BIOLOGICALLY-ACTIVE REP PROTEINS OF ADENOASSOCIATED VIRUS TYPE-2 PRODUCED AS FUSION PROTEINS IN ESCHERICHIA-COLI

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have b een shown in vitro to perform several activities related to AAV DNA re plication. The Rep78 and Rep68 proteins are likely to be involved in t he targeted integration of the AAV DNA into human chromosome 19, and t he full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To o btain sufficient quantities for facilitating the characterization of t he biochemical properties of the Rep proteins, the AAV rep open readin g frame was cloned and expressed in Escherichia coli as a fusion prote in with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-R ep78 proteins displayed the following activities reported for wild-typ e Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (te rminal resolution site) endonuclease activity, (iv) binding to a seque nce within the integration locus for AAV DNA on human chromosome 19, a nd (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacte rized the sequence requirements for Rep binding to the ITR and found t hat only the A and A' regions are necessary, not the hairpin form of t he ITR.