蛋白酶
细胞生物学
线粒体内膜
化学
生物
线粒体
生物化学
酶
作者
Elena Arutyunova,Laine Lysyk,Melissa Morrison,Cory L. Brooks,M. Joanne Lemieux
出处
期刊:Methods in molecular biology
日期:2021-01-01
卷期号:: 1-20
被引量:2
标识
DOI:10.1007/978-1-0716-1394-8_1
摘要
Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1 mg of pure PARL per liter of yeast culture.
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