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Membrane Proteins – Production and Functional Characterization - 1st Edition - ISBN: 9780128015216, 9780128016251

Membrane Proteins – Production and Functional Characterization, Volume 556

1st Edition

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Series Volume Editor: Arun Shukla
Hardcover ISBN: 9780128015216
eBook ISBN: 9780128016251
Imprint: Academic Press
Published Date: 7th April 2015
Page Count: 670
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Table of Contents

    <li>Preface</li> <li>Section I: Recombinant Expression of Membrane Proteins<ul><li>Chapter One: Engineering <i>Escherichia coli</i> for Functional Expression of Membrane Proteins<ul><li>Abstract</li><li>1 Introduction</li><li>2 Preparation of Erythromycin-Sensitive <i>E. coli</i> Strain</li><li>3 Preparation of Expression Plasmid</li><li>4 Selecting Cells for Better Expression</li><li>5 Characterizing Evolved Strains</li><li>6 Summary</li><li>Acknowledgments</li></ul></li><li>Chapter Two: ACEMBLing a Multiprotein Transmembrane Complex: The Functional SecYEG-SecDF-YajC-YidC Holotranslocon Protein Secretase/Insertase<ul><li>Abstract</li><li>1 Introduction</li><li>2 ACEMBLing the HTL Multiprotein Complex</li><li>3 Purifying the HTL</li><li>4 HTL Integrity and Activity</li><li>5 Discussion and Conclusions</li><li>Acknowledgments</li></ul></li><li>Chapter Three: Expression and Purification of OsVDAC4<ul><li>Abstract</li><li>1 Introduction</li><li>2 Equipment</li><li>3 Materials</li><li>4 Protocols</li><li>Acknowledgments</li></ul></li><li>Chapter Four: Membrane Protein Expression in <i>Lactococcus lactis</i><ul><li>Abstract</li><li>1 Introduction</li><li>2 Equipment and Materials</li><li>3 Buffers and Media</li><li>4 Protocol</li><li>5 Step 1: Cloning the Target Gene into pNZ8048 and Transformation into <i>L. lactis</i></li><li>6 Step 2: Growth of <i>L. lactis</i>, Expression of the Target Protein and Preparation of Membrane Vesicles</li><li>Acknowledgment</li></ul></li><li>Chapter Five: An Unconventional Anaerobic Membrane Protein Production System Based on <i>Wolinella succinogenes</i><ul><li>Abstract</li><li>1 Introduction</li><li>2 Methods</li><li>3 Purification of Proteins Expressed in <i>W</i>. <i>succinogenes</i></li><li>Acknowledgments</li></ul></li><li>Chapter Six: Membrane Protein Expression and Analysis in Yeast<ul><li>Abstract</li><li>1 Theory</li><li>2 Equipment</li><li>3 Materials</li><li>4 Protocol</li><li>5 Step 1: Transformation of <i>Y. lipolytica</i></li><li>6 Step 2: Small-Scale Membrane Protein Expression</li><li>7 Step 3: Membrane Protein Preparation</li><li>8 Step 4: Membrane Protein Analysis: Expression Levels</li><li>9 Step 5: Membrane Protein Analysis: Functionality</li></ul></li><li>Chapter Seven: Heterologous Expression of G-Protein-Coupled Receptors in Yeast<ul><li>Abstract</li><li>1 Introduction</li><li>2 Pichia pastoris</li><li>3 Saccharomyces cerevisiae</li><li>4 Which System to Choose?</li><li>5 Generating <i>P. pastoris</i> Expression Plasmids</li><li>6 Generating and Screening the <i>P. pastoris</i> Expression Clones</li><li>7 Some Considerations</li><li>8 Generating <i>S. cerevisiae</i> Expression Clones</li><li>9 In-Gel Fluorescence</li><li>10 Confocal Microscopy</li><li>11 Assessment of Protein Expression Levels</li><li>12 Yeast Cell Growth Assay</li><li>13 Optimization of the Assay Conditions</li><li>14 Outcome of the Functional Analysis</li><li>15 Conclusions</li><li>Acknowledgments</li></ul></li><li>Chapter Eight: Recombinant G Protein-Coupled Receptor Expression in <i>Saccharomyces cerevisiae</i> for Protein Characterization<ul><li>Abstract</li><li>1 Introduction</li><li>2 Plasmid Design</li><li>3 Characterization</li><li>4 Conclusion</li><li>Acknowledgments</li></ul></li><li>Chapter Nine: Baculovirus-Mediated Expression of GPCRs in Insect