Jeon Won Bae, Aceti David J, Bingman Craig A, Vojtik Frank C, Olson Andrew C, Ellefson Jason M, McCombs Janet E, Sreenath Hassan K, Blommel Paul G, Seder Kory D, Burns Brendan T, Geetha Holalkere V, Harms Amy C, Sabat Grzegorz, Sussman Michael R, Fox Brian G, Phillips George N
The Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Bobcock Drive, 53706-1549, USA.
J Struct Funct Genomics. 2005;6(2-3):143-7. doi: 10.1007/s10969-005-1908-7.
The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.
真核生物结构基因组学中心（CESG）已建立了以高通量模式纯化拟南芥蛋白的程序。重组蛋白在其N端与（His）₆-MBP标签融合，并在大肠杆菌中表达。使用自动AKTApurifier系统，融合蛋白首先通过固定化金属亲和色谱（IMAC）进行纯化。在通过TEV蛋白酶切割（His）₆-MBP标签后，通过减法第二次IMAC将（His）₆-MBP标签与目标蛋白分离。作为质量保证的一部分，所有纯化的蛋白都进行了基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和电喷雾电离质谱（ESI）分析，以确认目标蛋白的身份和完整性，并确定硒代甲硫氨酸（SeMet）以及¹⁵N和¹³C同位素的掺入情况。这些方案已成功用于提供适合通过X射线晶体学和核磁共振进行结构研究的高质量蛋白。
