Arutyunova E, Panigrahi R, Strisovsky K, Lemieux M J
Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada.
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Methods Enzymol. 2017;584:255-278. doi: 10.1016/bs.mie.2016.10.031. Epub 2016 Dec 13.
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
菱形蛋白酶是一种膜内酶，可水解脂质双分子层中跨膜蛋白的肽键。它们在关键生物学事件中发挥多种作用，并与多种疾病状态相关。在过去十年中，人们已经获得了大量关于这类迷人蛋白酶的结构和功能知识。结构分析和动力学分析都需要毫克级别的蛋白质，这对于像菱形蛋白酶这样的膜蛋白来说可能具有挑战性。在此，我们提供了一个详细的方案，用于优化从大肠杆菌（ecGlpG）、流感嗜血杆菌（hiGlpG）和斯氏普罗威登斯菌（AarA）中表达和纯化三种菱形蛋白酶。我们讨论了表达条件的优化，如诱导剂浓度、诱导时间和温度，以及纯化方案，并对每个步骤给出了精确细节。所提供的方案每升细菌培养物可产生1 - 2.5毫克的菱形酶，有助于膜内蛋白酶的结构和功能研究。