Cells<ul><li>Abstract</li><li>1 Introduction</li><li>2 Cell Maintenance</li><li>3 Recombinant Bacmid DNA</li><li>4 Transfection, Amplification, and Storage of Virus</li><li>5 Protein Expression</li></ul></li><li>Chapter Ten: Expression of Membrane Proteins in the Eyes of Transgenic <i>Drosophila melanogaster</i><ul><li>Abstract</li><li>1 Introduction</li><li>2 Protein Expression in Transgenic <i>Drosophila melanogaster</i></li><li>3 Protocol</li><li>4 Case Study: Signal Peptide Peptidase</li><li>5 Conclusions and Outlook</li></ul></li><li>Chapter Eleven: Rapid Method to Express and Purify Human Membrane Protein Using the <i>Xenopus</i> Oocyte System for Functional and Low-Resolution Structural Analysis<ul><li>Abstract</li><li>1 Introduction</li><li>2 Subcloning in pMJB08 and cRNA Production</li><li>3 Frog Surgery, Oocytes Preparation and Injection</li><li>4 Determination of Maximal Level Expression</li><li>5 Membrane Preparation and Detergent Screening</li><li>6 Membrane Protein Purification: Ion Metal Affinity Chromatography</li><li>7 Posttranslational Verification Using Deglycosylation</li><li>8 Determination of Surface Membrane Expression Using Surface Biotinylation</li><li>9 Functional Verification of Membrane Proteins in the Oocyte</li><li>10 Grids Preparation and Negative Staining</li><li>11 Image Processing</li><li>12 Conclusion</li><li>Acknowledgments</li></ul></li><li>Chapter Twelve: Expression of G Protein-Coupled Receptors in Mammalian Cells<ul><li>Abstract</li><li>1 Introduction</li><li>2 Materials and Supplies</li><li>3 Procedure</li><li>Acknowledgments</li></ul></li><li>Chapter Thirteen: Construction of Stable Mammalian Cell Lines for Inducible Expression of G Protein-Coupled Receptors<ul><li>Abstract</li><li>1 Introduction</li><li>2 Construction of Stable HEK293S Cell Lines Containing Integrated Genes Regulated by Tetracycline</li><li>3 Growth of Tetracycline-Inducible Stable Cell Lines in Suspension Culture</li><li>4 Purification of Membrane Proteins Tagged with the Antirhodopsin Rho-1D4 Antibody</li><li>Acknowledgments</li></ul></li><li>Chapter Fourteen: Rapid and Facile Recombinant Expression of Bovine Rhodopsin in HEK293S GnTI<sup>&#x2212;</sup> Cells Using a <i>PiggyBac</i> Inducible System<ul><li>Abstract</li><li>1 Theory</li><li>2 Protocol</li><li>3 Step 1: Subcloning of Wild-Type and Mutant Opsin cDNA into Plasmid, PB-T-PAF</li><li>4 Step 2: Transfection, Transient Expression, and Generation of Stable Bulk Cell Cultures</li><li>5 Step 3: Scale-Up of Bulk Cell Cultures in Roller Bottles</li><li>6 Step 4: Induction and Cell Harvesting</li><li>7 Step 5: Rhodopsin Purification and Spin Labeling</li><li>8 Conclusion</li><li>Acknowledgments</li></ul></li><li>Chapter Fifteen: Semliki Forest Virus-Based Expression of Recombinant GPCRs<ul><li>Abstract</li><li>1 Introduction</li><li>2 SFV Expression System</li><li>3 Expression of GPCRs</li><li>4 Summary</li></ul></li><li>Chapter Sixteen: Screening for Lipid Requirements of Membrane Proteins by Combining Cell-Free Expression with Nanodiscs<ul><li>Abstract</li><li>1 Introduction</li><li>2 General Considerations</li><li>3 Protocol: CF Lysate Preparation</li><li>4 Protocol: Preparation of NDs and Liposomes</li><li>5 Protocol: Optimizing the Efficiency of CF Expression</li><li>6 Protocol: CF Expression Reactions in the L-CF Mode</li><li>7 Protocol: Lipid Modulation of Membrane Protein Activity: The Example of MraY Translocases</li><li>8 Protocol: Purification of CF-Expressed Membrane Proteins</li><li>9 Conclusion and Outlook</li><li>Acknowledgments</li></ul></li></ul></li> <li>Section II: Reconstitution and Functional Characterization of Membrane Proteins<ul><li>Chapter Seventeen: Liposome Reconstitution and Transport Assay for Recombinant Transporters<ul><li>Abstract</li><li>1 Introduction</li><li>2 Reconsitution of Protein into Liposomes</li><li>3 