大鼠突变型硒蛋白W结合和未结合谷胱甘肽时的选择性产生。

Selective production of rat mutant selenoprotein W with and without bound glutathione.

作者信息

Bauman A T, Malencik D A, Barofsky D F, Barofsky E, Anderson S R, Whanger P D

机构信息

Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA.

出版信息

Biochem Biophys Res Commun. 2004 Jan 9;313(2):308-13. doi: 10.1016/j.bbrc.2003.11.133.

DOI:10.1016/j.bbrc.2003.11.133

PMID:14684161

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein.

摘要

对带有6x组氨酸标签的大鼠突变硒蛋白W（RMSW）重组形式进行基质辅助激光解吸/电离质谱（MALDI MS）和电喷雾电离质谱（ESI MS）分析，结果显示需氧生长条件下主要产生一种未结合谷胱甘肽的RMSW形式（10,305道尔顿），而厌氧条件下产生一种谷胱甘肽结合形式（305道尔顿）（10,610道尔顿）。除了镍-亚氨基二乙酸琼脂糖层析外，还采用丙酮沉淀和二乙氨基乙基纤维素层析程序实现了RMSW的纯化。从镍-亚氨基二乙酸琼脂糖柱洗脱后，还需要包括多价金属离子结合（PMIB）树脂层析和羧甲基纤维素层析在内的额外步骤，以保持纯化蛋白的溶解性。