Purification and characterization of an active form of the p78(Rep) protein of adeno-associated virus type 2 expressed in Escherichia coli

The 78-kDa product (p78(Rep)) of the rep gene of AAV-2 was expressed with an amino-terminal histidine-tag in Escherichia coli and was purified under denaturing conditions. After renaturation of the p78(Rep) protein by serial steps of dialysis, the biochemical activitites of the p78(Rep) protein were demonstrated, which include the ATP-dependent endonuclease and helicase activity as well as sequence-specific binding to the AAV-2 terminal repeat. These activities were retained when the protein was purified under denaturing conditions followed by renaturation. When compared with published data for p68(Rep), the helicase activity of p78(Rep) was stronger and the endonuclease acitvtiy was weaker. The p78(Rep) protein was able to inhibit HIV-1 replication after co-microinjection together with infectious proviral HIV-1 DNA into the nuclei of human cells, suggesting that p78(Rep) is necessary for inhibition of HIV-1 in vivo.