High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae.
High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae.
复制标题
酿酒酵母膜蛋白的高通量蛋白质生产。
DOI：
10.1007/978-1-4939-9624-7_11
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Hays,FranklinA
中科院分区：
文献类型：
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作者：
Johnson,JenniferM;Hays,FranklinA
本章概述了一个协议，以评估大规模的蛋白质生产和纯化选定的目标，从最初的中等通量克隆策略的可行性。因此，可以使用经验最小化方法评估大量潜在的候选蛋白质、突变体或表达变体。此外，该方案的一个关键输出是利用酵母作为有效筛选和生产纯化蛋白质的手段。该方案的主要焦点是多位整合膜蛋白的过表达，尽管方法可以同样应用于可溶性蛋白。该方案首先概述了高通量（无机器人）克隆表达蛋白到双标签酵母表达质粒。然后筛选这些膜蛋白的表达水平、去污剂溶解性、初始纯度和色谱特征。小规模和大规模的表达方法都将沿着发酵进行讨论。
This chapter outlines a protocol to assess viability for large-scale protein production and purification for selected targets from an initial medium-throughput cloning strategy. Thus, one can assess a broad number of potential candidate proteins, mutants, or expression variants using an empirically minimalistic approach. In addition, a key output from this protocol is utilization ofSaccharomyces cerevisiaeas a means for the efficient screening and production of purified proteins. The primary focus in this protocol is overexpression of polytopic integral membrane proteins though methods can be equally applied to soluble proteins. The protocol starts with outlining high-throughput (sans robotics) cloning of expression proteins into a dual-tag yeast expression plasmid. These membrane proteins are then screened for expression level, detergent solubilization, initial purity, and chromatography characteristics. Both small- and large-scale expression methods are discussed along with fermentation.