Falcicchio Pierpaolo, Levisson Mark, Kengen Servé W M, Koutsopoulos Sotirios
Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.
Methods Mol Biol. 2014;1129:487-96. doi: 10.1007/978-1-62703-977-2_34.
The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.
嗜热和超嗜热微生物的发现，它们在接近或高于100°C的环境温度下茁壮成长，彻底改变了我们对于生命能够存在的最高温度极限的看法。（超）耐热蛋白质的特性拓宽了我们的理解，并为解决生物物理学中最具挑战性的问题之一提供了新的机会：在“正常”蛋白质会发生剧烈结构变化的高温下，如何维持结构稳定性和生物学功能？在我们的实验室中，我们已经纯化并研究了许多耐热和超耐热蛋白质，试图确定热稳定性的分子基础。在这里，我们介绍在大肠杆菌中表达此类蛋白质和酶的方法，并提供过量生产和纯化的通用方案。能够生产在高温下仍保持其稳定性和活性的酶，为广泛的生物催化应用创造了令人兴奋的机会。
