Membrane Protein Protocols

Membrane Protein Protocols : Expression, Purification, and Characterization

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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.
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Product details

  • Hardback | 334 pages
  • 160 x 240 x 25mm | 698.54g
  • Totowa, NJ, United States
  • English
  • 2003 ed.
  • XXII, 334 p.
  • 1588291243
  • 9781588291240
  • 2,584,769

Back cover copy

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.
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Table of contents

Part I. Expression of Membrane Proteins

Expression and Purification of the Amphipathic Form of Rabbit Cytochrome b5 in Escherichia coli
Lucy Waskell

Dihydroorotate Dehydrogenase of Escherichia coli
Kaj Frank Jensen and Sine Larsen

A General Approach for Heterologous Membrane Protein Expression in Escherichia coli: The Uncoupling Protein, UCP1, as an Example
Alison Z. Shaw and Bruno Miroux

Expression of Membrane-Bound Iron-Sulfur Proteins
Dirk Schneider, Christian L. Schmidt, and Andreas Seidler

Heterologous Expression of Human Scavenger Receptor Class B Type I (SR-BI) in Pichia pastoris
Chang-Hoon Han, Moritz Werder, Wilk von Gustedt, and Helmut Hauser

Expression of Oligomeric Amiloride-Sensitive Epithelial Sodium Channel in Sf9 Insect Cells
U. Subrahmanyeswara Rao

Functional Expression of His-Tagged Rhodopsin Sf9 Insect Cells
Giel J.C.G.M. Bosman, Jenny van Oostrum, Githa Breikers, Petra H.M. Bovee-Geurts, Corne H.W. Klaassen, and Willem J. DeGrip

Part II. Detergent Selection in Membrane Protein Purification

Preparation of Glycerol Facilitator for Protein Structure and Folding Studies in Solution
Darren Manley and Joe D. O'Neil

Solubilization of Chemokine Receptors from Cell Membranes
Robert Staudinger and Juan C. Bandres

A Systematic Approach for the Solubilization of the Integral Membrane Protein Lysophospholipid: Acyl-CoA Acyltransferase (LAT)
Claus Kerkhoff and Volkhard Kaever

Part III. Membrane Protein Purification and Reconstitution

Purification of Omp50, a Minor Porin of Campylobacter jejuni
Jean Michel Bolla

Purification of Porins from Mycobacterium smegmatis
Christian Heinz, Eva Roth, and Michael Niederweis

Isolation of the Melanocortin 5 Receptor: From cDNA Sequence to Isolating an Integral Membrane Protein
Benjamin L.Clarke

Purification of Mammalian Serine Palmitoyltransferase, a Hetero-Subunit Enzyme for Sphingolipid Biosynthesis, by Affinity-Peptide Chromatography
Kentaro Hanada and Masahiro Nishijima

Purification of Phosphatidylglycerophosphate Synthase from Cultured Mammalian Cells
Kiyoshi Kawasaki and Masahiro Nishijima

Purification of Pancreastatin Receptor from Rat Liver Membranes
Victor Sanchez-Margalet, Jose Santos-Alvarez, and Sandra Diaz-Troya

Purification, Reconstitution, and Functional Characterization of Zinc Transporter from Rat Renal Brush Border Membranes
Rajendra Prasad

Isolation of Lipid Raft-Associated Proteolipids
Jaime Millan, Maria C. de Marco, Mohammed Qaidi, Alicia Batista, Fernando Martin-Belmonte, and Miguel A. Alonso

Purification of Membrane-Bound Catechol-O-Methyltransferase
Maria Joao Bonifacio and Patricio Soares-da-Silva

Purification and Characterization of Transporter Proteins from Human Erythrocyte Membrane
Da-Neng Wang, M. Joanne Lemieux, and Jonathan M. Boulter

Purification of the Human Erythrocyte PS-Stimulated Mg2+-ATPase: A Putative PS Flippase
Jill V. Lyles, Kathleen Cornely-Moss, Christine M. Smith, and David J. Daleke

Reconstitution and Assay of Biogenic Membrane-Derived Phospholipid Flippase Activity in Proteoliposomes
Sathyanarayana N. Gummadi, Sigrun Hrafnsdottir, Jane Walent, William E. Watkins, and Anant K. Menon

Expression, Purification, and Reconstitution of Rat Liver Carnitine Palmitoyltransferase I
Nicholas F. Brown

Part IV. Structural Analysis of Membrane Proteins

Crystallization in Lipidic Cubic Phases: A Case Study with Bacteriorhodopsin
Valentin I. Gordeliy, Ramona Schlesinger, Rouslan Efremov, Georg Buldt, and Joachim Heberle

Optical Biosensor Assay Using Retroviral Receptor Pseudotypes
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Review quote

"Investigators working with membrane proteins will want to acquire this book. This is a useful compilation of protocols. . .there is much of utility in this book"-Doody's Health Sciences Book Review Journal

"...a useful reference work for researchers investigating the structure and function of membrane proteins." -- Immunology News

"...provides good food for thought for new and advanced membrane protein researchers." - ChemBioChem

"...highly recommendable to people working in proteomics..." - Proteomics Journal
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