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利用分隔式自我复制（CSR）改造来自于Thermus thermophilus的I型DNA聚合酶

发布: 2022-04-23 840阅读 228评论

在基因元件（例如DNA聚合酶）的定向进化中，CSR（分隔式自我复制）和CPR（分隔式同伴复制）是一类重要的方法。目前利用野生型的Taq DNA聚合酶进行乳化PCR仍存在运行时间长，PCR产物产量低且由于抑制剂的干扰存在假阴性的等缺陷。

该篇论文改进了来自于Thermus thermophilus HB27的I型DNA聚合酶 Tth pol，使其能更高效地用于DNA聚合酶与其他基因元件的定向进化。

图一 CSR定向进化流程图

在这一工作中，研究者首先进行了以下4个步骤的改进和突变（如图二）：

i.删除N端5’-3’外切酶结构域；

ii.与DNA结合蛋白(Sso7d)融合表达；

iii.在Tth pol引入已知的能提高Taq pol活性的位点（626E, 707I, 708E, 743A）；

iv.优化密码子降低模板GC含量 （从68%降低至53%）

图二 N端5’-3’外切酶结构域，Sso7d融合表达，以及引入已知的能提高Taq pol活性的位点；

经过以上的改进和突变，Tth pol的扩增效率得到了显著提高，如图三所示：

 

图三 改进前Tthpol和改进后 Tth pol的扩增能力对比

然后，研究者又构建了随机突变文库并利用CSR进行进一步的筛选，以期望获得更高效率的Tth pol突变体，能实现利用更少的循环数取得良好的扩增效果，且表现更稳定。

图四 CSR筛选SDTthCs12RsEx pol的随机突变文库

经过4轮筛选，在DNA结合蛋白Sso7d上的K7T和S18T，以及在Tth DNA聚合酶上的E338K和E690D，四个点突变被筛选到，表明这些突变体能在更短的循环数内达到较好的扩增效果。

图五 筛选到的突变体的突变位点（预测）

然后，研究者通过定点突变构建了单独的突变体，并进行了乳化和非乳化PCR的验证。

图六 非乳化和乳化PCR的验证

研究者发现，无论是乳化和非乳化PCR，Tth pol的突变体E338K和E690D都能够以10s/kb的速度扩增长度为4.65 kb DNA，表明这两个突变体能在更短的循环数内达到较好的扩增效果，然而其机制尚不清楚。

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