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Protein Engineering, Vol. 13, No. 9, 645-654, September 2000
© 2000 Oxford University Press

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

B. Villbrandt1, H. Sobek2, B. Frey2 and D. Schomburg3

GBF (Gesellschaft für Biotechnologische Forschung), Department of Structure Research, Mascheroder Weg 1, D-38124 Braunschweig and 2 Roche Molecular Biochemicals, Nonnenwald 2, D-82377 Penzberg, Germany

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (Taq polymerase), which has no catalytic activity, has been exchanged for the 3'–5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (Tne polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the Taq polymerase and the Tne polymerase: chimera TaqEc1 (exchange of residues 292–423 from Taq polymerase for residues 327–519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292–423 of Taq polymerase for residues 295–485 of Tne polymerase) and chimera TaqTne2 (exchange of residues 292–448 of Taq polymerase for residues 295–510 of Tne polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50°C: high polymerase activity, processivity, 3'–5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'–5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60°C, decreased polymerase activity compared with the Taq polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72°C, processivity and less reduced thermostability compared with the chimera TaqTne1.


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