FIG. 8.

VapB-1 forms nontoxic complexes with VapC-1 in vitro. The antitoxin VapB-1 was cloned into pET24b as a single gene and purified using the MagneHis native protein purification protocol. Various amounts of purified VapB-1 were incubated with a constant amount of VapC-1 for 30 min prior to the addition of the total RNA substrate in RNase activity assays. A 4:1 ratio of VapB-1 to VapC-1 abrogates the RNase activity of VapC-1. Lanes 1 and 6, MagneHis protein elution buffer control; lanes 2 and 7, 0.2 μg of VapB-1; lanes 3 and 8, 0.4 μg of VapB-1 plus 0.1 μg of VapC-1 (4:1 ratio); lanes 4 and 9, 0.2 μg of VapB-1 plus 0.1 μg of VapC-1 (2:1 ratio); lanes 5 and 10, 0.1 μg of VapC-1 alone. For these assays, H. influenzae strain R2866 total RNA was used. Note the natural 23S fragmentation pattern, which differs from that of strain 86-028NP.