Villbrandt B, Sobek H, Frey B, Schomburg D
GBF (Gesellschaft für Biotechnologische Forschung), Department of Structure Research, Mascheroder Weg 1, Penzberg, Germany.
Protein Eng. 2000 Sep;13(9):645-54. doi: 10.1093/protein/13.9.645.
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 degrees 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 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.
无催化活性的嗜热栖热菌DNA聚合酶（TAQ聚合酶）的间隔结构域，已被嗜温同源菌大肠杆菌DNA聚合酶I（E.coli pol I）和嗜热同源菌那不勒斯栖热袍菌DNA聚合酶（TNE聚合酶）的3'-5'核酸外切酶结构域所替换。利用E.coli pol I的Klenow片段的三维（3D）结构以及TAQ聚合酶和TNE聚合酶的间隔结构域与聚合酶结构域的3D模型，构建了三种嵌合DNA聚合酶：嵌合体TaqEc1（将TAQ聚合酶的292 - 423位残基替换为E.coli pol I的327 - 519位残基）、嵌合体TaqTne1（将TAQ聚合酶的292 - 423位残基替换为TNE聚合酶的295 - 485位残基）和嵌合体TaqTne2（将TAQ聚合酶的292 - 448位残基替换为TNE聚合酶的295 - 510位残基）。嵌合体TaqEc1在50℃的中间温度下表现出双亲聚合酶的特征：高聚合酶活性、持续合成能力、3'-5'核酸外切酶活性和校对功能。相比之下，嵌合体TaqTne1和TaqTne2没有显著的3'-5'核酸外切酶活性和校对功能。嵌合体TaqTne1的最适温度为60℃，与TAQ聚合酶相比，聚合酶活性降低，持续合成能力下降。嵌合体TaqTne2在72℃时表现出高聚合酶活性、持续合成能力，与嵌合体TaqTne1相比，热稳定性降低程度较小。
