Eijsink Vincent G H, Bjørk Alexandra, Gåseidnes Sigrid, Sirevåg Reidun, Synstad Bjørnar, van den Burg Bertus, Vriend Gert
Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, PO Box 5040, N-1432 As.
J Biotechnol. 2004 Sep 30;113(1-3):105-20. doi: 10.1016/j.jbiotec.2004.03.026.
During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.
在过去15年里，不断有报告描述通过引入突变来稳定的蛋白质。这些报告涵盖了从对诸如T4溶菌酶和芽孢杆菌RNA酶等小酶的开创性理性设计工作到蛋白质设计和定向进化的一段时间。与此同时，对天然存在的超稳定蛋白质的纯化和表征增进了我们对蛋白质稳定性的理解。在此过程中，人们提出了许多合理的蛋白质稳定策略，其中一些（例如通过引入脯氨酸或二硫键实现的熵稳定）具有相当高的成功率。另一方面，比较研究和定向进化方面的努力表明，有许多突变策略可导致高稳定性，其中一些策略难以定义和合理化。该领域的最新进展包括越来越意识到蛋白质表面对稳定性的重要性，以及通常极少量的突变就能使稳定性大幅提高的观念。另一个进展涉及这样一种观念，即在高温下可逆且完全展开的小纯蛋白质的“实验室稳定性”与“工业稳定性”之间存在根本差异，“工业稳定性”通常由部分展开过程以及某种不可逆失活过程（例如聚集）所决定。只要对热失活机制有足够的了解，就可以成功且高效地对酶进行合理稳定化。
