Panwar Pankaj, Lemieux M Joanne
Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Curr Protoc Protein Sci. 2014 Apr 1;76:29.9.1-29.9.25. doi: 10.1002/0471140864.ps2909s76.
Rhomboid proteases are membrane-embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC).
菱形蛋白酶是嵌入膜中的蛋白酶，可切割跨膜蛋白的肽键。它们在细胞信号传导事件中发挥多种作用。来自流感嗜血杆菌的菱形蛋白酶GlpG（hiGlpG）是具有六个跨膜区段的典型菱形蛋白酶形式。在本单元中，介绍了使用pBAD载体中的araBAD启动子系统优化hiGlpG表达的详细方案。优化参数包括诱导剂浓度、诱导温度和时间。对这些关键因素的优化导致开发出一种方案，经离子金属亲和色谱（IMAC）纯化后可产生1.6至2.5毫克/升的蛋白质。进一步的纯化可包括尺寸排阻色谱（SEC）。
