Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States.
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States; Genetics Institute, University of Florida, Gainesville, FL, United States.
Methods Enzymol. 2021;659:315-326. doi: 10.1016/bs.mie.2021.08.002. Epub 2021 Sep 2.
Tandem affinity purification is a useful strategy to isolate multisubunit complexes of high yield and purity but can be limited when working with halophilic proteins that are not properly expressed in Escherichia coli. Halophilic proteins are desirable for bioindustrial applications as they are often stable and active in organic solvents; however, these proteins can be difficult to express, fold, and purify by traditional technologies. Haloarchaea provide a useful alternative for expression of halophilic proteins. These microorganisms use a salt-in strategy to maintain homeostasis and express most of their proteins with halophilic properties and low pI. Here, we provide detailed protocols for the genetic modification, expression and tandem affinity purification of "salt-loving" multisubunit complexes from the haloarchaeon Haloferax volcanii. The strategy for isolation of affinity tagged 20S proteasomes that form cylindrical proteolytic nanomachines of α1, α2 and β subunits is described.
串联亲和纯化是一种从大肠杆菌中高效、高纯度地分离多亚基复合物的有用策略，但当涉及到不能正确表达的嗜盐蛋白时，该策略可能会受到限制。嗜盐蛋白在生物工业应用中很有价值，因为它们通常在有机溶剂中稳定且具有活性；然而，这些蛋白质用传统技术进行表达、折叠和纯化可能会比较困难。盐杆菌可以作为表达嗜盐蛋白的替代方法。这些微生物使用盐进策略来维持体内平衡，并表达大多数具有嗜盐特性和低等电点的蛋白质。在这里，我们提供了来自嗜盐古菌盐火菌的详细方案，用于“嗜盐”多亚基复合物的遗传修饰、表达和串联亲和纯化。描述了形成 α1、α2 和 β 亚基的圆柱形蛋白水解纳米机器的亲和标记 20S 蛋白酶体的分离策略。
