Purification of mammalian DNA polymerases: DNA polymerase α

Teresa S.F. Wang, William C. Copeland, Lars Rogge, Qun Dong

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)77-84
Number of pages8
JournalMethods in enzymology
Volume262
Issue numberC
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Purification of mammalian DNA polymerases: DNA polymerase α'. Together they form a unique fingerprint.

  • Cite this