Preparation of cell lysates. The lysate was centrifuged at 12,000 g for 2 min, and the freshly prepared supernatant was used in the in vitro complementation assay. The cells were lysed by adding 2-3 drops of chloroform. The debris was removed by centrifuging at 15,000 rpm for 10 min. 10 plaque forming units per ml.
Complementation assay in vitro .
Isolation and purification of the recombinant protein. The cell suspension was sonicated for 2 min, and cellular debris was removed by centrifuging for 10 min at 12,000 g . The precipitate was centrifuged under the same conditions. After centrifugation, the pellet containing protein was resuspended in 10 mM Tris-HCl buffer, pH 7.8, and dialyzed against the same buffer overnight. The protein was eluted using the same buffer, and the fractions were analyzed by electrophoresis.