Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.
Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Methods Mol Biol. 2021;2178:469-478. doi: 10.1007/978-1-0716-0775-6_29.
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 are 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，在环境温度接近或高于 100°C 的条件下茁壮成长，彻底改变了我们对生命存在的最高温度极限的认识。（超）耐热蛋白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”蛋白发生剧烈结构变化的高温下，结构稳定性和生物功能如何得以维持？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，试图确定热稳定性的分子基础。在这里，我们提供了在大肠杆菌中表达此类蛋白和酶的方法，并提供了一种用于大量生产和纯化的通用方案。能够生产在高温下保持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。
