the manufacturer’s recommendation (see Material and Methods). While the control CRM without template DNA did not show any agglutination reaction

at MG-132 manufacturer all, agglutination of anti-TDH antibody coated beads was observed in all serial dilutions of CRMs with TDH templates. All CRMs were tested in five serial twofold dilutions (Table 3). In all CRMs de novo synthesized proteins showed a positive agglutination reaction. To test if functional toxins were synthesized in the cell-free systems, their hemolytic activity was determined. To this end, aliquots of the CRMs and SNs were taken and spotted directly on blood agar plates containing rabbit erythrocytes. To quantify the hemolytic activity of the mature proteins and their tagged derivatives click here aliquots of supernatants (SNs) all SNs were adjusted to contain the same amount of soluble toxins (120 μg/ml). After 20 h of incubation at 37 °C clear zones of hemolysis were visible on the plates. The weakest hemolytic activity was observed in case of the double tagged toxin (Strep- and 6xHis-tagged) and the largest zone was caused by the untagged mTDH. Neither the control CRM (no template control reaction, NTC) nor the CRMs from tagged preprotein

derivatives produced visible hemolysis except preTDH2, where a very small zone of hemolysis was visible ( Fig. 5 spot 5). To quantify hemolytic activity of synthesized TDH and its different variants a semiquantitative hemolysis assay was performed. Twofold serial dilutions taken from the SN containing the soluble mature toxins and their tagged derivatives were incubated with 4% rabbit erythrocytes. Lysis of erythrocytes was determined photometrically and confirmed that

the mature toxin without any additional tag has the highest activity as it showed hemolysis in the highest dilution containing 0.09 μg protein (Fig. 6). The C-terminal His-tagged toxin was the derivative with the second highest hemolytic activity, while the two other toxin derivatives (Strep-mTDH and Strep-mTDH-His) showed the weakest activity. Recent studies indicate that tetramer formation is indispensable for hemolytic activity (Yanagihara et al., 2010) which others means that cell-free synthesized TDH monomers are able to oligomerize to intact and functional tetramers in solution. Further results suggest that the adhesion of TDH to erythrocytes depends on two processes: binding and post-binding (Tang et al., 1994). Postbinding effects, which take place after binding to the cell membrane and prior to lysis of the cell, are specified as e.g. toxin oligomerization, permeabilization of the cell membrane and insertion into the membrane. Also TDH induced phosphorylation of specific membrane proteins has been demonstrated to be important for hemolysis (Yoh et al., 1996).