具有两个氨基酸取代的 Tth DNA 聚合酶的锰离子非依赖性逆转录酶活性。

Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions.

机构信息

Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China.

Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, 222005, China.

出版信息

Protein Pept Lett. 2023;30(3):193-200. doi: 10.2174/0929866530666230216113348.

DOI:10.2174/0929866530666230216113348

PMID:36797604

Abstract

BACKGROUND

The DNA polymerase of Thermus thermophilus (Tth pol) presents reverse transcriptase activity with Mn, and can be used for one-step RT-qPCR. However, Mn would reduce amplification fidelity and cause nonspecific products.

OBJECTIVE

Eliminating the Mn dependence of the reverse transcriptase activity of Tth pol by point mutations.

METHODS

We constructed three variants I640F, I709K, and I640F/I709K, and measured their DNA polymerase and reverse transcriptase activities without Mn. Their enzymatic characteristics and PCR inhibitor resistance were also tested. Finally, these variants were applied in one-step RT-qPCR.

RESULTS

All three variants presented reverse transcriptase activity with Mg only and increased DNA polymerase activity. The variants, except I709K, showed no significant difference in thermostability, optimal pH, optimal NaCl concentration, storage stability and PCR inhibitor resistance compared to the wild type. Variant I640F/I709K had good performance in one-step RT-qPCR with Mg only, whereas both variants with single substitution exhibited nonspecific amplification.

CONCLUSION

We successfully constructed three Tth pol variants possessing Mn independent reverse transcriptase activity. The variant I640F/I709K was suitable for one-step RT-qPCR because of its good performance.

摘要

背景

嗜热栖热菌（Thermus thermophilus）的 DNA 聚合酶（Tth pol）具有 Mn 依赖性的逆转录酶活性，可用于一步法 RT-qPCR。然而，Mn 会降低扩增保真度并导致非特异性产物。

目的

通过定点突变消除 Tth pol 逆转录酶活性对 Mn 的依赖。

方法

构建了三个变体 I640F、I709K 和 I640F/I709K，并在无 Mn 的情况下测量了它们的 DNA 聚合酶和逆转录酶活性。还测试了它们的酶学特性和抗 PCR 抑制剂能力。最后，将这些变体应用于一步法 RT-qPCR。

结果

所有三个变体均仅在 Mg 存在下具有逆转录酶活性，并提高了 DNA 聚合酶活性。除 I709K 外，与野生型相比，这些变体在热稳定性、最适 pH 值、最适 NaCl 浓度、储存稳定性和抗 PCR 抑制剂能力方面没有显著差异。与野生型相比，变体 I640F/I709K 在仅用 Mg 的一步法 RT-qPCR 中表现出良好的性能，而两个单取代变体均表现出非特异性扩增。