Lin Chiann-Tso, Moore Priscilla A, Auberry Deanna L, Landorf Elizabeth V, Peppler Terese, Victry Kristin D, Collart Frank R, Kery Vladimir
Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Protein Expr Purif. 2006 May;47(1):16-24. doi: 10.1016/j.pep.2005.11.015. Epub 2005 Dec 15.
Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.
蛋白质晶体学、绘制蛋白质相互作用图谱以及其他功能基因组学方法需要纯化许多不同的蛋白质，每种蛋白质都要有足够的产量和纯度，以用于后续的高通量应用。为了有效地满足这一需求，有必要开发强大、自动化的高通量蛋白质表达和纯化流程。我们开发并比较了两种基于在大肠杆菌（E. coli）中表达细菌基因来自动纯化重组蛋白的替代工作流程。第一种是过滤分离方案，其中目的蛋白在800毫升大肠杆菌培养物的大量培养物中表达，然后使用Ni-NTA琼脂糖（Qiagen）通过过滤纯化进行分离。第二种是较小规模的磁分离方法，其中目的蛋白在25毫升大肠杆菌培养物的小体积培养物中表达，然后使用带有MagneHis Ni2+琼脂糖（Promega）的96孔纯化系统进行分离。两种工作流程都提供了相当的蛋白质平均产量，以在600纳米处测量的细菌培养物每光密度单位计，约为8微克纯化蛋白。我们讨论了这些自动化工作流程的优点和局限性，它们每次纯化运行可提供纯度超过90%且产量在100微克至45毫克范围内的蛋白质，以及优化这些方案的策略。
