Protein Secretion

In this book, leading experts provide robust and well-established protocols to elucidate the multiplicity of tools that have been developed to study protein sorting, membrane targeting, transmembrane crossing, and secretion across multiple membranes.

Over the past thirty years, many elegant genetic and biochemical approaches have been combined in order to advance the study of protein secretion and the necessary navigation through cell membranes, yet, despite this progress, less than two hundred membrane protein structures are known, nowhere near the complete inventory that the discovered protein export systems suggest. In Protein Secretion: Methods and Protocols , leading experts in the field provide robust, well-established protocols to elucidate the multiplicity of tools that have been developed to study protein sorting, membrane targeting, transmembrane crossing, and secretion across multiple membranes. With examples involving both prokaryotic and eukaryotic organisms, the volume covers subjects ranging from bioinformatics and proteomics to fundamental enzymology and genetics to cell biology, structural analyses, and biophysics. Written in the highly successful Methods in Molecular Biology ™ series format, chapters contain introductions to their respective topics, lists of the key materials and reagents, step-by-step, readily reproducible protocols, and detailed notes on troubleshooting and avoiding known pitfalls.

Comprehensive and dependable, Protein Secretion: Methods and Protocols focuses on well-characterized paradigms so that scientists studying a vast array of subjects from biochemistry and genetics to biotechnology and biopharmaceuticals can benefit and expand upon their vital research.

Comprehensive guide to developing, running, and analyzing assays to monitor protein secretion from single proteins to proteome scale Presents a modular approach to experimental design, covering model biological systems, screening modalities, detection technologies, and bioinformatics tools Covers a wide range of cell-based and in vitro assay formats and systems Includes supplementary material: sn.pub/extras

Inhalt
The Extraordinary Diversity of Bacterial Protein Secretion Mechanisms.- In Vitro and In Vivo Approaches to Studying the Bacterial Signal Peptide Processing.- Membrane Insertion of Small Proteins.- Membrane Protein Insertion in E. coli.- Study of Polytopic Membrane Protein Topological Organization as a Function of Membrane Lipid Composition.- In Vivo Analysis of Protein Translocation to the Escherichia coli Periplasm.- Sorting of Bacterial Lipoproteins to the Outer Membrane by the Lol System.- Purification and Functional Reconstitution of the Bacterial Protein Translocation Pore, the SecYEG Complex.- Reconstitution of the SecY Translocon in Nanodiscs.- In Vitro Assays to Analyze Translocation of the Model Secretory Preprotein Alkaline Phosphatase.- Characterization of Interactions Between Proteins Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.- Analysis of Tat Targeting Function and Twin-Arginine Signal Peptide Activity in Escherichia coli.- Site-Specific Cross-Linking of In Vitro Synthesized E. coli Preproteins for Investigating Transmembrane Translocation Pathways.- Tracking the Secretion of Fluorescently Labeled Type III Effectors from Single Bacteria in Real Time.- Comparative Analysis of Cytoplasmic Membrane Proteomes of Escherichia coli Using 2D Blue Native/SDS-PAGE.- Using Hidden Markov Models to Discover New Protein Transport Machines.- Bioinformatics Predictions of Localization and Targeting.- The Chloroplast Protein Import Machinery: A Review.- Measurement of the Energetics of Protein Transport Across the Chloroplast Thylakoid Membrane.- In Vitro Dissection of Protein Translocation into the Mammalian Endoplasmic Reticulum.- In Vitro Reconstitution of the Selection, Ubiquitination, and Membrane Extraction of a Polytopic ERAD Substrate.- Studying the ArfGAP-Dependent Conformational Changes in SNAREs.- Studying Endoplasmic Reticulum Function In Vitro Using siRNA.- High-Quality Immunofluorescence of Cultured Cells.- TrappingOxidative Folding Intermediates During Translocation to the Intermembrane Space of Mitochondria: In Vivo and In Vitro Studies.- Native Techniques for Analysis of Mitochondrial Protein Import.