Assaying Transport</li><li>4 Summary</li><li>Acknowledgment</li></ul></li><li>Chapter Eighteen: Lipid Reconstitution and Recording of Recombinant Ion Channels<ul><li>Abstract</li><li>1 Introduction</li><li>2 Liposome Reconstitution of Ion Channels</li><li>3 Liposome Flux Assays</li><li>4 Electrical Recording of Reconstituted Protein</li><li>5 Summary</li><li>Acknowledgments</li></ul></li><li>Chapter Nineteen: Reconstitution of Membrane Proteins: A GPCR as an Example<ul><li>Abstract</li><li>1 Introduction</li><li>2 Production of Liposomes</li><li>3 Reconstitution into Liposomes</li><li>4 Reconstitution into Nanodiscs</li><li>5 Reconstitution into Lipodisqs</li><li>6 Determination of Lipid-to-Protein Ratio</li><li>7 Concentration Methods</li><li>8 Summary</li><li>Acknowledgments</li></ul></li><li>Chapter Twenty: Ion Channel Reporter for Monitoring the Activity of Engineered GPCRs<ul><li>Abstract</li><li>1 Introduction</li><li>2 ICCR Design</li><li>3 Functional Characterization</li><li>4 Current Advantages and Limitations of the ICCR Technology</li><li>Acknowledgments</li></ul></li><li>Chapter Twenty-One: Methods for Labeling Skeletal Muscle Ion Channels Site-Specifically with Fluorophores Suitable for FRET-Based Structural Analysis<ul><li>Abstract</li><li>1 Introduction</li><li>2 Fluorescent Protein (FP) Fusions</li><li>3 Labeling of Tetracysteine (Tc) Tags by Biarsenical Fluorophores</li><li>4 Labeling of Poly-Histidine Tags by Fluorescent NTA Conjugates</li><li>5 Three-Way FRET Using a Combination of Site-Specific Labeling Methods</li><li>6 Summary</li><li>Acknowledgments</li></ul></li><li>Chapter Twenty-Two: Structural and Functional Studies of NirC from <i>Salmonella typhimurium</i><ul><li>Abstract</li><li>1 Introduction</li><li>2 Production and Isolation of <i>St</i>NirC</li><li>3 Structural Analysis of <i>St</i>NirC</li><li>4 Functional Assays for <i>St</i>NirC</li><li>5 Conclusions</li><li>Acknowledgments</li></ul></li><li>Chapter Twenty-Three: Surface Plasmon Resonance Analysis of Seven-Transmembrane Receptors<ul><li>Abstract</li><li>1 Introduction</li><li>2 Surface Plasmon Resonance</li><li>3 SPR Approaches to FBDD</li><li>4 SPR Applications for GPCRs</li><li>5 SPR Applications for Allosteric Compounds</li><li>6 SPR Fragment Screening of Thermostabilized GPCRs</li><li>7 SPR Fragment Screening of Fully Functional GPCRs</li><li>8 Confirmation of SPR Fragment Hits</li><li>9 Conclusion</li></ul></li><li>Chapter Twenty-Four: Cross-linking Strategies to Study Peptide Ligand&#x2013;Receptor Interactions<ul><li>Abstract</li><li>1 Introduction</li><li>2 Photochemical Cross-linking</li><li>3 Chemical Cross-linking</li><li>4 Cross-Linking by Unnatural Amino Acid Replacement into the Receptor</li><li>5 Identifying the Cross-link Site</li><li>6 Concluding Remarks</li><li>Acknowledgments</li></ul></li><li>Chapter Twenty-Five: From Recombinant Expression to Crystals: A Step-by-Step Guide to GPCR Crystallography<ul><li>Abstract</li><li>1 Introduction</li><li>2 Target Selection, Construct Design, and Protein Engineering</li><li>3 Recombinant Expression and Selection of Host System</li><li>4 Functional Solubilization and Homogenous Purification</li><li>5 Approaches for Crystallogenesis and Optimization</li><li>6 Conclusion and Future Perspectives</li><li>Acknowledgments</li></ul></li><li>Chapter Twenty-Six: Structure-Based Biophysical Analysis of the Interaction of Rhodopsin with G Protein and Arrestin<ul><li>Abstract</li><li>1 Introduction</li><li>2 Preparation of Proteins and Peptides</li><li>3 Protein X-ray Crystallography</li><li>4 <i>UV/Vis</i> Absorption Spectroscopy: &#x201C;Extra Meta II&#x201D;</li><li>5 Site-Directed Fluorescence Spectroscopy</li><li>6 FTIR Difference Spectroscopy</li><li>7 Molecular Dynamics Simulations</li><li>Acknowledgments</li></ul></li></ul></li> <li>Author Index</li> <li>Subject Index</li>


