Figure 4

(A) Contact dependent hemolysis of Rv1694. Rv1694 (with and w/o His-tag) and mock vector transformed E. coli were examined for contact dependent lysis of rabbit RBC. E. coli (~10⁷) cells and rabbit erythrocytes (~10⁵) were mixed and briefly centrifuged to ensure close contact between bacteria and RBC. The resultant mixture of cells was incubated at 37°C for 24 to 30 hours. Degree of lysis was monitored by measuring the absorbance at 540 nm of a cell-free supernatant. UI indicates uninduced E. coli and groups labeled with 1-5 were harvested time in hour(s) from induction (with 1.0 mM IPTG) time point. Error bars represent standard deviation of two independent experiments. (B) Hemolytic activity of the purified Rv1694: Specific hemolytic activity of purified Rv1694 (40 μg/ml) was carried out by two-fold serial dilution of Rv1694 which was mixed with rabbit RBC (1.5%). After 24 hrs of incubation, the absorbance was measured at 540 nm for release of hemoglobin. At ~50% hemolysis, the protein concentration was 18.0 μmug/ml. In presence of thiol-reducing agent, the hemolytic activity was 3.6 fold lesser than the maximum activity of Rv1694. Total haemoglobin release was obtained by lysing the RBC with deionized water. Error bars represent standard deviation from three independent experiments. (C) Hemolytic activity of Rv1694 generated by coupled in vitro transcription and translation: Rv1694 generated by coupled in vitro transcription and translation (~5.0 out of 50.0 μl reaction mix) was mixed with 100 μl of 0.3% rRBC in a 96 well plate as described earlier and the absorbance at 590 nm for rRBC lysis was noted every 10 minutes. As a positive control, we have also translated staphylococcal α-HL and followed its hemolysis.