International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 3.2, p. 92

Section 3.2.1. Introduction

J. A. Ernst,a,b D. G. Yansurac and C. M. Kothd*

aDepartment of Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,bDepartment of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,cDepartment of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA, and dDepartment of Structural Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA
Correspondence e-mail:  koth.christopher@gene.com

3.2.1. Introduction

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Integral membrane proteins constitute about a third of the proteome of most organisms but less than 1% of all entries in protein structural databases (Berman et al., 2002[link]). This disparity is largely due to inherent difficulties in their expression, solubilization and purification. The production of sufficient protein for structural studies can be challenging for any target, but several obstacles are unique to membrane proteins. For example, proper insertion into the membrane relies on host cellular machinery that may be limiting or incompatible. Exceeding this capacity can lead to cell death or the accumulation of aggregated and inactive protein within the cell (Geertsma et al., 2008[link]). Also, post-translational modifications such as glycosylation, acylation and sulfation may not be faithfully reproduced (Grisshammer & Tate, 1995[link]). As a further complication, even if sufficient expression can be achieved, most membrane-protein structural studies require that the target be extracted from the cellular membrane using detergents. However, detergents can adversely affect protein structure and function, as well as influence the outcome of crystal trials (Engel et al., 2002[link]; Lemieux et al., 2003[link]; Prive, 2007[link]). Fortunately, if a strategy for purifying sufficient quantities of a given membrane-protein target can be established, crystallization strategies largely mimic the standard techniques for soluble proteins (Newby et al., 2009[link]).

Despite the aforementioned challenges, high-resolution structures of almost 200 unique membrane proteins have now been solved (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html ), the vast majority using protein produced by recombinant methods (Willis & Koth, 2008[link]). The rate of new structure determinations has also increased dramatically over the last few years, mimicking the exponential growth of soluble protein structures in the early 1980s. Space limitations here preclude a thorough review of all possible membrane-protein production methods. However, an examination of the successful expression, solubilization and purification strategies that have led to membrane-protein structures reveals that, in many cases, remarkably similar methods have been used (Carpenter et al., 2008[link]; Willis & Koth, 2008[link]; Newby et al., 2009[link]). Guided by these methods, the following sections detail a rational and consensus `first-attempt' strategy that has worked for a broad range of membrane targets with only minor variations in technique (Dobrovetsky et al., 2005[link], 2007[link]; Lunin et al., 2006[link]). It must be noted that while this represents an evidence-guided approach, the methods provided herein will not work for every membrane protein and, in fact, will fail for many. Thus, prioritized lists of alternative strategies are provided for those targets in which the initial expression or isolation attempts do not succeed or problems are encountered.

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