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Full Description
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.
Contents
Expression of Membrane Proteins.- Expression and Purification of the Amphipathic Form of Rabbit Cytochrome b5 in Escherichia coli.- Dihydroorotate Dehydrogenase of Escherichia coli.- A General Approach for Heterologous Membrane Protein Expression in Escherichia coli.- Expression of Membrane-Bound Iron-Sulfur Proteins.- Heterologous Expression of Human Scavenger Receptor Class B Type I (SR-BI) in Pichia pastoris.- Expression of Oligomeric Amiloride-Sensitive Epithelial Sodium Channel in Sf9 Insect Cells.- Functional Expression of His-Tagged Rhodopsin in Sf9 Insect Cells.- Detergent Selection in Membrane Protein Purification.- Preparation of Glycerol Facilitator for Protein Structure and Folding Studies in Solution.- Solubilization of Chemokine Receptors from Cell Membranes.- A Systematic Approach for the Solubilization of the Integral Membrane Protein Lysophospholipid.- Membrane Protein Purification.- Purification of Omp50, a Minor Porin of Campylobacter jejuni.- Purification of Porins from Mycobacterium smegmatis.- Isolation of the Melanocortin 5 Receptor.- Purification of Mammalian Serine Palmitoyltransferase, a Hetero-Subunit Enzyme for Sphingolipid Biosynthesis, by Affinity-Peptide Chromatography.- Purification of Phosphatidylglycerophosphate Synthase from Cultured Mammalian Cells.- Purification of Pancreastatin Receptor from Rat Liver Membranes.- Purification, Reconstitution, and Functional Characterization of Zinc Transporter from Rat Renal Brush Border Membranes.- Isolation of Lipid Raft-Associated Proteolipids.- Purification of Membrane-Bound Catechol-O-Methyltransferase.- Purification and Characterization of Transporter Proteins from Human Erythrocyte Membrane.- Purification of the Human Erythrocyte PS-Stimulated Mg2+-ATPase.- Reconstitution and Assay of Biogenic Membrane-Derived Phospholipid Flippase Activity in Proteoliposomes.- Expression, Purification, and Reconstitution of Rat Liver Carnitine Palmitoyltransferase I.- Structural Analysis of Membrane Proteins.- Crystallization in Lipidic Cubic Phases.- Optical Biosensor Assay Using Retroviral Receptor Pseudotypes.
