This chapter discusses the purification of mammalian DNA polymerases, DNA polymerase α. DNA polymerase α plays a key role in the initiation and replication of the mammalian chromosome. The production of a panel of monoclonal antibodies against human DNA polymerase α allowed the development of a three-step immunoaffinity purification protocol. The monoclonal antibodies were used as the immunoligand bound to a protein A-Sepharose 4B resin to purify the DNA polymerase α from cultured human cells. This protocol yielded an immunocomplex of four polypeptides and a stoichiometric amount of monoclonal antibody, which has both the DNA polymerase ct and DNA primase activities. Identification of the DNA polymerase a catalytic polypeptide led to the isolation of its full-length cDNA by the reverse genetic approach. The efficient single-step immunoaffinity purification protocol gives a high yield of DNA polymerase α with high specific activity and with no monoclonal antibody (MAb) present in the enzyme fraction. The enzymatic properties of the DNA polymerase α purified by one-step protocol are identical to those of the four-subunit DNA polymerase α- primase purified by the three-step IgG-protein A immunoaffinity protocol.
ASJC Scopus subject areas
- Molecular Biology