Membrane Protein Protocols: Expression, Purification, and Characterization (Methods in Molecular Biology (228))

Knowledge of the three-dimensional structure of a protein is absolutely required for the complete understanding of its function. The spatial orientation of amino acids in the active site of an enzyme demonstrates how substrate specificity is defined, and assists the medicinal chemist in the design of s- cific, tight-binding inhibitors. The shape and contour of a protein surface hints at its interaction with other proteins and with its environment. Structural ana- sis of multiprotein complexes helps to define the role and interaction of each individual component, and can predict the consequences of protein mutation or conditions that promote dissociation and rearrangement of the complex. Determining the three-dimensional structure of a protein requires milligram quantities of pure material. Such quantities are required to refine crystallization conditions for X-ray analysis, or to overcome the sensitivity limitations of NMR spectroscopy. Historically, structural determination of proteins was limited to those expressed naturally in large amounts, or derived from a tissue or cell source inexpensive enough to warrant the use of large quantities of cells. H- ever, with the advent of the techniques of modern gene expression, many p- teins that are constitutively expressed in minute amounts can become accessible to large-scale purification and structural analysis.

Recent developments in molecular biology and biochemistry have made it possible to determine the three-dimensional structure of membrane proteins and so come to a full understanding of their function. In Membrane Protein Protocols: Expression, Purification, and Characterization, researchers at major universities and research centers around the world describe in detail the key techniques that have proven successful in the study of receptors and transport proteins. The book provides examples of how different membrane proteins can be overexpressed in both prokaryotic and eukaryotic expression systems, how natural and overexpressed proteins can be solubilized from their host membranes, and how the solubilized protein can be purified in active form. Each protocol contains step-by-step instructions to ensure success, troubleshooting advice, lists of reagents, and tips on avoiding pitfalls.
Timely and highly practical, Membrane Protein Protocols: Expression, Purification, and Characterization offers investigators proven methods to determine the three-dimensional structure of membrane receptors and transport proteins, as well as proven practical help in the discovery of new clinical targets for the design of novel pharmaceutical agents useful in treating cancer, bacterial and viral infection, and many metabolic diseases.