Membrane Proteins – Production and Function Characterization a volume of Methods in Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines.

Key Features

  • Volume of Methods In Enzymology
  • Contains a collection of a diverse array of topics in the area of membrane protein biology ranging from recombinant expression, isolation, functional characterization, biophysical studies and crystallization


These two volumes of Methods In Enzymology should be very useful to any researcher - including graduate students, post-doctoral fellows, and faculty members - working in the area of structure and function of membrane proteins.


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© Academic Press 2015
7th April 2015
Academic Press
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About the Series Volume Editor

Arun Shukla

Arun Shukla

Dr. Arun K. Shukla obtained his M.Sc. (Master in Science) from the Center for Biotechnology at the Jawaharlal Nehru University in New Delhi, India. Dr. Shukla did his Ph.D. from the Department of Molecular Membrane Biology at the Max Planck Institute of Biophysics in Frankfurt, Germany. His Ph.D. research work was focused on structural studies of G Protein-Coupled Receptors (GPCRs).

Dr. Shukla subsequently carried out his post-doctoral work in the Department of Medicine at the Duke University in North Carolina, USA. During his post-doctoral research work, Dr. Shukla focused on understanding the biophysical and structural basis of ß-arrestin mediated regulation of GPCRs and non-canonical GPCR signaling. Dr. Shukla has served as an Assistant Professor in the Department of Medicine at the Duke University in Durham, North Carolina, USA.

Dr. Shukla is currently an Assistant Professor in Department of Biological Sciences and Bioengineering at the Indian Institute of Technology, Kanpur, India. Dr. Shukla is also an Intermediate Fellow of the Wellcome Trust-DBT India Alliance. The research program in Dr. Shukla’s laboratory is focused on understanding the molecular mechanism of activation, signaling and regulation of G Protein-Coupled Receptors.

Affiliations and Expertise

Indian Institute of Technology, Kanpur, India