Malgapo Martin Ian P, Linder Maurine E
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2019;2009:179-189. doi: 10.1007/978-1-4939-9532-5_14.
DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.
DHHC酶是一类整合膜蛋白，可催化将棕榈酸（一种16碳脂肪酸）翻译后添加到蛋白质的半胱氨酸残基上。尽管多年来已鉴定出的棕榈酰化蛋白库大幅增长，但对DHHC蛋白的生化和机制研究因大量纯化该酶存在固有困难而受到挑战。在此，我们描述了制备带有六组氨酸序列和FLAG表位标签的重组DHHC蛋白的方案，这些标签有助于纯化。该程序已在纯化该酶家族的多个成员中成功测试；以DHHC3及其催化无活性的半胱氨酸突变体DHHS3为例。使用去污剂十二烷基麦芽糖苷（DDM）从全细胞裂解物中提取重组蛋白，并进行双柱纯化。通过尺寸排阻色谱（SEC）检查纯化蛋白的均一性和单分散性。400 mL Sf9昆虫细胞培养物感染后的制备物通常可产生0.5 mg的DHHC3和1.0 mg的催化无活性的DHHS3。通过SEC，两种形式在浓度高达1 mg/mL时均呈现单分散状态。
