ABSTRACT
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-ß-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.
Subject(s)
Cell Membrane/chemistry , Detergents/chemistry , Glucosides/chemistry , Membrane Proteins/isolation & purification , Alkylation , Animals , Cell Fractionation , Cerebral Cortex/chemistry , Chemical Precipitation , Chromatography, Liquid , Crystallization , Escherichia coli/chemistry , Membrane Proteins/chemistry , Oxidation-Reduction , Proteolysis , Saccharomyces cerevisiae/chemistry , Swine , Tandem Mass SpectrometryABSTRACT
Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large-scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high-value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.
