Kim Jared, Ng Ho Leung
Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii.
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas.
Curr Protoc Protein Sci. 2017 Nov 1;90:29.19.1-29.19.10. doi: 10.1002/cpps.40.
This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as n-dodecyl-β-maltoside (DDM). Detergent extracts are then centrifuged, heat precipitated, and filtered to remove insoluble, thermally unstable, and/or aggregated proteins. Samples are then prepared for analysis by mass spectrometry: proteins are precipitated by methanol/chloroform extraction and subjected to reduction, alkylation, and protease digestion. The resulting peptides are passed through a detergent removal column, desalted, rehydrated in 0.1% formic acid (v/v), and identified by LC-MS/MS. © 2017 by John Wiley & Sons, Inc.
本单元通过筛选来自大肠杆菌、酿酒酵母和猪脑组织的膜蛋白以获取有利于结晶的生化特性，从而应对与膜蛋白晶体学相关的几个关键挑战。首先，获取组织样本或细胞沉淀。分离细胞、洗涤，然后通过超声处理、珠磨法或手动匀浆进行裂解。通过离心从裂解物中分离膜蛋白，并溶解在适合结晶的温和去污剂中，如正十二烷基-β-麦芽糖苷（DDM）。然后将去污剂提取物离心、热沉淀并过滤，以去除不溶性、热不稳定和/或聚集的蛋白质。接着制备样品用于质谱分析：通过甲醇/氯仿萃取沉淀蛋白质，并进行还原、烷基化和蛋白酶消化。所得肽段通过去污剂去除柱，脱盐，在0.1%甲酸（v/v）中复水，并通过液相色谱-串联质谱（LC-MS/MS）进行鉴定。© 2017约翰威立父子公司版权所有
