DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction.
DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction.
复制标题
DNA聚合酶杂种源自Furococus of Feriosus和Thermoccus kodakarensis的家族-B酶：改善聚合酶链反应的性能。
DOI：
10.3389/fmicb.2014.00224
复制
发表时间：
2014
影响因子：
5.2
通讯作者：
Connolly BA
中科院分区：
文献类型：
--
作者：
Elshawadfy AM;Keith BJ;Ee Ooi H;Kinsman T;Heslop P;Connolly BA
聚合酶链式反应(PCR)在生物科学领域得到了广泛的应用，其中古生菌B家族DNA聚合酶因其高度的热稳定性和保真度而成为首选。尽管Kodakarensis的类似蛋白(Tkod-Pol)具有更好的聚合酶扩增性能，但来自Furiosus的热球菌酶(PFU-Pol)比Kodakarsis的类似蛋白(Tkod-Pol)更常被使用。在此对两种聚合酶进行了综合比较，证实了Tkod-Pol：(1)更快地扩展了引物模板；(2)具有更高的加工性；(3)在常规和实时PCR中表现出了优越的性能。然而，Tkod-Pol的热稳定性不如PFU-Pol，两种酶的保真度相同。为了了解Tkod-Pol的良好性质，我们制备了杂化蛋白。用单突变、双突变和三突变将存在于Tkod-Pol的“分叉点”(核酸外切酶和聚合酶通道的交界处)的精氨酸定位在Pfu-Pol的相应位置，略微改善了聚合酶链式反应的性能。负责双链DNA结合的PFU-Pol拇指结构域已经完全被Tkod-Pol的Tkod-Pol结构域取代，再次提供了更好的PCR特性。结合“分叉点”和拇指互换突变导致了聚合酶链式反应能力的显著提高，保持了高保真度，并保持了与PFU-POL相关的卓越的热稳定性。然而，即使是精氨酸/拇指互换突变体在聚合酶链式反应中也低于Tkod-Pol，这表明在PFU-Pol框架内可以进一步改进。这项工作的意义在于观察到，通过混合来自密切相关的古生菌聚合酶的成分，很容易获得聚合酶链式反应性能的改善，这一方法未来可能会通过使用更多来自这些生物体的聚合酶来扩展。
The polymerase chain reaction (PCR) is widely applied across the biosciences, with archaeal Family-B DNA polymerases being preferred, due to their high thermostability and fidelity. The enzyme from Pyrococcus furiosus (Pfu-Pol) is more frequently used than the similar protein from Thermococcus kodakarensis (Tkod-Pol), despite the latter having better PCR performance. Here the two polymerases have been comprehensively compared, confirming that Tkod-Pol: (1) extends primer-templates more rapidly; (2) has higher processivity; (3) demonstrates superior performance in normal and real time PCR. However, Tkod-Pol is less thermostable than Pfu-Pol and both enzymes have equal fidelities. To understand the favorable properties of Tkod-Pol, hybrid proteins have been prepared. Single, double and triple mutations were used to site arginines, present at the “forked-point” (the junction of the exonuclease and polymerase channels) of Tkod-Pol, at the corresponding locations in Pfu-Pol, slightly improving PCR performance. The Pfu-Pol thumb domain, responsible for double-stranded DNA binding, has been entirely replaced with that from Tkod-Pol, again giving better PCR properties. Combining the “forked-point” and thumb swap mutations resulted in a marked increase in PCR capability, maintenance of high fidelity and retention of the superior thermostability associated with Pfu-Pol. However, even the arginine/thumb swap mutant falls short of Tkod-Pol in PCR, suggesting further improvement within the Pfu-Pol framework is attainable. The significance of this work is the observation that improvements in PCR performance are easily attainable by blending elements from closely related archaeal polymerases, an approach that may, in future, be extended by using more polymerases from these organisms.
登录
查看更多内容
影响因子：
5.6
作者：
Firbank, Susan J.;Wardle, Josephine;Connolly, Bernard A.
通讯作者：
Connolly, Bernard A.
影响因子：
16.8
作者：
Hogg, Matthew;Osterman, Pia;Johansson, Erik
通讯作者：
Johansson, Erik
影响因子：
1.1
作者：
Matsukawa, Hiroaki;Yamagami, Takeshi;Ishino, Yoshizumi
通讯作者：
Ishino, Yoshizumi
DOI：
10.1073/pnas.96.7.3600
发表时间：
1999-03-30
影响因子：
11.1
作者：
Hopfner, KP;Eichinger, A;Angerer, B
通讯作者：
Angerer, B
影响因子：
3.5
作者：
LUNDBERG, KS;SHOEMAKER, DD;MATHUR, EJ
通讯作者：
MATHUR, EJ