海藻糖对热不稳定酶的热稳定化及热激活作用及其在全长cDNA合成中的应用
Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA.
作者信息
Carninci P, Nishiyama Y, Westover A, Itoh M, Nagaoka S, Sasaki N, Okazaki Y, Muramatsu M, Hayashizaki Y
机构信息
Genome Science Laboratory, Tsukuba Life Science Centre, Institute of Physical and Chemical Research (RIKEN), Ibarak, Japan.
出版信息
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):520-4. doi: 10.1073/pnas.95.2.520.
Abstract
The advent of thermostable enzymes has led to great advances in molecular biology, such as the development of PCR and ligase chain reaction. However, isolation of naturally thermostable enzymes has been restricted to those existing in thermophylic bacteria. Here, we show that the disaccharide trehalose enables enzymes to maintain their normal activity (thermostabilization) or even to increase activity at high temperatures (thermoactivation) at which they are normally inactive. We also demonstrate how enzyme thermoactivation can improve the reverse transcriptase, reaction. In fact, thermoactivated reverse transcriptase, which displays full activity even at 60 degrees C, was powerful enough to synthesize full length cDNA without the early termination usually induced by stable secondary structures of mRNA.
摘要
热稳定酶的出现推动了分子生物学的巨大进步，例如聚合酶链式反应（PCR）和连接酶链式反应的发展。然而，天然热稳定酶的分离一直局限于嗜热细菌中存在的那些酶。在此，我们表明二糖海藻糖能使酶保持其正常活性（热稳定），甚至在高温下（热激活）增加活性，而在这些高温下它们通常是无活性的。我们还展示了酶的热激活如何改善逆转录酶反应。事实上，热激活的逆转录酶即使在60摄氏度时也能显示出完全活性，其强大到足以合成全长互补DNA（cDNA），而不会出现通常由信使核糖核酸（mRNA）的稳定二级结构诱导的早期终止。
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