High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.

Abstract:

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda 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-amino-acid 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 full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. 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.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Stoffel fragment Lawyer FC1993 Molecular Weight 6.13E+04 Dalton
Stoffel fragment Lawyer FC1993 Cloned or native Cloned in E. coli
Stoffel fragment Lawyer FC1993 5-3' Exonuclease No
Stoffel fragment Lawyer FC1993 Specific Activity 3.69E+05 Other
Stoffel fragment Lawyer FC1993 Vmax 130 /second Reaction: Nucleotide incorporation; Substrate: dNTPs; Experimental conditions: Temp (75°C)
Taq pol I Lawyer FC1993 Molecular Weight 9.4E+04 Dalton
Taq pol I Lawyer FC1993 Cloned or native Cloned in E. coli
Taq pol I Lawyer FC1993 5-3' Exonuclease Yes
Taq pol I Lawyer FC1993 Full length or truncated Full length
Taq pol I Lawyer FC1993 Specific Activity 2.92E+05 Other
Taq pol I Lawyer FC1993 Vmax 150 /second Reaction: Nucleotide incorporation; Substrate: dNTPs; Experimental conditions: Temp (75°C)

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