Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5' --> 3' exonuclease domain-deleted Tfi DNA polymerase, named 5' --> 3' Exo(-) Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg(-1) protein. The 5' --> 3' exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5' --> 3' Exo(-) Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.