High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquatlcus DNA Polymerase and a Truncated Form Deficient in 5' to 3' Exonuclease Activity

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-aminoacid construct produced 1 2 % of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For fulllength 94-kD Taq Pol I, yield was 3.26x 107 units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 • 106 units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75~ to 80~ 2 4 mM MgClz and 10-55 mM KCI. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCI2 is the preferred cofactor compared to MnCI z, CoCIz, and NiCI 2. The fulllength Taq Pol I has an activity halflife of 9 min at 97.5~ The Stoffel fragment has a half-life of 21 min at 97.5~ Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.